Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein

Hideyuki Takahashi; Naoshi Dohmae; Kwang Sik Kim; Ken Shimuta; Makoto Ohnishi; Shigeyuki Yokoyama; Tatsuo Yanagisawa

doi:10.1371/journal.pone.0237883

. 2020 Aug 31;15(8):e0237883. doi: 10.1371/journal.pone.0237883

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein

Hideyuki Takahashi ^1,^*, Naoshi Dohmae ², Kwang Sik Kim ³, Ken Shimuta ¹, Makoto Ohnishi ¹, Shigeyuki Yokoyama ^4,⁵, Tatsuo Yanagisawa ^4,⁵

Editor: Nediljko Budisa⁶

PMCID: PMC7458321 PMID: 32866169

Abstract

Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNA^Pyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNA^Pyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein.

Introduction

Neisseria meningitidis is a fastidious Gram-negative microorganism that generally exists in the non-invasive so-called “carrier state” at a rate of 0.4–25% in human populations [1, 2]. However, N. meningitidis exhibits the abilities to cross the epithelial layer of the upper respiratory tract, infiltrate the bloodstream, evade the defenses of the human immune system, adhere to the endothelial layers of peripheral and brain vessels, cross the brain-blood barrier, and replicate in cerebrospinal fluid [3]. Although epidemiological analyses previously suggested that a human genetic polymorphism [4] and climate conditions [5] are important for predicting the outcomes of infection, the reasons why invasive meningococcal diseases (IMD) occur in some individuals, but not in others remain unclear [6].

Classical analyses revealed the factors exposed on meningococcal surfaces, such as the polysaccharide capsule, pili, Opa, and Opc, (reviewed in [7–9]), and genome mining with whole-genome sequencing (WGS) identified the following meningococcal pathogenic factors: NhhA, NadA [10], App [11], NalP [12], MspA [13], TspA [14], adhesion complex protein (ACP) [15], and fHbp [16]. Among the factors described above, meningococcal pili, which are mainly composed of the major pilin, PilE, and three minor pilins, PilV, PilX, and ComP, play the most important roles in the meningococcal infection processes involved in interactions with human epithelial and endothelial cells (reviewed in [17, 18]). To elucidate the molecular mechanisms underlying meningococcal pathogenesis in more detail, functional genomics by linking genotypes to phenotypes have allowed investigation of the relationships between gene transcript abundance or deficiencies and the capacity to function under various physiological conditions to be investigated, by genome-wide signature-tagged mutagenesis (STM) with an infant rat infection model [19], with human cultured cells [20], and in different media [21], microarrays with infection in human cultured cells [22], comparative genomics [23, 24], transcriptomics [25–28], and proteomics [29–31]. However, the mechanisms by which N. meningitidis causes septicemia and meningitis in humans cannot be completely explained by these characterized factors [32].

Although progressive WGS recently identified many genes in the meningococcal genome, more than half of those in the annotated open reading frame have remained as function-unknown hypothetical proteins. One of the difficulties associated with the characterization of meningococcal hypothetical proteins may be the limited number of analytical methods applicable to N. meningitidis, including tools to directly manipulate meningococcal components [33, 34]. Under these conditions, we also conducted STM to identify the factors responsible in the sequence type (ST)-2032 N. meningitidis strain, known for its high infectious ability in human brain microvascular endothelial cells (HBMEC) [35]. Among the STM mutants with highly defective infectious abilities in HBMEC, we identified PilV [36] of the meningococcal pilus and GltT-GltM glutamate transporter [37, 38] as meningococcal pathogenic factors for infection in host cells. However, several STM mutants were identified as disruptants of hypothetical genes, and we noted that, one disruptant of a hypothetical gene annotated as NMB1345 in the N. meningitidis MC58 genome [39], exhibited largely defective internalization ability into HBMEC. However, the deduced amino acid sequence of NMB1345 lacked homology with any function-known proteins from other species. We previously attempted to examine protein-protein interactions using already existing methods, such as the two-hybrid system or a pull-down assay, to find some clues about the function of the NMB1345 protein but were unsuccessful. Therefore, more powerful strategies are needed to elucidate the function of the NMB1345 protein in N. meningitidis.

One of the appropriate methods for our study is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNA^Pyl pair from Methanosarcina mazei (MmPylRS/T) [40]. The genetic code expansion allows ncAAs containing fluorophores, photocaged groups, photocrosslinkers, metal ion-chelating groups, and other groups to be site-specifically incorporated into target proteins in vivo in a broad range of species from Escherichia coli to eukaryotic systems, such as Caenorhabditis elegans, fruit flies, and mice (reviewed in [41]), thereby enabling in vivo examinations of biological functions. Protein photocrosslinking via the incorporation of photoreactive ncAAs has emerged as a superior strategy for identifying physiological interaction partner(s) and their functions [42, 43], and in combination with mass spectrometry (MS), these approaches have resolved biological issues that are difficult or impossible to address using the majority of currently available methods [42]. However, the question of whether the orthogonality of the tRNA^Pyl-PylRS pair is specific or compatible with the translational system in N. meningitidis has not been addressed.

In the present study, we applied genetic code expansion to N. meningitidis and developed a method for the genetic incorporation of photoreactive ncAAs into N. meningitidis in order to identify the physiological function of the NMB1345 protein. After establishing this method, we performed site-specific incorporation with one of the most widely used photoreactive amino acids, p-benzoyl-L-phenylalanine (pBPa) [44], into the NMB1345 protein in N. meningitidis. By using crosslinking in N. meningitidis followed by purification and MS analyses of endogenous proteins crosslinked to the NMB1345 protein, we found that the NMB1345 protein specifically interacted with PilE, a major component of meningococcal pili, and thus designated NMB1345 as PilE associated molecule A (PamA) in the present study. We further identified the amino acid residue of PilE crosslinked to PamA K273 and further found that the site-directed PilE mutation reduced both the interaction with PamA and the ability to internalize into HBMEC. Considering these results, we speculated that PamA plays an important role in the formation of meningococcal pili. This is the first study to successfully incorporate ncAAs into N. meningitidis in order to determine the physiological function of a protein with an unknown function and structure in pathogenic bacteria.

Materials and methods

Bacterial growth conditions

N. meningitidis strains HT1125 [35] and its derivatives, H44/76 [45] as well as derivatives harboring IncQ plasmids (Table 1) were routinely grown on GC agar plates at 37 ^oC in a 5% CO₂ atmosphere [46]. E. coli strains were grown in Luria-Bertani (LB) broth liquid cultures (Becton- Dickinson) or on LB plate (LB liquid medium containing 1.5% agar) or in MagicMedia (Invitrogen) at the indicated temperatures. When required, antibiotics were added at the following concentrations: kanamycin at 150 μg/ml, chloramphenicol at 5 μg/ml, erythromycin at 4 μg/ml, and spectinomycin at 75 μg/ml for N. meningitidis; kanamycin at 50 μg/ml, chloramphenicol at 25 μg/ml, and ampicillin at 50 μg/ml for E. coli. The N. meningitidis and E. coli strains used in the present study are listed in Table 2.

Table 1. IncQ plasmids introduced into N. meningitidis strains which results were shown in this study.

Plasmid	Relative properties	Antibiotic selection marker	References
pHT128	Derivative of pGSS33, an IncQ broad-host -range vector	Tet, Cml	[33]
pHT1212	Derivative of pHT128 carrying a lacI^q-P_tac-MmPylRS[Y306A/Y384F] and two P_lpp-tRNA^pyl genes	Tet, Cml	This study
pHT1262	Derivative of pHT128 carrying a lacI^q-MmPylRS([A302T/N346T/C348T/W417C]) and two P_lpp-tRNA^pyl genes	Tet, Cml	This study
pHT936	Derivative of pHT128 carrying lacI^q-P_tac-gst⁺ genes	Cml	This study
pHT1355	Derivative of pHT1212 carrying a P_tac-gst E51amb gene	Cml	This study
pHT1356	Derivative of pHT1212 carrying a P_tac-gst F52amb gene	Cml	This study
pHT1357	Derivative of pHT1262 carrying a P_tac-gst E51amb gene	Cml	This study
pHT1358	Derivative of pHT1262 carrying a P_tac-gst F52amb gene	Cml	This study
pHT1263	Derivative of pHT1262 carrying a pamA⁺-His₆ gene	Cml	This study
pHT1270	Derivative of pHT1262 carrying a pamA K208amb-His₆ gene	Cml	This study
pHT1274	Derivative of pHT1262 carrying a pamA K278amb-His₆ gene	Cml	This study
pHT1276	Derivative of pHT1262 carrying a pamA K309amb-His₆ gene	Cml	This study
pHT1279	Derivative of pHT1262 carrying a pamA K341amb-His₆ gene	Cml	This study
pHT1282	Derivative of pHT1262 carrying a pamA K382amb-His₆ gene	Cml	This study
pHT1284	Derivative of pHT1262 carrying a pamA K395amb-His₆ gene	Cml	This study
pHT1388	Derivative of pHT1262 carrying a pamA K278amb-StrepTag₂-His₆ gene	Cml	This study

Open in a new tab

Tet and Cml stand for tetracycline and chloramphenicol resistance marker, respectively.

Table 2. Strains used in this study.

N. meningitidis strains
HT1125
Strain	Genotype	Parent strain	Reference
HT1125	ΔsiaB-ΔsiaD::kan (ST-2032)	[35]
HT1572	ΔsiaB-ΔsiaD::kan pamA::Tn-spc	HT1125	This study
HT1736	ΔsiaB-ΔsiaD::kan pamA::Tn-spc ggt::pamA⁺-cat	HT1572	This study
HT1822	ΔsiaB-ΔsiaD::kan ΔpamA::spc	HT1125	This study
HT2224	ΔsiaB-ΔsiaD::kan ΔpamA::spc ggt::pamA⁺-cat	HT1822	This study
HT2215	ΔsiaB-ΔsiaD::kan pamA’-lacZ-ermC	HT1822	This study
HT2217	ΔsiaB-ΔsiaD::kan pamA’-phoA-ermC	HT1822	This study
HT1744	ΔsiaB-ΔsiaD::kan pilE⁺-cat (translational fusion)	HT1125	This study
HT2167	ΔsiaB-ΔsiaD::kan pilE I12A-cat (translational fusion)	HT1125	This study
HT2218	ΔsiaB-ΔsiaD::kan pilF⁺-FLAG-ermC	HT1125	This study
HT2219	ΔsiaB-ΔsiaD::kan pilF⁺-FLAG -ermCΔpamA::spc	HT1822	This study
HT2136	ΔsiaB-ΔsiaD::kan pilP⁺-FLAG -ermC	HT1125	This study
HT2137	ΔsiaB-ΔsiaD::kan pilP⁺-FALG-ermCΔpamA::spc	HT1822	This study
HT2132	ΔsiaB-ΔsiaD::kan pilM⁺-HA -ermC	HT1125	This study
HT2133	ΔsiaB-ΔsiaD::kan pilM⁺-HA -ermC ΔpamA::spc	HT1822	This study
HT2211	ΔsiaB-ΔsiaD::kan pilX⁺-FLAG-ermC	HT1125	This study
HT2212	ΔsiaB-ΔsiaD::kan pilX⁺-FLAG -ermC ΔpamA::spc	HT1822	This study
HT1688	ΔsiaB-ΔsiaD::kan pilE::ermC	HT1125	[36]
HT1822	ΔsiaB-ΔsiaD::kan ΔpilV::ermC	HT1125	[36]
HT2230	ΔsiaB-ΔsiaD::kan pilE⁺-cat pilX⁺-FLAG-ermC	HT1744	This study
HT2231	ΔsiaB-ΔsiaD::kan pilE I12A-cat pilX⁺-FLAG-ermC	HT2167	This study
H44/76
Strain	Genotype	Parent strain	Reference
H44/76	wild type strain (ST-32, Serogroup B)		[33]
HT1001	H44/76 gyrA (Nal^R)	H44/76	This study
HT1940	ΔpamA::spc	H44/76	This study
HT2095	ΔpamA::spc pilE⁺-FLAG-ermC	HT1940	This stud
HT2014	ΔpamA::spc pilE::ermC	HT1940	This study
HT2162	ΔpamA::spc pilE⁺-ermC (translational fusion)	HT1940	This study
HT2166	ΔpamA::spc pilE I12A-ermC (translational fusion)	HT1940	This study
HT1015	pilE::ermC	H44/76	[35]
Escherichia coli strains
Strain	Genotype		References
JM109	endA1 gyrA96 hsdR17(rk^-mk⁺) mcrB⁺ recA1 relA1 supE44 thi-1Δ(lac-proAB) F'[traD36 proAB lacI^qZΔM15]		Nippon gene
BL21	F–dcm ompT hsdSB(rB–mB–) gal		NEB
BL21(DE3)	F–dcm ompT hsdSB(rB–mB–) gal λ(DE3)		NEB

Open in a new tab

Production of anti-PamA protein rabbit antiserum

All PCR experiments were performed with PrimeSTAR Max GXL DNA polymerase (Takara Bio, Japan). The approximately 3.5-kb PCR fragment containing the pamA gene amplified from N. meningitidis HT1125 genomic DNA with the primer set (NMB1345-21 and NMB1345-2) (S1 Table) was cloned into the pTWV228 vector (Takara Bio, Japan), resulting in pHT922 (S2 Table), which is the ancestral plasmid in the present study. A ΔN-pamA gene, in which the N-terminal hydrophobic region consisting of the first 23 amino acids was removed (S1 Fig), was amplified by PCR with the primer set (NMB1345(pET303)-1(NsiI) and NMB1345(pET303)-2(BH)) (S1 Table) and cloned into the same site of the pET303/CT-His expression plasmid (Invitrogen) to construct pHT934 (S3 Table). Regarding the production of the ΔN-PamA protein for rabbit immunization, a 1 L culture of E. coli strain BL21 (NEB) harboring the plasmid was grown in MagicMedia at 37°C overnight, and the cells were collected. The subsequent purification of the recombinant protein and generation of polyclonal rabbit antibodies to the putative hydrophilic domain of the PamA protein were performed as described previously [46].

Construction of meningococcal mutants

In order to construct the N. meningitidis pamA deletion mutant, the 6.3-kb PCR fragment was amplified with the primers (NMB1345-3 and NMB1345-4) (S1 Table) from pHT922, in which a 1.5-kb DNA fragment containing the coding region of the pamA gene was completely removed, and ligated with a spectinomycin-resistance gene (spc) after phosphorylation to construct pHT930. A 3-kb DNA fragment, in which the pamA structural gene was replaced with the spc gene, was amplified with the primers (NMB1345-21 and NMB1345-2) (S1 Table) from pHT930 and transformed into N. meningitidis strain HT1125, and spectinomycin-resistant (Spc^R) clones were selected, resulting in the ΔpamA mutant HT1822 (Table 2).

The ΔpamA mutant complemented with the pamA⁺ gene at the ggt allele was constructed as follows: A chloramphenicol-resistance gene (cat) was inserted into the SmaI site downstream of the pamA gene of pHT922 to generate pHT923 (S2 Table). A 4.6-kb PCR fragment containing the pamA⁺-cat gene was amplified from pHT923, in which the meningococcal ggt structural gene was cloned in pTWV228 with primers (M13-RV-ggt-5′and M13-47-ggt-3′) (S1 Table) and cloned into the BstXI site of pHT195 (at the middle of the ggt coding region) by In-Fusion cloning (Clontech), resulting in the plasmid pHT924. A 6.3-kb DNA fragment containing the ggt::pamA⁺-cat genes was amplified from pHT924 with primers (ggt-3 and ggt-4) (S1 Table), transformed into N. meningitidis ggt::pamA⁺-cat mutants, and chloramphenicol-resistant (Cml^r) clones were selected, resulting in the pamA deletion mutant HT2224, which were ectopically complemented with the pamA⁺ genes expressed from their own promoter at the ggt allele (Table 1).

Since we have demonstrated that the pilE-cat translational fusion could work as well as the wild-type PilE⁺ N. meningitidis strain [36], an N. meningitidis I12A pilE mutant, in which Ile at position 12 was substituted with Ala, was constructed as described previously. In brief, pHT872 (pilE⁺-cat translational fusion on pGEM-3z) (S2 Table) [36] was site-directed mutagenized with primers (pilE-I12A-1 and pilE I12A-2) (S1 Table) to construct pHT1536 (S2 Table). After confirming its sequence, a 1.5-kb PCR fragment containing the pilE I12A-cat gene was amplified from pHT1536 by a primer set (T7 and SP6) (S1 Table) and transformed into HT1125. Cml^r clones were selected and the mutation was confirmed by direct sequencing as for the pilE I12A-cat N. meningitidis mutant HT2167 (Table 1).

To construct the pilE⁺-ermC or pilE I12A-ermC translational fusion in N. meningitidis strain H44/76 harboring the IncQ plasmid, a 0.9-kb DNA fragment from HT1125 chromosomal DNA containing the structural gene of the wild-type pilE gene and its upstream region was amplified with both primers (pGEM-3z-(SmaI)-2(15mer)-pilE-51 and pilE-52) (S1 Table). A 0.2-kb DNA fragment of the pilE downstream region was also amplified with a primer set (pilE-53 and pilE-18) (S1 Table), and a 0.75-kb DNA fragment of the ermC gene containing the SD sequence was amplified from pHT24 with a primer set (ermC-21 and ermC-22) (S1 Table). The three PCR fragments were cloned into the SmaI site of pGEM-3z to construct pHT1504 (S2 Table). The N. meningitidis I12A pilE mutant was constructed as described above using the primer set (pilE-I12A-1 and pilE I12A-2) (S1 Table) with pHT1504 as the template. After the sequence was confirmed, a 1.5-kb PCR fragment containing the pilE I12A-ermC genes was amplified from pHT1536 (S2 Table) with universal primers (T7 and SP6) and transformed into HT1125. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilE I12A-ermC N. meningitidis mutant HT2166 (Table 1). A 1.5-kb PCR fragment containing the pilE⁺-ermC genes amplified from pHT1504 (S2 Table) or the pilE I12A-ermC genes amplified from pHT1536 (S2 Table) by universal primers (T7 and SP6) was transformed into HT1940. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilE⁺-ermC HT2162 or pilE I12A-ermC HT2166 N. meningitidis mutants in the ΔpamA::spc genetic background (Table 1).

The addition of a FLAG tag to the pilE gene at the 3′-terminus on its chromosomal locus was achieved as follows: a 1.6-kb PCR fragment containing the pilE allele in H44/76 was amplified with a primer set (pilE-11and pilE-12) (S1 Table), and after phosphorylation, it was cloned into pMW119 to construct pHT1419 (S2 Table). A 5.8-kb PCR fragment was amplified with a primer set (pilE-17 and FLAG’(15mer)-pilE-18) (S1 Table) from pHT24, and a 1-kb PCR fragment containing the ermC gene was also amplified with a primer set (pilE-17’(15mer)-M13-47 and FLAG-RV-M) (S1 Table) from pHT24. The two PCR fragments were ligated by In-Fusion Cloning to construct pHT1420, in which the FLAG tag was fused to the pilE structural gene followed by an ermC selection marker (S2 Table). A 2.6-kb PCR fragment containing the pilE^+-FLAG-ermC genes was amplified from pHT1420 with a primer set (pilE-11 and pilE-12) and transformed into HT1940. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilE⁺-FLAG-ermC N. meningitidis mutant HT2095 (Table 1).

The pilE insertional mutant HT2014 was constructed by transforming HT1940 with 500 ng of HT1015 (H44/76 pilE::ermC) [35] chromosomal DNA, followed by the selection of the Erm^r strain HT2014 (Table 1).

The addition of a FLAG tag to the pilF gene at the 3′-terminus on its chromosomal locus was performed as follows: a 1.8-kb PCR fragment containing the pilF allele in the H44/76 N. meningitidis strain was amplified with a primer set (pMW(SmaI)-up(15mer)-pilF-1 and pMW(SmaI)-down(15mer)-pilF-2) (S1 Table) and cloned into the SmaI site of pMW119 by In-Fusion cloning to construct pHT1449 (S1 Table). A 6-kb PCR fragment amplified with a primer set (pilF-3 and FLAG’(15mer)-pilF-4) (S1 Table) from pHT1499 and a 1-kb PCR fragment containing the ermC gene amplified with a primer set (pilF-3’(15mer)-M13-47 and FLAG-RV-M) (S1 Table) from pHT24 were ligated by In-Fusion Cloning to construct pHT1457, in which the FLAG tag was fused to the pilF structural gene followed by an ermC selection marker (S1 Table). A 2.8-kb PCR fragment containing the pilF⁺-FLAG-ermC genes was amplified from pHT1457 with a primer set (pMW(SmaI)-up(15mer)-pilF-1 and pMW(SmaI)-down(15mer)-pilF-2) (S1 Table), and then transformed into HT1125 and HT1822. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilF⁺-FLAG-ermC N. meningitidis mutants HT2218 and HT2219, respectively (Table 1).

A 3.9-kb DNA fragment containing partial pilM-pilN⁺-pilO⁺-pilP⁺ genes in H44/76 N. meningitidis was amplified with a primer set (pMW(SmaI)-up(15mer)-pilMNOP-3 and pMW(SmaI)-down(15mer)-pilMNOP-4) (S1 Table) and cloned into the SmaI site of pMW119 by In-Fusion cloning to construct pHT1450 (S2 Table).

The addition of a FLAG tag to the pilP gene at the 3’ terminus on its chromosomal locus was achieved as follows: a 7.2-kb DNA fragment amplified with a primer set (pilP-3 and FLAG’(15mer)-pilP-2) (S1 Table) from pHT1450 and a 1-kb PCR fragment containing the ermC gene amplified with a primer set (pilP-3’(15mer)-M13-47 and FLAG-RV-M) (S1 Table) from pHT24 were ligated by In-Fusion cloning to construct pHT1454 (S2 Table), in which the FLAG tag was fused to the pilP structural gene at the 3’ terminus followed by an ermC selection marker. A 4.9-kb PCR fragment containing the pilP⁺-FLAG-ermC genes was amplified from pHT1454 with a primer set (pMW(SmaI)-up(15mer)-pilMNOP-3 and pMW(SmaI)-down(15mer)-pilMNOP-4)(S1 Table) and transformed into HT1125 and HT1822. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilP⁺-FLAG-ermC N. meningitidis mutants HT2136 and HT2137, respectively (Table 1).

The addition of the HA tag to the pilM gene at the 3’ terminus on its chromosomal locus was achieved; however, the ORFs of the pilM (1.1 kb), pilN (0.6 kb), and pilO (0.6 kb) genes were very close or overlapped, and thus an ermC selection marker was inserted downstream of the pilO gene. A 7.1-kb PCR fragment containing the partial pilM gene and pMW119 from pHT1450 was amplified with a primer set (pilO-3 and HA’-pilM-2) (S1 Table). A 1.2-kb PCR fragment was amplified with a primer set (HA’(15mer)-pilN-1 and pilO-2) from pHT1450 and a 1-kb ermC gene fragment was also amplified with a primer set (pilO-2’(15mer)-M13-RV and pilO-3(15mer)-M13-47) (S1 Table) from pHT24. The three PCR fragments were ligated by In-Fusion Cloning to construct pHT1453, in which the HA tag was fused to the pilM structural gene with an ermC selection marker (S2 Table). A 2.6-kb PCR fragment containing the pilM^+-HA pilN⁺pilO⁺-ermC-pilP genes was amplified from pHT1453 with a primer set (pMW(SmaI)-up(15mer)-pilMNOP-3 and pMW(SmaI)-down(15mer)-pilMNOP-4) (S1 Table) and transformed into HT1125 and HT1822. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilM⁺-HA-ermC N. meningitidis mutants HT2132 and HT2133, respectively.

The addition of a FLAG tag to the pilX gene at the 3’ terminus on its chromosomal locus was achieved as follows: a 0.5-kb pilX DNA region containing the 400-bp upstream and 160-bp downstream regions of the pilX ochre codon in H44/76 was amplified with a primer set (pMW(SmaI)-up(15mer)-pilX-3 and pMW(SmaI)-down(15mer)-pilX-6) (S1 Table), and then cloned into the SmaI site of pMW119 by In-Fusion cloning to construct pHT1594. A 4.7-kb PCR fragment amplified with a primer set (M13-47′(15mer)-pilX-5 and FLAG′(15mer)-pilX-8) (S1 Table) from pHT1594, and a 1-kb PCR fragment containing the ermC gene amplified with a primer set (FLAG-RV-M and M13-47) (S1 Table) from pHT24 were ligated by In-Fusion Cloning to construct pHT1597, in which the FLAG tag was fused to the pilX structural gene followed by an ermC selection marker (S2 Table). A 1.5-kb PCR fragment containing partial pilX^-FLAG-ermC genes was amplified from pHT1597 with a primer set (pMW(SmaI)-up(15mer)-pilX-3 and pMW(SmaI)-down(15mer)-pilX-6) (S1 Table) and transformed into HT1125 and HT1822. Erm^r clones were selected and the mutation was confirmed by direct sequencing as for the pilX⁺-FLAG-ermC N. meningitidis mutants HT2211 and HT2212, respectively (Table 1).

pamA-lacZ translational fusion N. meningitidis mutants were constructed as follows: a 3-kb PCR fragment containing the lacZ gene was amplified from E. coli strain W3110 [47] chromosomal DNA with the primers (lacZ-21 and M13-RV’(15mer)-lacZ-22) (S1 Table) and a 1-kb ermC gene fragment was amplified with universal primers (M13-RV and M13-47) (S1 Table) from pHT24 [33]. The two PCR fragments were cloned into a 7.2-kb DNA fragment (designated Fragment C) amplified from pHT922 containing the upstream and downstream regions of the pamA gene with the first 408-bp region including the N-terminal membrane spanning region (designated pamA′) with a primer set (M13-47′(15mer)-nmb1345-61 and lacZ′-21(15mer)-nmb1345-62) (S1 Table) by In-Fusion cloning to construct pH1605 (S2 Table and Fig 2B).

Fig 2 — (A) Western blot analyses of subcellular fractions of the PamA protein in N. *meningitidis* strain HT1125. A whole-cell lysate periplasmic fraction (P) isolated by CHCl₃ shock, cytoplasmic and periplasmic fraction (CP), IM-enriched fraction, and outer membrane (OM)-enriched fraction were prepared by the sonication method from the N. *meningitidis* strain, as described previously [45]. Western blotting with anti-PamA (left) and anti-LptA (right) [55] rabbit antisera. LptA is a control for the proper isolation of the IM-rich fraction. Black and gray arrows show PamA and LptA, respectively. (B) Membrane topology of the PamA protein using the *pamA*′*-lacZ* and *pamA*′*-phoA* fused N. *meningitidis* strain. The hatched region consists of the 20 amino acid residues corresponding to the putative membrane-spanning region (S1 Fig). The first 408-bp region of the *pamA* gene was fused to the *lacZ* or *phoA* gene, and the gene was incorporated into the *pamA* locus on the meningococcal chromosome. (C) β-Galactosidase and alkaline phosphatase activities. (D,E) Western blot of spheroplasted N. *meningitidis* strain H44/76 harboring pHT936 (an IncQ plasmid expressing the *gst*⁺ gene, Table 1) followed by a treatment with Proteinase K. Aliquots of the sample were analyzed by Western blotting with anti-GST mAb (D) and anti-PamA rabbit serum (E). Black and gray arrows show PamA and GST, respectively.

pamA′-phoA translational fusion N. meningitidis mutants were constructed as follows: a 1.5-kb PCR fragment containing the phoA gene was amplified from E. coli strain W3110 chromosomal DNA with the primers (phoA-21 and M13-RV’(15mer)-phoA-2) (S1 Table), and a 1-kb ermC gene fragment was amplified with universal primers (M13-RV and M13-47) from pHT24 [33]. The two PCR fragments were cloned into Fragment C by In-Fusion Cloning to construct pH1603 (S2 Table). PCR fragments amplified with nmb1345-5 and nmb1345-21 (S1 Table) from pHT1605 containing the pamA′-lacZ-ermC-downstream region of the pamA gene or from pHT1603 containing the pamA′-phoA-ermC-downstream region of the pamA gene were transformed to HT1125, and erythromycin-resistant clones were selected, as for the pamA′-lacZ::ermC or pamA′-phoA::ermC N. meningitidis mutants on its chromosome HT2215 and HT2217, respectively (Table 1 and Fig 2B).

Assessment of host cell-associated and internalized bacteria

The infection of HBMEC with N. meningitidis was performed as described previously [46]. Results are expressed as means ± the standard deviation (SD), and bacterial numbers were statistically compared using the two-tailed Student’s t-test.

Western blotting

SDS-PAGE and Western blotting were performed as described previously [48].

Observations of meningococci and ezrin accumulation by immunofluorescence staining

HBMEC monolayer preparations, N. meningitidis infections of these layers, and the preparation of immunostaining samples were performed as described previously [46]. Infected HBMEC and attached meningococci were observed using an ECLIPSE E600 microscope (Nikon) with a 100× oil immersion objective.

Construction of IncQ plasmids for N. meningitidis

The mutated M. mazei pylT (tRNA^Pyl) and mutated pylRS genes for mAzZLys (PylRS[Y306A/Y384F]) [49] and pBPa (PylRS A302T/N346T/C348T/W417C]) [50] (MmpBpaRS) were derived from pCDF-Pyl-Fx1 [49]. The four point mutations were generated by three-step mutations with the following three primer sets (A302-T and A302-7-R) (S1 Table), (N346T-C348T-F and N346-C348-R) (S1 Table), and (W417C-F and W417C-R) (S1 Table) using the Mutagenesis Basal Kit (Takara Bio). The plasmid constructed from pCDF-Pyl-Fx1 as described above was named pCDF-Pyl-Fx3 in this study (S3 Table). A 2-kb PCR fragment containing one copy of the pylRS gene (Y306A/Y384F) and two copies of the pylT genes expressed from the E. coli lpp promoter [49] (pylRS (Y306A/Y384F)/T) from pCDF-Pyl-Fx1 or a 2-kb PCR fragment containing one copy of the MmpBPaRS genes and two copies of the pylT genes expressed from the E. coli lpp promoter [49] (MmpBPaRS/T) from pCDF-Pyl-Fx3 were amplified by PCR with a primer set (ptac-12-pylRS-1 and M13-47-reverse-lppT-2) (S1 Table), and then cloned into pTTQ18, an expression vector with a strong tac promoter (P_tac) controlled by the LacI protein [51] using In-Fusion cloning to construct pHT1208 and pHT1261, respectively (S2 Table). The PCR fragment containing the lacI^q-P_tac-pylRS[Y306A/Y384F]/T or lacI^q-P_tac-MmpBPaRS/T genes was amplified by PCR with primers (M13-47 reverse and ptac-12) (S1 Table), and after phosphorylation, the fragment was cloned into the blunted SacI and PstI sites of pHT128, an IncQ plasmid [33], resulting in pHT1212 (lacI^q-P_tac-pylRS[Y306A/Y384F]/T) and pHT1262 (lacI^q-P_tac-MmpBPaRS/T), respectively (Table 1 and S4 Table).

PCR fragments containing the gst gene derived from pGEX-6P1 (GE Healthcare) with an amber codon at position Glu 51 (gst E51) or Phe 52 (gst F52) were constructed as follows: the gst E51 or gst F52 mutation was introduced by site-directed mutagenesis with two sets of primers (gst E51amb-1 and gst E51amb-2 for gst E51) (gst F52amb-1 and gst F52amb-2 for gst F52) (S1 Table) using the PrimeSTAR Mutagenesis Basal Kit. After the sequence was confirmed, a 0.5-kb PCR fragment containing Ptac-gstE51amb or Ptac-gstF52amb was amplified with a primer set (tetA-1-gst-up and gst-down-tetA-2) (S1 Table) and cloned into the SalI site of pHT1212 or pHT1216 to construct pHT1355, pHT1356, pHT1357, and pHT1358, respectively (Table 1).

To construct a series of amber mutations in the pamA gene, the His₆ tag was initially introduced into the 3’ terminus of the native pamA gene as follows: a 7.2-kb PCR fragment was amplified from pHT922 with a primer set (NMB1345-1 and NMB1345-2-(His)₆) (S1 Table), phosphorylated, and then self-ligated to construct pHT1213 (Table 1).

Amber mutation series (pamA K-amb) plasmids (see S1 Fig) were constructed by site-directed mutagenesis of pHT1213 (S2 Table) with primer sets (S1 Table). The pamA K-amb gene on pTWV228 was confirmed by sequencing and the plasmid is listed in S2 Table. A 2.2-kb PCR fragment containing the pamA K-amb gene was amplified with a primer set (tetA-NMB1345-up and tetA-NMB1345-down) (S1 Table) from the pTWV228-based plasmids listed in S2 Table, and cloned into the SalI site of pHT1216 or pHT1262 by In-Fusion cloning to construct IncQ plasmids containing the lacI^q-P_tac-pylRS[Y306A/Y384F]/T and pamA K-amb genes (pHT1212) or lacI^q-P_tac-MmpBPaRS/T and pamA K-amb genes (pHT1262) listed in Table 1.

The addition of StrepTag₂-His₆ synthetic DNA (S3 Fig) to the pamA K278amb gene was achieved as follows: a 6.5-kb PCR fragment amplified from pHT1242 with a primer set (spacer-1(15mer)-nmb1345-12 and His₆′(15mer)-nmb1345-11) (S1 Table) was ligated to StrepTag₂-His₆ synthetic DNA by In-Fusion cloning to construct pHT1293 (S2 Table). A 2.4-kb PCR fragment containing the pamAK278amb-StrepTag₂-His₆ gene was amplified with a primer set (tetA-NMB1345-up and tetA-NMB1345-down) (S1 Table) from pHT1293 and cloned into the SalI site of pHT1262 by In-Fusion cloning, to construct pHT1388 (Table 1).

The introduction of IncQ plasmids into N. meningitidis strains was performed as described previously [33].

UV crosslinking of whole N. meningitidis cells expressing the PamA Lys-amber (pamA K-amb) mutants by pyrrolysine-based amber suppression with pBPa

The N. meningitidis H44/76 ΔpamA::spc mutant (HT1940) (Table 2) harboring IncQ plasmids containing lacI^q -Ptac-MmpBPaRS/T genes and pamA K-amber genes (Table 1) was cultivated on GC agar plates containing 1 mM IPTG and 1 mM pBPa at 37°C in 5% CO₂ for 18 hours. To identify the optimal pamA K-amb mutants for pyrrolyl-based amber suppression followed by photocrosslinking, N. meningitidis transformants grown one-fourth of a GC plate were suspended in 50 μl PBS, transferred into the wells of a 96-well plate/strain, and irradiated with 365 nm UV using a FL-365-SD UV lamp stand (Opticode, Japan) at a distance of 2 cm on ice for 2 hours. After harvesting, UV-irradiated bacteria were resuspended in 400 μl of urea buffer (50 mM Tris-HCl pH7.5, 8 M urea, 1% Triton X-100, and 100 mM NaCl) and then transferred into a 1.5-mL tube. Bacteria were disrupted by sonication, and the soluble fraction was obtained by centrifugation. PamA K-amb-His₆ proteins with expression suppressed by pBPa were purified with 200 μL TALON resin (Clontech). The purified fraction (approx. 250 μl) was precipitated by TCA/acetone at -80 ^oC overnight, and the precipitate was finally dried and suspended in 25 μl 1 × SDS buffer. Aliquots were analyzed by SDS-PAGE.

Identification of endogenous proteins crosslinked with PamA K278(pBPa) in N. meningitidis

To identify endogenous protein(s) crosslinked to PamA K278(pBPa) in N. meningitidis, an synthetic DNA fragment containing twin Strep-Tag (StrepTag₂) [52] was further introduced into pHT1274, resulting in pHT1388 (S3 Fig and Table 2). N. meningitidis HT1940 harboring pHT1388 was grown on approximately 200 GC agar plates containing 1 mM IPTG and 1 mM pBPa at 37°C in 5% CO₂ for 18 hours. The bacteria from 3 GC agar plates were suspended in 0.5 mL PBS, transferred into the wells of a 12-well plate (more than 50 wells in total), and irradiated with 365 nm UV on ice for 2 hours. The irradiated bacteria from 2 wells were transferred into a 15-mL tube and harvested by centrifugation. The collected bacteria were suspended in 5 mL IP Lysis buffer (25 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) and disrupted by sonication followed by centrifugation at 12,000 rpm at 4°C for 30 min. The supernatant (soluble fraction) was collected into one tube. Crosslinked proteins were purified by two column chromatography steps using Ni-Sepharose High Performance (GE Healthcare) and Strep-Tactin Sepharose (Iba, Germany). The purified fraction was concentrated approximately 70-fold using a VIVASPIN TURBO 15 (MW 50k) Centrifuge Filter unit (Sartorius), to a final volume of approximately 100 μL. An aliquot of the sample was analyzed by 4–12% NuPAGE (Invitrogen), and immunoblotting with an anti-His₆ monoclonal antibody (mAb) (Wako, Japan). All proteins were also visualized using the CBB staining kit (APRO SCIENCE, Japan) and bands that were also detected by immunoblotting were excised. These bands were subjected to trypsin digestion and peptide extraction. Extracted peptides were dried and resuspended in H₂O and 0.1% (w/w) trifluoroacetic acid for MS. The LC-MS/MS analysis was performed with an Advance nanoLC (Michrom Biosources) coupled to an LTS Mass Spectrometer (Thermo Fisher Scientific). Proteins were identified by running MASCOT (http://www.matrixscience.com) against the NCBI database (NCBInr 20160202). Candidates with molecular masses that were consistent with the difference between the crosslinked complexes and the PamA protein were selected. The accuracy of each interaction between PamA K278(pBPa) and the candidate was further examined by constructing N. meningitidis HT1940 harboring pHT1388 mutants, in which the FLAG tag was added to the candidate gene on its chromosome, as in the pilX-FLAG N. meningitidis strain (Table 1) described above.

Identification of the counterpart of the amino acid residue of PilE crosslinked with PamA K278(pBPa)

A DNA fragment containing ΔN-pamA K278amb was amplified from pHT1242 with a primer set (NMB1345(pGEX)-1(BH) and NMB1345(pGEX)-2(XhoI)) (S1 Table) and cloned into the same sites of pMAL-c2 (NEB) to construct pHT1473 (S3 Table). A 0.5-kb PCR fragment containing the Gln₆-ΔN-PilE gene, in which the first 9 amino acids (PilE leader peptides in N. meningitidis) were replaced with 6 glutamine residues (Gln₆) (see the Results section), was amplified with a primer set ((Gln)₆-pilE-45 and pCDF-1b(PacI)(15mer)-pilE-44) (S1 Table) from H44/76 N. meningitidis chromosomal DNA, and designated as fragment B. To clone the PCR fragment into pCDF-1b-kan (S3 Table) [53], a 3.6-kb DNA fragment was amplified from pCDF-1b-kan with a primer set (pCDF-1b (downstream of PacI) and (Gln)₆(15mer)-pCDF-1b-2) (S1 Table) and ligated to PCR fragment B by In-Fusion Cloning to construct pHT1447 (S3 Table), in which the ΔN-(Gln)₆-PilE protein was expressed from the T7 promoter (P_T7). A 0.6-kb PCR fragment containing the P_T7- ΔN-(Gln)₆-pilE gene was amplified from pHT1447 with a primer set (pSTV28(SmaI-5′)-pCDF-1b-1 and pSTV28(SmaI-3′)-pCDF-1b-2) (S1 Table) and cloned into the SmaI site of pSTV28 (Takara Bio) by In-Fusion Cloning to construct pHT1474 (S3 Table). E. coli BL21(DE3) cells harboring pCDF-Pyl-Fx3, pHT1473, and pHT1474 were cultivated in 100 mL MagicMedia containing 1 mM pBPa at 30°C overnight. The harvested bacteria were suspended in 1.25 mL PBS and divided into 5 aliquots (0.25 mL/tube), transferred into the wells of a 24-well plate/aliquot, and irradiated with 365 nm UV light on ice for 2 hours. UV-irradiated bacteria were collected again in one tube and disrupted by suspension in 1 mL B-PER (Thermo) containing protease inhibitor cocktail (Nacalai Tesque, Japan), followed by centrifugation at 12,000 rpm at 4°C for 30 min. The MBP-ΔN-PamA K278(pBPa) protein crosslinked to Gln₆-ΔN-PilE [as well as uncrosslinked MBP-ΔN-PamA K278(pBPa)] was purified with amylose resin (NEB). The purified fraction was concentrated approximately 70-fold using a VIVASPIN TURBO 15 (MW 50k) Centrifuge Filter unit (Sartorius) to a final volume of approximately 250 μL. An aliquot of the sample was analyzed by 4–12% NuPAGE (Invitrogen), and the bands corresponding to the complex containing MBP-ΔN-PamA K278(pBPa) crosslinked to Gln₆-ΔN-PilE were excised. In addition, an uncrosslinked band corresponding to MBP-ΔN-PamA K278(pBPa) was isolated for a comparative analysis. The band corresponding to the Gln₆-ΔN-PilE protein was also excised. The amino acid residue of Gln₆-ΔN-PilE crosslinked to ΔN-PamA K278(pBPa) was identified by comparing peptides from the three bands as described previously [54].

Transmission Electron Microscopy (TEM)

Negatively stained samples were prepared for TEM as described previously [55]. Electron microscopy was performed with a Hitachi H-7600 Transmission Electron Microscope.

Measurement of the ratio of pilus components to PilE in purified pili

Meningococcal pili were prepared as described previously [36]. The relative amounts of PilF-FLAG, PilP-FLAG, PilM-FLAG, PilV, and PilX-FLAG to PilE in purified pili were examined by Western blotting with anti-FLAG mAb (Wako, Japan), anti-PilV [36], or anti-PilE rabbit serum [33] with appropriate dilutions using a quantification program in Fusion Capt 17 in Fusion Solo 7S (M&S Instruments). Results are expressed as means ± SD. The adhered and internalized bacterial numbers and the relative amounts of pilus components to PilE in purified pili were compared using the two-tailed Student’s t-test, and P values of <0.05 were considered significant.

Results

Genetic evidence that a hypothetical protein annotated as NMB1345 is important for N. meningitidis internalization into HBMEC

During the course of our research on the infectious abilities of the ST-2032 N. meningitidis strain mutagenized by STM to HBMEC, we found that a transposon mutant, in which the gene annotated as NMB1345 (renamed as PamA in this study) in the N. meningitidis MC58 genome [39] was disrupted and resulted in defective infectious ability in HBMEC (Fig 1A). To confirm the relationship between the mutation and infectious defect in the mutant, we constructed a null mutant (HT1822 ΔpamA::spc) and an ectopically complemented strain with the wild-type pamA⁺ gene at the ggt locus (HT2224) and examined their infectious activities in HBMEC (Fig 1B). While the insertional and null mutants adhered to HBMEC less efficiently than the wild-type strain HT1125, the number of internalized bacteria largely decreased to approximately 1/100 of HT1125 and the defect in internalized bacterial numbers was recovered in HT2224. These results indicate that the mutation in the pamA gene affects meningococcal infection in HBMEC, particularly internalization.

Fig 1 — (A) Schematic representation of the wild-type, insertion, and deletion mutants in the *pamA* gene and ectopic complementation of the *pamA*⁺ gene at the *ggt* locus in N. *meningitidis* strains. (B) Effect of *ΔpamA* mutation on N. *meningitidis* infection of HBMEC. The adherence (left) and internalization (right) of *pamA N*. *meningitidis* mutants to HBMEC, and the effects of complementation of the *pamA*⁺ gene in the *pamA* deletion mutant on bacterial infection. Each value is the mean ± standard error of the mean (CFU per 10⁴ HBMEC) from at least four experiments. Open, filled, light gray, and dark gray bars indicate the bacterial number of N. *meningitidis* wild-type *pamA*⁺ (HT1125), *pamA*::*Tn-spc* (HT1572), and *ΔpamA*::*spc* (HT1822), and *pamA*^- mutants in which the *pamA*⁺ gene was ectopically complemented (HT1736 and HT2224), respectively (see S1 Table). *P<0.01, #P<0.05, significantly different from the *pamA*⁺ strain or *pamA*^- mutants complemented with the *pamA*⁺ gene. (C) Immunofluorescence microscopy showing the accumulation of ezrin beneath N. *meningitidis*-infected HBMEC. The HBMEC monolayer was infected with wild-type *pamA*⁺ (middle-left), *ΔpamA* (middle-right), and *ΔpamA/pamA*⁺ (right) N. *meningitidis* strains. A non-infected HBMEC monolayer is also shown in the left panels. Bacteria and HBMEC were observed by phase-contrast microscopy (upper panels). Ezrin was immunostained with an anti-ezrin mAb and Alexa Fluor 488-conjugated rabbit anti-mouse IgG (green channel, lower panels).

We also investigated host cell cytoskeleton rearrangements caused by meningococcal infection by monitoring the localization of ezrin, because the accumulation of ezrin beneath meningococci on HBMEC is required for bacterial internalization into host cells [36, 37] (Fig 1C). While ezrin was widely distributed throughout non-infected cells, it was condensed at the site of bacterial attachment in HBMEC infected with wild-type N. meningitidis strain HT1125. In contrast, ezrin condensation was not observed in cells infected with the ΔpamA N. meningitidis mutant HT1822, but was detected in HT2224 (ΔpamA/pamA⁺). These results are consistent with previous findings, and strongly suggest that PamA participates in the meningococcal-elicited bacteria-induced reorganization of the host cell cytoskeleton upon N. meningitidis infection.

Characterization of the biochemical properties of PamA in N. meningitidis

The deduced amino acid sequence of the PamA protein consists of 516 residues (S1 Fig) with a predicted molecular mass of 57 kDa. The hydrophobicity analysis indicated that the PamA protein had a one membrane-spanning region at its N terminus (S1 Fig), suggesting that the PamA protein is localized at the membrane in N. meningitidis. To clarify the localization in N. meningitidis, cellular proteins were biochemically fractionated using a differential detergent solubilization method [45] and analyzed by Western blotting (Fig 2A). LptA, a meningococcal inner membrane (IM) protein [55], was largely detected in the 4% Triton X-100 soluble (IM-enriched) fraction, supporting the proper fractionation in this experiment. Under the same conditions, the PamA protein was mainly enriched in the IM-rich fraction. This result suggests that PamA is located at the IM in N. meningitidis.

We also investigated the membrane topology of the PamA molecule in N. meningitidis by constructing N. meningitidis mutants, in which the first N-terminal 136 amino acid residues of PamA including the membrane-spanning region, termed pamA′, were fused to the lacZ or phoA gene [56] on the chromosome (Fig 2B)(see Materials and Methods). While the wild-type HT1125 and pamA′-lacZ N. meningitidis HT2215 strains (Table 2) did not exhibit β-galactosidase activity, the pamA′-phoA N. meningitidis strain HT2217 (Table 2) showed alkaline phosphatase activity (Fig 2C). Since the alkaline phosphatase activity of PhoA was detected only in the periplasmic space [57], these genetic results suggest that the hydrophobic region of the PamA protein faces the periplasmic side. We also biochemically examined the membrane topology, using a Proteinase K digestion experiment with the spheroplasted N. meningitidis strain expressing glutathione S-transferase (GST) from an IncQ plasmid (Fig 2D and 2E). Under the condition where the cellular protein GST was mostly protected from Proteinase K digestion (Fig 2D), PamA was largely digested (Fig 2E). Collectively, these results suggest that PamA is localized at the IM and faces the periplasmic space in N. meningitidis.

Incorporation of ncAAs into proteins in living N. meningitidis

To attempt the application of genetic code expansion to N. meningitidis for the incorporation of ncAAs with a photocrosslinker into the PamA protein, we used two mutated MmPylRS/T genes to genetically encode ncAAs in N. meningitidis. A previous study reported that a modified MmPylRS gene for E. coli, MmPylRS[Y306A/Y384F] [49] is applicable to N^ε-(m-azidobenzyloxycarbonyl)-L-lysine (mAzZLys) (Fig 3A, left), which is a photocrosslinker excited by ultraviolet (UV) irradiation [58], and MmPylRS([A302T/N346T/C348T/W417C]) [50] (MmpBPaRS) is optimized for p-benzoyl-L-phenylalanine (pBPa) (Fig 3A, right), which is also one of the widely used photocrosslinkers [44].

We examined the incorporation of mAzZLys and pBPa in N. meningitidis by biochemically monitoring the suppression of the amber codon at position Glu51 or Phe52 of GST (GST E51amb or GST F52amb) by Western blotting (Fig 3B). The expression of both the gst E51amb and gst F52amb mutants was suppressed by pBPa, while the gst F52amb mutant was not suppressed by mAzZLys. The suppression efficiency could be considered as approximately 25% of the wild-type GST protein with pBPa in the gst E51amb mutant, and 37% and 35% with mAzZLys and pBPa in the gst F52amb mutant, respectively. Since the incorporation efficiency of ncAAs is approximately 35–50% in E. coli [59], this result suggested that genetic code expansion by the two modified MmPylRS/T genes was efficiently conducted in N. meningitidis (Fig 3B). These results obtained by biochemical and genetic pyrrolysine-based amber suppression suggest that mAzZLys and pBPa are incorporated into the GST protein in N. meningitidis. The proper incorporation of pBPa into a protein in N. meningitidis, as determined by an MS analysis, was examined with PamA K278 (pBPa)(see a later section, S5 Fig) since the amounts of GST proteins in N. meningitidis were too low to purify.

We further examined the UV crosslinking by using whole bacterial cells (Fig 3C). Longer UV irradiation increased the amount of the dimer form of GST, and the majority of GST F52(pBPa) was fixed as the dimer form for 2 hours because GST is a dimeric enzyme [44, 60, 61]. On the other hand, the dimerization of the GST E51amb(mAzZLys) and GST E51(pBPa) proteins was not observed by photocrosslinking, while GST E51(mTmdZLys) dimerized in E. coli [62], indicating that the processes involved in UV crosslinking with ncAAs do not always function well in N. meningitidis. Collectively, these results demonstrated that physiological transient protein-protein interactions can be detected by crosslinking via the incorporation of photoreactive ncAAs in N. meningitidis and showed that the incorporation of ncAAs by pyrrolysine-based amber suppression could be performed in N. meningitidis.

Incorporation of a genetically encoded photocrosslinking amino acid into the PamA protein to identify the accompanying protein in N. meningitidis

Since it was not possible to predict the three-dimensional structure of PamA by Phyre2 [63], due to the lack of homology to any other protein identified to date, we were unable to speculate which amino acid residues are exposed on the outside of the PamA molecule in N. meningitidis. Therefore, the identities of suitable amino acid residues for the incorporation of photoreactive ncAAs into the PamA protein remained unclear. Thus, in the present study, we focused on the lysine (Lys or K) residue for amber (UAG) substitution because lysine is primarily located on protein surfaces [64, 65]. Forty Lys residues are present in the PamA protein (S1 Fig), and we constructed three pamA amber mutants (designated as pamA K-amb) at K148 (pamA K148amb), K273 (pamA K273amb), and K388 (pamA K388amb) to estimate the proper position(s) in the PamA protein for ncAAs incorporation. N. meningitidis strain H44/76 ΔpamA (HT1940) expressing the MmPylRS[Y306A/Y384F]/T genes and one of the three pamA K-amb genes on an IncQ plasmid was cultivated in the presence of mAzZLys, and whole bacterial extracts were analyzed by Western blotting (S2A Fig). The full length and a sufficient amount of PamA K148amb by incorporation of mAzZLys [(PamA K148(mAzZLys)], PamA K273(mAzZLys), and PamA K388(mAzZLys) were only observed in the presence of mAzZLys, suggesting that mAzZLys is incorporated into the PamA protein by pyrrolysine-based amber suppression in N. meningitidis. We also examined UV-irradiated whole bacterial cells by Western blotting, faint bands appeared to correspond to proteins crosslinked to PamA (mAzZLys), were detected in the PamA K273(mAzZLys) and PamA K388(mAzZLys) samples (S2B Fig). The same result was also obtained for N. meningitidis strain HT1940 expressing the MmpBPaRS/T genes and one of the three pamA K-amb genes on an IncQ plasmid, cultivated in the presence of pBPa (S3 Fig). These results indicate that the Lys residues located from position Lys148 to the carboxy-terminal end (S1 Fig) were localized on the surface of the PamA molecule in N. meningitidis. Moreover, the amber suppression appeared to be more efficiently for PamA K273(pBPa) and PamA K388(pBPa) with pBPa than with mAzZLys (Fig 4 and S3 Fig). Thus, the photocrosslinking experiments by pyrrolysine-based amber suppression described hereafter were conducted with the pBPa and MmpBPaRS/T genes.

Fig 4 — UV crosslinking of Pam K208amb, K278amb, K309amb, K341amb, K395amb, and K382amb mutants expressed by pyrrolysine-based amber suppression with pBPa in *ΔpamA N*. *meningitidis*. Bacteria that grew on one-fourth of a GC agar plate in the presence of 1 mM pBPa were crosslinked by UV light irradiation for 2 hours and then the PamA K(pBPa)-His₆ protein was purified. An aliquot was analyzed by anti-His₆ mAb. The black arrow shows the full-length PamA K-amb protein expressed by pyrrolysine-based amber suppression with pBPa, and the gray arrow indicates protein complexes crosslinked between PamA K(pBPa) and unidentified endogenous proteins in N. *meningitidis*.

To confirm the proper position(s) for the incorporation of pBPa into the PamA protein, we constructed 26 pamA K-amber mutants, in which each Lys residue located from position Lys148 to the carboxy-terminal end in the PamA protein was replaced with the amber codon (S1 Fig, Table 1 and S4 Table). UV-irradiated N. meningitidis strain HT1940 cells expressing the MmpBPaRS/T and pamA K-amber-His₆ genes were analyzed by Western blotting (S3 Fig). Some PamA(pBPa) mutants seemed to be crosslinked to some endogenous protein in N. meningitidis (shown in red in S3 Fig), and the more detailed analysis revealed that the following six pamA K-amber mutants with expression suppressed by pBPa [K208(pBPa), K278(pBPa), K309(pBPa), K341(pBPa), K382(pBPa), and K395(pBPa)] appeared to be crosslinked to some endogenous proteins in N. meningitidis (shown in red in Fig 4) while K309(pBPa) was eliminated. In the present study, we focused only on the proteins crosslinked to PamA K278(pBPa), because the three bands with the highest intensities were found in the PamA K278(pBPa) sample.

To characterize the 150-, 140-, and 85-kDa protein complexes that contained an endogenous protein crosslinked to PamA K278(pBPa), we added a Twin-Strep tag (Strep₂) [66] and His₆ tag at the carboxy-terminal end (S4 Fig) and purified the three corresponding protein complexes from N. meningitidis strain HT1940 harboring pHT1388 grown on approximately 200 plates as described in the Materials and Methods. During the purification process, we also obtained the purified PamA K278 (pBPa) protein expressed in N. meningitidis. The incorporation of pBPa at K278amb of PamA-Strep₂ -His₆, was confirmed by MS analyses (S5 Fig). This result demonstrates that an ncAA was selectively incorporated into a protein in N. meningitidis.

The three corresponding protein complexes were subjected to a gel-based proteomic analysis using MS (Fig 5A and 5B). A compilation of the results obtained using MASCOT (MATRIX SCIENCE, Japan) suggested several candidates, and we selected those with higher scores and numbers of matched peptides for more reliable results in the present study (Table 3). Since the molecular mass of PamA-Strep₂-His₆ is approximately 62 kDa due to the addition of Strep₂-His₆ at the carboxy-terminal end (S4 Fig), the bands corresponding to 150- and 140-kDa were presumed to contain counterparts with a molecular mass of approximately 100 kDa. PamA is listed as the top score of the MASTCOT analysis (Table 3) for the 150 and 140 kDa bands, providing support for the presence of this protein in the two bands. On the other hand, we did not identify any candidate with a deduced molecular mass from 80 to 90 kDa by MASCOT (Table 3). To examine the possibility that the observed molecular mass on SDS-PAGE was different from the calculated molecular mass in the database, we constructed N. meningitidis mutants that expressed the FLAG-tagged proteins of 5 candidates with estimated molecular masses of 75–160 kDa for 150 kDa, and those of 9 candidates with estimated molecular masses of 48–74 kDa (shown in bold in Table 3). Crosslinked samples from the N. meningitidis mutants expressing the FLAG-tagged protein, MmpBPaRS/T and PamA K278(pBPa)-Strep₂-His₆, were examined by Western blotting; however, none of these candidates were crosslinked to PamA K278(pBPa) in N. meningitidis. Considering these results, the two proteins presented by the 150- and 140-kDa bands with the highest intensities on Western blotting apparently correspond to the homo-trimer of the PamA protein (see Discussion and S12 Fig).

Table 3. Summary of the protein candidates crosslinked to PamA in N. meningitidis identified by mass spectrometry.

1. Protein identified from the photocrosslinking products for a molecular weight around 150kDa
Accession number	Protein name	*N. meningitidis* strain**	Estimated Molecular weight	Number of matched peptides	MASCOT score	Sequence coverage
gi\|325136153	hypothetical protein NMBM0579_0886	NMBM0579_0886	57kDa	102	1843	25%
gi\|325144519	hypothetical protein	NMBM01240013_0931	57kDa	99	1758	24%
gi\|325140435	hypothetical protein	CU385	57kDa	93	1718	24%
gi\|488174415	hypothetical protein		57kDa	93	1920	22%
gi\|485353984	DNA-directed RNA polymerase, β subunit	73696	150kDa	24	1436	22%
gi\|488170470	hypothetical protein		57kDa	71	1394	20%
gi\|6977941	App protein		160kDa	7	425	7%
gi\|915797040	adhesin		160kDa	6	390	6%
gi\|83270238	AusI		158kDa	4	288	4%
gi\|389605917	NAD(P) transhydrogenase subunit alpha	α522	54kDa	2	162	2%
gi\|488141712	carbon starvation protein A		75kDa	3	146	3%
gi\|896381024	hypothetical protein		57kDa	8	119	2%
gi\|488158877	hypothetical protein		147kDa	2	109	2%
gi\|254672742	alanine or glycine: cation symporter, AGCS family	α275	45kDa	2	109	1%
2. Protein identified from the photocrosslinking products for a molecular weight around 140kDa.
Accession number	Protein name	*N. meningitidis* strain**	Estimated Molecular weight	Number of matched peptides	MASCOT score	Sequence coverage
gi\|325136153	hypothetical protein NMBM0579_0886	NMBM0579_0886	57kDa	80	1852	25%
gi\|120866758	hypothetical protein NMC1281	FAM18	56kDa	69	1863	23%
gi\|501178984	DNA-directed RNA polymerase β subunit		154kDa	28	1808	27%
gi\|325140435	hypothetical protein NMBCU385_0826	CU385	57kDa	72	1803	22%
gi\|325144519	hypothetical protein	NMBM01240013_0931	57kDa	78	1782	24%
gi\|488171514	hypothetical protein		57kDa	70	1656	23%
gi\|488170470	hypothetical protein		57kDa	54	1267	18%
gi\|488186495	hypothetical protein		147kDa	12	800	12%
gi\|488186095	immunoglobulin A1 protease		121kDa	9	653	9%
gi\|488151215	peptidylprolyl isomerase		56kDa	8	500	8%
gi\|316985319	filamentous hemagglutinin family N-terminal domain protein	H44/76	261kDa	8	496	8%
gi\|488154218	heme biosynthesis operon protein HemX		48kDa	7	490	7%
gi\|488151215	peptidylprolyl isomerase		56kDa	8	465	8%
gi\|488146114	D-lactate dehydrogenase		64kDa	5	363	5%
gi\|389605276	UPF0141 inner membrane protein yhjW		62kDa	6	351	5%
gi\|488170091	peptidase		200kDa	3	202	3%
gi\|120866107	putative protein-export membrane protein	FAM18	66kDa	3	176	3%
gi\|732853	IgA1 protease		54kDa	3	168	3%
gi\|488141712	carbon starvation protein A		74kDa	3	165	3%
gi\|316985875	4Fe-4S binding domain protein		146kDa	3	150	3%
gi\|389605917	NAD(P) transhydrogenase subunit α		54kDa	2	140	2%
gi\|488141355	arginine decarboxylase		71kDa	2	129	2%
gi\|349520	pilus structural subunit, partial		17kDa	2	128	2%
gi\|488143525	protein translocase component YidC		60kDa	2	125	2%
gi\|254670993	1-deoxy-D-xylulose 5-phosphate synthase		69kDa	2	114	2%
gi\|304337210	methionine-R-sulfoxide reductase		59kDa	2	112	2%
gi\|4838369	NatD		52kDa	2	109	2%
3. Protein identified from the photocrosslinking products for a molecular weight around 85kDa.
Accession number	Protein name	*N. meningitidis* strain**	Estimated Molecular weight	Number of matched peptides	MASCOT score	Sequence coverage
gi\|2460281	outer membrane protein Omp85		88kDa	63	2799	41%
gi\|488170971	membrane protein		88kDa	58	2599	38%
gi\|325136153	hypothetical protein NMBM0579_0886		57kDa	63	1774	24%
gi\|325144519	hypothetical protein NMBM01240013_0931		57kDa	60	1692	23%
gi\|488171514	hypothetical protein		57kDa	56	1619	22%
gi\|488174415	hypothetical protein		57kDa	56	1577	22%
gi\|325141117	LPS-assembly protein LptD	CU385	87kDa	21	1440	19%
gi\|488147360	elongation factor G		77kDa	27	1398	20%
gi\|325141117	putative organic solvent tolerance protein	CU385	87kDa	20	1149	18%
gi\|409107079	type IV pilus biogenesis and competence protein PilQ		80kDa	17	973	15%
gi\|209363328	hemoglobin receptor, partial		87kDa	16	909	14%
gi\|488154195	ATP-dependent Clp protease ATP-binding subunit ClpA		85kDa	21	905	16%
gi\|30017077	TonB-dependent siderophore receptor FetA		73kDa	14	793	13%
gi\|120866913	RNA polymerase sigma factor		75kDa	11	616	10%
gi\|325142240	2-oxoglutarate dehydrogenase E1 component		102kDa	10	496	8%
gi\|488150134	GNAT family N-acetyltransferase		89kDa	7	421	7%
gi\|496712676	fimbrial protein		18kDa	9	307	4%
gi\|645213765	peptidase		88kDa	5	295	5%
gi\|402319479	hypothetical protein NMEN93004_1215		18kDa	6	294	5%
gi\|488144915	phosphoenolpyruvate synthase		87kDa	5	293	5%
gi\|254673965	putative efflux system transmembrane protein		113kDa	5	249	4%
gi\|496706569	transferrin-binding protein 2		74kDa	5	239	4%
gi\|896272027	pilus assembly protein PilQ		75kDa	5	222	4%
gi\|45245	periplasmic iron-binding protein		34kDa	4	204	4%
gi\|121052755	GTP pyrophosphokinase		86kDa	3	199	3%

Open in a new tab

Proteins shown in italics corresponds to the PamA protein itself.

Proteins shown in bold indicates the candidates examined for crosslinking to PamA K278(pBPa) in N. meningitidis by constructing the FLAG-tagged candidate mutants, but the crosslinking could not be detected.

The protein shown underlined is PilE crosslinked to PamA K278(pBPa) in N. meningitidis, which was confirmed in this study.

We then characterized the 85-kDa band, which could contain an approximately 20 kDa endogenous protein crosslinked to PamA K278(pBPa). LC-MS/MS followed by MASCOT analyses revealed two possible candidates, a fimbrial protein (17 kDa) and the hypothetical protein NMEN93004_1215 (18 kDa) (Table 3). The BLAST homology search indicated that while we could not find any protein corresponding to NMEN93004_1215 in the databases, the fimbrial protein (accession number gi|496712676) was 95% identical to the protein NMBH4476_0018 annotated as “type IV pilus assembly protein PilA” in N. meningitidis strain H44/76 with the highest E-value (Table 3). Further computational analyses revealed that the “PilA protein” annotated in GenBank was 96% identical to PilE, a major component of meningococcal pili (reviewed in [17]). To confirm that the 85-kDa complex was composed of PilE and K278(pBPa)-Strep₂-His₆, we constructed the pilE-FLAG N. meningitidis strain HT2095 and examined the crosslinked samples by Western blotting with an anti-FLAG mAb (Fig 5C). The analysis revealed that PilE-FLAG was present in the 85-kDa band, with dependence on pBPa and UV irradiation (Fig 5C). We further examined crosslinked samples from the wild-type (HT1940 harboring pHT1388) and pilE-insertional mutant (HT2014 harboring pHT1388) by Western blotting with anti-PilE rabbit serum [33] (Fig 5D). The 85-kDa band was also observed with anti-PilE rabbit serum in the pilE⁺ genetic background only with pBPa and UV irradiation. Considering these results, we conclude that the 85-kDa band contains the PilE protein, indicating that PamA interacts with PilE in N. meningitidis.

Mapping the amino acid residue in PilE that crosslinks to the PamA protein

To further clarify the interaction between PamA and PilE, we attempted to identify the amino acid residue of PilE that crosslinks with PamA K278(pBPa). Since mapping was difficult and tedious [42], particularly for the endogenous proteins, the examination was conducted with recombinant proteins in E. coli.

We examined the interaction of two truncated recombinants, ΔN-PamA K278amb fused to maltose-binding protein (MBP) (MBP-ΔN-PamA K278amb) and ΔN-PilE with six glutamines (Gln₆) in E. coli (S6 Fig). The crosslinked complex between MBP-ΔN-PamA K278(pBPa) and Gln₆-ΔN-PilE was purified from E. coli and investigated using a gel-based proteomic analysis with LC-MS/MS (S7–S9 Figs) [42]. The peptides containing pBPa (IEVGK*[pBPa]LAFSTK), corresponding to aa 639 to 650 of MBP-ΔN-PamA K278(pBPa) (corresponding to aa 274 to 284 of native PamA), from crosslinked and uncrosslinked samples were analyzed by MS/MS. As shown in S9 Fig, the sequence was read from the annotated b and y ions of a crosslinked peptide until isoleucine (Ile) at position 19. This result indicated that the region around Ile19 in Gln₆-ΔN -PilE is the site crosslinked to K278(pBPa) in MBP-ΔN-PamA.

Genetic elucidation of the interaction between PamA and PilE in N. meningitidis and its important for meningococcal internalization into HBMEC

Ile at position 19 in Gln₆-ΔN-PilE corresponded to position 12 in native PilE in N. meningitidis (S10 Fig) because prepilin is processed by the prepilin peptidase PilD during translocation at the IM in N. meningitidis [36, 67]. Multiple alignments showed that the N-terminal region of PilE around Ile12 is highly conserved among neisserial species, including N. gonorrhoeae and N. lactamica, implicating the importance of the region in PilE (S10A Fig) [68].

To further examine the functional interaction between PamA and the region containing the PilE I12 residue in N. meningitidis, the meningococcal pilE I12A-cat mutant HT2167 was constructed (Table 1), and we monitored its infectious ability and the accompanying host cell’s cytoskeleton rearrangement by the localization of ezrin. The pilE I12A N. meningitidis mutant was less efficiently internalized into HBMEC, while the adhesion remained unchanged (Fig 6A), and ezrin did not accumulate beneath the pilE I12A-cat mutant (Fig 6B). Western blotting with UV-irradiated samples purified from HT2167 expressing the MmpBPaRS/T and pamA K278amb genes also confirmed that the interaction between PilE and PamA K278(pBPa) is markedly reduced by the pilE I12A mutation (Fig 6C), providing genetic evidence for the interaction between PamA and PilE in N. meningitidis. Furthermore, since the phenotypes appeared to be mostly identical to that of the ΔpamA N. meningitidis mutant (Figs 1 and 6A), these results indicated that PamA and PilE interact functionally with each other in N. meningitidis and that this interaction is important for meningococcal infection in HBMEC.

Fig 6 — (A) A I12A mutation in the *pilE* gene affects N. *meningitidis* internalization into HBMEC. Adherence (white) and internalization (gray) of N. *meningitidis* strains in HBMEC. Each value is the mean ± standard error of the mean (CFU per 10⁴ HBMEC) from at least four experiments. The bars indicate the bacterial numbers of N. *meningitidis* wild-type *pamA*⁺ (HT1125, left), *ΔpamA*::*spc* (HT1822, middle-left), *pilE*⁺*-cat* (HT1744 middle-right), and *pilE I12A-cat* (HT2167, right), respectively (see S1 Table). *P<0.01, **P<0.02, significantly different from the *pamA*⁺ strain or *pilE*⁺*-cat* strain. (B) Immunofluorescence microscopy showing the accumulation of ezrin beneath N. *meningitidis* strains. The HBMEC monolayer was infected with wild-type *pilE*⁺*-cat* (middle) and *pilE I12A-cat* (right) N. *meningitidis* strains. A non-infected HBMEC monolayer is also shown in the left panels. Bacteria and HBMEC were observed by phase-contrast microscopy (upper panels). Ezrin was immunostained with anti-ezrin mAb and Alexa Fluor 488-conjugated rabbit anti-mouse IgG (green channel, lower panels). (C) PilE I12A was crosslinked to PamA K278(pBPa) less efficiently than wild-type PilE in N. *meningitidis*. Black and red arrows show the crosslinked complex between PamA K278(pBPa) and PilE, and the gray arrow indicates an unidentified band that crossreacted with anti-PilE rabbit serum. +/- indicates the presence or absence of the *pilE* gene, respectively.

Characterization of pili in the ΔpamA N. meningitidis mutant

The results described above suggested that PamA affects the functions of meningococcal pili, resulting in a negative impact on meningococcal infection in HBMEC. To further clarify the effects of PamA on pili, we investigated the function of pili in N. meningitidis. Electron microscopy with negative staining showed that the ΔpamA N. meningitidis mutant was similarly piliated to the wild-type strain HT1125 (S11A Fig, left and middle-left). Meningococcal pili are known to play a role not only in the infection of host cells [17, 69], but also in meningococcal natural competence, switching motility, and aggregation [70, 71]. An examination of pilus function in natural competence and aggregation revealed that the pili of the ΔpamA N. meningitidis mutant retain similar functions to those of the wild-type pilE N. meningitidis strain (S11B and S11C Fig). These results suggest that PamA does not participate in pilus formation itself or in functions related to natural competence and aggregation. It is important to note that the pilE I12A-cat N. meningitidis mutant is also as functional as the wild-type pilE-cat N. meningitidis strain while its piliation number its apparently smaller possibly due to translational fusion with the cat gene (S11 Fig and Fig 6).

We examined the effects of PamA on meningococcal pili in more detail. Although the main component of the meningococcal pilus is PilE, its formation requires more than 20 proteins [72–74]. To investigate the effects of the pamA mutation on pili, we initially assessed the pilus protein levels in HT1125 and HT1822 using LC-MS/MS with Tandem Mass Tag (TMT) labeling [38] (S5 Table), and found that the amounts of some pilus components appeared to differ between HT1125 and HT1822. However, accurate comparisons of the protein amounts, particularly that for PilE, by TMT are methodologically difficult because N. meningitidis has 8 non-expressed pilS genes [75], according to the deduced amino acids recorded as Protein in the database. Therefore, we directly monitored the contents of pilus components, particularly for three minor pilins: PilX for aggregation in and adherence to human cultured cells [71, 76, 77], PilV to trigger plasma membrane reorganization [36, 71, 78], and ComP for natural competence [79]. PilF, PilM, and PilP, located on the meningococcal inner membrane for pilus formation [8, 67, 69, 80, 81], were also monitored. The relative amounts of the components to the PilE subunit in purified pili were measured by Western blotting followed by quantification (Fig 7). The relative ratios of PilF, PilP, and PilM to PilE did not significantly differ between the ΔpamA and wild-type N. meningitidis strains. On the other hand, in ΔpamA pili the relative amount of PilV increased, whereas that of PilX decreased (Fig 7A). It is important to note that the amount of ComP was too low in the whole cell extract or purified pili to detect by WB, even when the comP-FLAG N. meningitidis strain or anti-ComP rabbit antiserum was used (data not shown). We also confirmed that the expressions of PilE, PilV, and PilX-FLAG were not affected by the ΔpamA mutation (Fig 7B). Considering these results, the pamA mutation may cause unusual proportions of PilV and PilX to PilE in meningococcal pili, probably by improper sorting of PilE during pili formation, which might affect meningococcal infection in human cultured cells.

Fig 7 — (A) Ratio of pilus components to PilE in purified pili. The relative amounts of PilF-FLAG (HT2218 and HT2219), PilP-FLAG (HT2136 and HT2137), PilM-HA (HT2132 and HT2133), PilV (HT1125 and HT1822), and PilX-FLAG (HT2211 and HT2212) to PilE in purified pili were measured by Western blotting with appropriately diluted samples followed by quantification of the band intensity using the 30-kDa band in the molecular marker as an internal golden standard. The values obtained were divided by the value of PilE in the same sample. The significance of differences was examined by the t-test. (B, C, D) The *pamA* mutation does not affect the expression of the PilE (B), PilV (C), or PilX-FLAG (D) protein in N. *meningitidis*. Bacterial extracts equivalent to OD₆₀₀ value of 0.0025 (for PilE) or 0.05 (for PilV and PilX-FLAG) were analyzed by Western blotting with anti-PilE and anti-PilV rabbit sera, and an anti-FLAG mAb, respectively. Black arrows indicate PilE, PilV, and PilX-FLAG, respectively.

Discussion

While progressive genome sequencing has recently provided novel insights into N. meningitidis, more than 50% of the annotated ORFs remain as “hypothetical proteins”, and their biological functions are currently unclear. This may be due to an incomplete understanding of meningococcal pathogenesis, which is largely attributable to the lack of analytical methods for N. meningitidis [34]. While the results of our genetic analyses indicated that the hypothetical protein NMB1345 (renamed PamA in the present study) in N. meningitidis plays an important role in meningococcal internalization into human cultured cells, the novel methodologies currently available for meningococci did not provide any insights into the function of PamA in N. meningitidis. Therefore, the incorporation of photoreactive ncAAs by pyrrolysine-based amber suppression was applied to a meningococcal protein with an unknown function, which implicated its physiological function in N. meningitidis.

The incorporation of ncAAs by pyrrolysine-based amber suppression has been used for site-specific incorporation into target proteins in vivo in a broad range of species from E. coli to eukaryotic systems (reviewed in [41]). However, regarding pathogenic bacteria, only a few studies have successfully incorporated ncAAs into EPEC [82], Shigella flexinelli [82], Salmonella typhimurium [83], M. tuberculosis [84], and B. cereus [85]. Moreover, these bacteria were only subjected to examinations of the incorporation of ncAAs into a well-characterized protein (e.g. GST or GFP), with a low molecular mass (approx. 10 kDa) and a known function [86], or a peptide [85], produced from expression vectors. Moreover, X-ray structure information for target proteins will also be advantageous for photoreactive ncAAs incorporation because the proper positions to incorporate ncAAs may be more easily predicted. Even when the X-ray structure of a target protein is unknown, a lower molecular mass is more advantageous because it is easier to scan the optimal site for ncAA incorporation in the target protein. Therefore, genetic photocrosslinker incorporation has not yet been applied to analyze a protein with unknown function or three-dimensional structure is unknown. Furthermore, some disadvantages in the application of photocrosslinking ncAAs incorporation combined with MS to Neisseria species are the very few expression vectors for neisseriae [33, 34] [87] and the limited culture methods in liquid medium due to autolysis [88]. To the best of our knowledge, this is the first study to demonstrate the incorporation of ncAAs into N. meningitidis. Moreover, after observing that ncAA-incorporated GST proteins are crosslinked as the dimer form in N. meningitidis (Fig 3E), we incorporated pBPa into the PamA protein in N. meningitidis (S5 Fig) and found that this meningococcal hypothetical protein with no physiological and structural information is closely associated with PilE in N. meningitidis.

Our strategy of incorporating a genetic photocrosslinker coupled with MS in N. meningitidis revealed that the meningococcal hypothetical protein PamA is associated with PilE in N. meningitidis. Further physicochemical analyses with PamA and PilE recombinants in E. coli identified the crosslinked regions between PamA K278(pBPa) PilE (S7–S9 Figs), and genetic analyses using the pilE I12A N. meningitidis mutant suggested that the interaction between PamA and PilE in N. meningitidis is functionally important for meningococcal pilus formation and infection to HBMEC (Fig 6). While further studies are needed, the PamA interaction with the N-terminal region of PilE around the Ile12 residue (S10 Fig) may be important to maintain the function of meningococcal pili by PamA in N. meningitidis.

The function of meningococcal PamA has not been clearly discerned in this study. The present results showed that PamA is not required for pilus formation itself (S11A Fig) but participates to keep optimal proportions of PilV and PilX to PilE in meningococcal pili (Fig 7). This result does not seem to contradict previous findings showing that the proper ratio of PilV and PilX to PilE in pili leads to pilus conformational changes that trigger plasma membrane reorganization [36, 71, 78, 76, 77]. However, we could not eliminate the possibility that more essential dysregulation(s) other than the imbalance of PilV and PilX to PilE in meningococcal pili by the pamA mutant were not identified in this study. We focused on three bands crosslinked with PamA K278(pBPa) with the highest intensity among the five PamA K-amb (pBPa) mutants, while the interaction between PamA and PilE found was identified from a band with the lowest intensity (Fig 4). On the other hand, the two protein complexes with the highest intensities (approximately 150 and 140 kDa) crosslinked to PamA K278(pBPa) were not identified in any protein other than PamA itself by peptide mass fingerprinting (Table 3). Although further analyses are needed, the two bands appeared to correspond to the homotrimer of PamA because intramolecular crosslinking was also detected in the MBP-ΔN-PamA K278(pBPa) recombinant protein in E. coli (S12 Fig). The endogenous proteins crosslinked to the PamA K208(pBPa), PamA K341(pBPa), PamA K382(pBPa), and PamA K395(pBPa) mutants have yet to be identified (Fig 4) and their characterization will provide more information about the novel function of PamA in N. meningitidis.

Surprisingly, unidentified genes involved in pilus function are still being discovered, although more than 20 proteins involved in pilus synthesis and function have already been identified by genetic [72–74] and WGS [32, 39] analyses. Therefore, our strategy using genetic screening by STM combined with genetically-encoded photocrosslinking amino acid incorporation and an MS analysis, could be a powerful method to identify new pathogenic factors in N. meningitidis. BLAST searches revealed pamA gene homologues in only three neisserial species: N. meningitidis, N. gonorrhoeae, and N. lactamica (S13 Fig). N. meningitidis and N. gonorrhoeae are closely related bacterial pathogens that share many common genes with a high degree of sequence identity (typically greater than 95%), and DNA relatedness studies clustered commensal N. lactamica with these two pathogenic Neisseria spp. [89]. Since the sequences of the pamA genes appeared to be more divergent among the three species than within N. meningitidis (S13 Fig), the pamA gene may have been acquired at a later stage of evolution by the three neisserial species, and the restricted possession of the pamA gene in the three neisserial species may reflect the physiological function of PamA.

While further studies are needed to clarify the functions of PamA in Neisseria, it is important to note that the application of new methodology to N. meningitidis provided novel insights into the physiological function of a meningococcal hypothetical protein with no obvious information such as its predicted biological function or three-dimensional structure. Our established system for ncAA incorporation by pyrrolysine-based amber suppression into N. meningitidis will facilitate a broad range of N. meningitidis studies, particularly the mapping of protein-protein interaction networks or characterization of the functions of hypothetical proteins in vivo. Nevertheless, as shown in the present study, in vivo research involving the combination of STM and photoreactive ncAAs incorporation by pyrrolysine-based amber suppression with an MS analysis will reveal meningococcal virulence factors that are difficult to identify using most currently available methods. The ncAA incorporation system described in the present study may become one of the strongest driving advancements for studies on meningococcal biology, pathogenesis, and vaccine development.

Supporting information

S1 Fig. Nucleotide sequence and deduced amino acid sequence of the pamA gene in N. meningitidis HT1125 strain.

(GenBank Accession No LC511747). Lysine (K) residues are shown in red and the K residues replaced with amber codons in this study are indicated in bold red. Positions of K residues are shown as blue numbers. The grey bar indicates the putative hydrophobic region identified by SOSUI [90], which region were deleted to construct the PamA recombinants in E. coli in this study.

(PDF)

Click here for additional data file.^{(427.8KB, pdf)}

S2 Fig. Photocrosslinking of PamA K148(mAzZLys), K273(mAzZLys), and K388(mAzZLys) in N. meningitidis.

(A) Detection of PamA K148amb, K273 and K388amb expressed by pyrrolysine-based amber suppression with mAzZLys in N. meningitidis. Bacteria grown on one-fourth of a GC agar plate in the presence or absence of 0.3 mM mAzZLys and extracts equivalent to OD₆₀₀ of 0.1 were analyzed by Western blotting with anti-PamA rabbit serum. +/- indicates the presence or absence of mAzZLys. The black arrow shows the full-length PamA protein. (B) Detection of complexes containing PamA K(mAzZLys) crosslinked to an endogenous protein in N. meningitidis. Bacteria grown on one-fourth of a GC agar plate in the presence of 0.3 mM mAzZLys were crosslinked by UV light irradiation, and mixed with SDS buffer. Aliquots were analyzed by Western blotting with an anti-His₆ mAb. +/- indicates presence or absence of mAzZLys (upper), treatment or nontreatment with UV irradiation (lower). Black and grey arrows show the full-length PamA protein and putative complexes corresponding to PamA K(mAzZLys) crosslinked to an endogenous protein, respectively.

(PDF)

Click here for additional data file.^{(500.1KB, pdf)}

S3 Fig. PamA K278(pBPa) protein crosslinks with endogenous proteins in N. meningitidis.

UV crosslinking of 27 PamA K-amb mutants expressed by pyrrolysine-based amber suppression with pBPa in ΔpamA N. meningitidis. The black arrow shows the full-length PamA K-amb protein expressed by pyrrolysine-based amber suppression with pBPa. PamA K-amb mutants shown in red were subjected to a more detailed analysis in Fig 4.

(PDF)

Click here for additional data file.^{(1.3MB, pdf)}

S4 Fig. Schematic structure of the C-terminal region of PamA K278-Strep₂-His₆.

(A) Amino acid sequence of the C-terminal region of PamA K278-Strep₂-His₆. (B) Nucleotide sequence of the C-terminal region of PamA K278-Strep₂-His_6. The same colors in A and B corresponds to the same region in amino acid (A) and nucleotide (B) sequences.

(PDF)

Click here for additional data file.^{(32.5KB, pdf)}

S5 Fig. Mass spectrometry analysis of PamA K278amb expressed by the pyrrolysine-based amber suppression with pBPa in N. meningitidis.

(A) Amino acid sequence of PamA with Strep₂-His₆ tag at C-terminus. The tryptic peptide containing a ncAA, pBPa is highlighted in red and the pBPa at position 278 is represented as X. (B) The incorporation of pBPa at position 278 was confirmed by MALDI-TOF MS analysis of the tryptic peptide IEVGXLAFSTK (X represents pBPa). The observed (obsd) molecular masses agreed well with the calculated (calcd) masses. (C) MALDI-TOF MS/MS analysis of the tryptic peptide of shown in B. Tandem mass spectrum of the peptide IEVGXLAFSTK (X = pBPa). The sequence can be read from the annotated b (red) or y (blue) ion series.

(PDF)

Click here for additional data file.^{(83.1KB, pdf)}

S6 Fig. Examination of purified MBP-ΔN-PamA K278(pBPa) and Gln₆-ΔN-PilE recombinant proteins from E. coli strains for LC-MS/MS analysis.

Recombinant proteins crosslinked in E. coli were purified with amylose resin and concentrated to 500 μl as described in the Materials and Methods. Aliquots (approximately 1 μl) were fractionated by SDS-PAGE and analyzed by staining with a CBB staining kit (A), and Western blotting with anti-MBP (B) and anti-His₆ (C) mAbs. Prestained molecular mass standards are shown by grey numbers with asterisks, since the apparent molecular masses were different from the actual masses due to abnormal mobility in the gel. +/- indicates treatment or non-treatment with UV irradiation, respectively. Black, blue and red arrows indicate PamA ΔN-K278amb (no pyrrolysine-based amber suppression with pBPa), PamA ΔN-K278(pBPa)-Strep₂-His₆ (full-length) and the complex crosslinked between PamA K278(pBPa)-Strep₂-His₆ and ΔN-Gln₆-PilE, respectively. (D) Schematic figure of the crosslinking between PamA ΔN-K278(pBPa)-Strep₂-His₆ and Gln₆-ΔN-PilE recombinant proteins in E. coli.

(PDF)

Click here for additional data file.^{(661.3KB, pdf)}

S7 Fig. Analysis of amino acid residues (region) in PilE crosslinked to PamA K278 (pBPa) by LC-MS/MS.

(A) Liquid chromatogram of uncrosslinked MBP-ΔN-PamA K278(pBPa) and the Gln₆-ΔN-PilE protein (upper), and the complex crosslinked between MBP-ΔN-PamA K278(pBPa) and Gln₆-ΔN-PilE (lower). The fraction eluted at 12.38 min from the uncrosslinked sample contained the peptide IEVGK(pBPa)LAFSTK corresponding to position 639 to 650 of MBP-ΔN-PamA K278(pBPa) (positions 274 to 284 for native PamA), determined by an MS analysis. (B) Enlarged liquid chromatogram after 14 min of elution. The fraction eluted at 17.32 min from the crosslinked sample contained the peptide MQQQQQQFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQK, corresponding to the first 51 amino acids of Gln₆-ΔN-PilE at the N terminus, which was crosslinked to the peptide IEVGK(pBPa)LAFSTK from the MBP-ΔN-PamA K278(pBPa) protein. (C) MS spectrum of the LC fraction eluted at 17.32 min that contained the cross-linked peptide between MQQQQQQFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQK from Gln₆-ΔN-PilE and IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa)(obsd:1380.9367 [M+H]⁵⁺).

(PDF)

Click here for additional data file.^{(68.8KB, pdf)}

S8 Fig. MS/MS spectrum of the peptide IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa).

The sequence was read from the annotated b and y ion series; b3, y4, y5, y6, y7, y8, y9, and y10 ions were observed.

(PDF)

Click here for additional data file.^{(28.3KB, pdf)}

S9 Fig. MS/MS spectrum of the cross-linked peptide between MQQQQQQFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQK from Gln₆-ΔN-PilE and IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa)(obsd:1380.9367 [M+H]⁵⁺).

The sequence was read from the annotated b and y ion series; b3, y4, y5, y6, y7, y8, y9, and y10 ions of the peptide IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa) (shown in red), and b3, b6, b9, b10, b11, b12, b13, b14, b15, b16, b17, b18, y4, y5, y6, y7, y8, y9, y10, y11, y12, y13, y14, y15, y16, y17, y18, y19, y20, y21, y22, y23, y24, y25, y26, y27, y28, y29, y30, y31, and y32 ions of the peptide MQQQQQQFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQK from Gln₆-ΔN-PilE (shown in blue) were observed.

(PDF)

Click here for additional data file.^{(48.8KB, pdf)}

S10 Fig. Sequence alignment between neisserial PilE and crystal structure of full-length gonococcal PilE.

(A) Amino acid sequence alignment of PilE proteins from N. meningitidis (Nm), N. gonorrhoeae (Ng) and N. lactamica (Nl). The N-terminal half of the α-helix (α1-N) responsible for pilin assembly is shown by red (residues 1–14; α1:1–14) and cyan (residues 15–28; α1:15–28) bars, respectively. The αβ-loop, protruding from the globular domain to form a ridge on the subunit surface is indicated as a yellow bar. The D-region containing the hypervariable loop that protrudes as a second ridge on the globular domain is indicated as a light green bar. Red numbers indicate the positions of amino acids at which the functions of α1-N are divided. Ile at position 12 is indicated by a red vertical arrow. N. meningitidis HT1125 PilE (DDBJ accession no. AB698857), N. meningitidis H44/76 PilE (GenBank accession no. CP002420), N. gonorrhoeae MS11 PilE (EMBL accession no. CAI08338) and N. lactamica NLA_1780 type IV pilus assembly protein PilA (GenBank accession no. FN995097). (B) X-ray crystal structure of gonococcal PilE [91]. α1:1–14, α1:15–28, αβ-loop and D-region are shown in red, cyan, yellow and light green, respectively (the colors in B correspond to those in A). Ile at position 12 is indicated as a space-filling molecule.

(PDF)

Click here for additional data file.^{(159.5KB, pdf)}

S11 Fig. Characterization of pili.

(A) Electron micrographs showing piliation of N. meningitidis strains HT1125 (wild-type), HT1822 (ΔpamA), HT1744 (pilE⁺-cat), and HT2167 (pilE I12A-cat). Upper and lower panels show magnifications of 20,000 and 50,000, respectively. Scale bars shown in black (upper) and white (lower) represent 200 nm. (B) Quantification of the competence for DNA transformation in N. meningitidis wild-type, and pilE^-, ΔpamA, pilE⁺-cat and pilE I12A-cat mutants. Equivalent numbers of recipient cells were transformed using 0.5 μg of chromosomal DNA purified from the Nal^R (nalidixic acid resistant) strain HT1001, and Nal^R transformants were counted. Results are expressed as numbers of Nal^R transformants per 1 μg DNA and ± standard deviation from at least 8 independent experiments. (C) Aggregation as assessed by phase-contrast microscopy. Aggregates of N. meningitidis strains HT1125 (wild-type), HT1156 (pilE^-), HT1822 (ΔpamA), HT1744 (pilE⁺-cat) and HT2167 (pilE I12A-cat) were observed after 4 hours of incubation in RPMI containing 10% fetal bovine serum.

(PDF)

Click here for additional data file.^{(4.5MB, pdf)}

S12 Fig. Detection of homomeric crosslinking between two MBP-ΔN-PamA K278(pBPa).

MS/MS spectrum of the peptide IEVGK(pBPa)LAFSTK (at positions 274 to 284 for native PamA) crosslinked to LNELVNLVTDLQIGAFINPNGSIAPS (at positions 247 to 273 for native PamA) in MBP-ΔN-PamA K278(pBPa). The crosslinked site was analyzed by SIM-XL (http://patternlabforproteomics.org/sim-xl). The sequence can be read from the annotated b and y ion series: b3, y3, y4, y5, y6 ions of the peptide IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa) (shown in red), and b3, b4, b5, b6, b7, b8, b10, b13, b19, b21, y4, y5, y6, y7, y8, y9, y10, y11, y12, y13, y14, y15 ions of a peptide LNELVNLVTDLQIGAFINPNGSIAPS from MBP-ΔN-PamA K278(pBPa) (blue) were observed, respectively. We could not confirm whether pBPa at position 278 was crosslinked to Leu at position 257 or Gln at position 258 (shown in yellow overlay).

(PDF)

Click here for additional data file.^{(282.5KB, pdf)}

S13 Fig. Phylogenetic tree showing the relation of pamA genes among neisserial species: N. meningitidis, N. gonorrhoeae and N. lactamica.

The pamA genes (the genes encoding “conserved hypothetical protein”) from three neisserial species were analyzed by Molecular Evolutionary Genetics Analysis (MEGA) X [92]. The evolutionary history was inferred using the Neighbor-Joining method [93]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [94] and are in the units of the number of base substitutions per site. The robustness of the NJ method was tested by bootstrapping with 500 replicates of data, and the percentages are shown at the nodes. This analysis involved 19 nucleotide sequences of genes encoding N. meningitidis HT1125 PamA (DDBJ accession no. LC511747), N. meningitidis H44/76 conserved hypothetical protein (GenBank accession no. CP002420), N. meningitidis MC58 hypothetical protein (GenBank accession no. AE002098), N. meningitidis M04-240196 conserved hypothetical protein (GenBank accession no. CP002423), N. meningitidis alpha14 conserved hypothetical protein (GenBank accession no. AM889136), N. meningitidis NZ-05/33 conserved hypothetical protein (GenBank accession no. CP002424), N. meningitidis M01-240149 conserved hypothetical protein (GenBank accession no. CP002421), N. meningitidis alpha710 hypothetical protein (GenBank accession no. CP001561), N. meningitidis 053442 conserved hypothetical protein (GenBank accession no. CP000381), N. meningitidis 8013 conserved hypothetical protein (GenBank accession no. FM999788), N. meningitidis M01-240355 conserved hypothetical protein (GenBank accession no. CP002422), N. meningitidis 510612 hypothetical protein (GenBank accession no. CP007524), N. meningitidis WUE2694 conserved hypothetical protein (GenBank accession no. FR774048), N. meningitidis G2136 conserved hypothetical protein (GenBank accession no. CP002419), N. meningitidis Z2491 hypothetical protein (GenBank accession no. AL157959), N. meningitidis FAM18 hypothetical protein (GenBank accession no. AM421808), N. gonorrhoeae FA1090 hypothetical protein (RefSeq accession no. NC_002946), N. gonorrhoeae NCCP11945 conserved hypothetical protein (GenBank accession no. CP001051) and N. lactamica putative secreted protein (GenBank accession no. FN995097).

(PDF)

Click here for additional data file.^{(20.2KB, pdf)}

S1 Table. Oligonucleotides used in this study.

(DOCX)

Click here for additional data file.^{(69.8KB, docx)}

S2 Table. Plasmids to construct N. meningitidis mutants.

(DOCX)

Click here for additional data file.^{(22KB, docx)}

S3 Table. Plasmids for production of protein recombinants expressed in E. coli.

(DOCX)

Click here for additional data file.^{(16.2KB, docx)}

S4 Table. IncQ plasmids for N. meningitidis strains which results were shown in supporting information.

(DOCX)

Click here for additional data file.^{(16.4KB, docx)}

S5 Table. Analysis of Pili components by Tandem Mass Tag labeling.

Pili isolated from N. meningitidis strains HT1125 (wild-type) and HT1822 (ΔpamA) were subjected to trypsin digestion. Labelling of the peptides and analysis by LC-MS/MS were performed as described previously [38]. Proteins were identified by running MASCOT against the NCBI database (NCBInr 20160202). Ratio of the protein levels were expressed as the value of relative amount in HT1822 divided into those in HT1125. The results from four independent experiments were shown in this table.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S1 File

(PDF)

Click here for additional data file.^{(18.6MB, pdf)}

Acknowledgments

We thank Dr. Masatomo Morita for advice about phylogenetic analyses.

Data Availability

All relevant data are within the paper and its Supporting Information files. The nucleotide sequence of PamA from N. meningitidis strain HT1125 has been deposited in the DNA Data Bank of Japan (DDBJ) (accession code: LC511747).

Funding Statement

This work was supported by JSPS KAKENHI Grant Numbers 15K08485 and 19K07550 (to H.T.), AMED under Grant Number 18fk0108071j0101 (to H.T.), by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from AMED under Grant Number JP16am0101022 (to S.Y.), and under Grant Number JP17am0101081 (to S.Y.).

References

1.Takahashi H, Haga M, Sunagawa T, Saitoh T, Kitahara T, Matsumoto S, et al. Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens. J Infect Chemother. 2016;22(7):501–4. 10.1016/j.jiac.2015.12.016 [DOI] [PubMed] [Google Scholar]
2.Chang Q, Tzeng YL, Stephens DS. Meningococcal disease: changes in epidemiology and prevention. Clinical epidemiology. 2012;4:237–45. 10.2147/CLEP.S28410 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Coureuil M, Jamet A, Bille E, Lecuyer H, Bourdoulous S, Nassif X. Molecular interactions between Neisseria meningitidis and its human host. Cell Microbiol. 2019;21(11):e13063 10.1111/cmi.13063 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Brouwer MC, Read RC, van de Beek D. Host genetics and outcome in meningococcal disease: a systematic review and meta-analysis. The Lancet Infectious diseases. 2010;10(4):262–74. 10.1016/S1473-3099(10)70045-1 [DOI] [PubMed] [Google Scholar]
5.Codjoe SN, Nabie VA. Climate change and cerebrospinal meningitis in the Ghanaian meningitis belt. International Journal of Environmental Research and Public Health. 2014;11(7):6923–39. 10.3390/ijerph110706923 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Peak IR, Srikhanta Y, Dieckelmann M, Moxon ER, Jennings MP. Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis. FEMS Immunol Med Microbiol. 2000;28(4):329–34. 10.1111/j.1574-695X.2000.tb01494.x [DOI] [PubMed] [Google Scholar]
7.Virji M. Pathogenic neisseriae: surface modulation, pathogenesis and infection control. Nat Rev Microbiol. 2009;7(4):274–86. 10.1038/nrmicro2097 [DOI] [PubMed] [Google Scholar]
8.Coureuil M, Lecuyer H, Bourdoulous S, Nassif X. A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers. Nat Rev Microbiol. 2017;15(3):149–59. 10.1038/nrmicro.2016.178 [DOI] [PubMed] [Google Scholar]
9.Schubert-Unkmeir A. Molecular mechanisms involved in the interaction of Neisseria meningitidis with cells of the human blood-cerebrospinal fluid barrier. Pathog Dis. 2017;75(2). 10.1093/femspd/ftx023 [DOI] [PubMed] [Google Scholar]
10.Capecchi B, Adu-Bobie J, Di Marcello F, Ciucchi L, Masignani V, Taddei A, et al. Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells. Mol Microbiol. 2005;55(3):687–98. 10.1111/j.1365-2958.2004.04423.x [DOI] [PubMed] [Google Scholar]
11.Serruto D, Adu-Bobie J, Scarselli M, Veggi D, Pizza M, Rappuoli R, et al. Neisseria meningitidis App, a new adhesin with autocatalytic serine protease activity. Mol Microbiol. 2003;48(2):323–34. 10.1046/j.1365-2958.2003.03420.x [DOI] [PubMed] [Google Scholar]
12.van Ulsen P, van Alphen L, ten Hove J, Fransen F, van der Ley P, Tommassen J. A Neisserial autotransporter NalP modulating the processing of other autotransporters. Mol Microbiol. 2003;50(3):1017–30. 10.1046/j.1365-2958.2003.03773.x [DOI] [PubMed] [Google Scholar]
13.Turner DP, Marietou AG, Johnston L, Ho KK, Rogers AJ, Wooldridge KG, et al. Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis. Infect Immun. 2006;74(5):2957–64. 10.1128/IAI.74.5.2957-2964.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Oldfield NJ, Bland SJ, Taraktsoglou M, Dos Ramos FJ, Robinson K, Wooldridge KG, et al. T-cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells. Cell Microbiol. 2007;9(2):463–78. 10.1111/j.1462-5822.2006.00803.x [DOI] [PubMed] [Google Scholar]
15.Hung MC, Heckels JE, Christodoulides M. The adhesin complex protein (ACP) of Neisseria meningitidis is a new adhesin with vaccine potential. MBio. 2013;4(2). 10.1128/mBio.00041-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Fletcher LD, Bernfield L, Barniak V, Farley JE, Howell A, Knauf M, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun. 2004;72(4):2088–2100. 10.1128/iai.72.4.2088-2100.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Coureuil M, Jamet A, Bille E, Lecuyer H, Bourdoulous S, Nassif X. Molecular interactions between Neisseria meningitidis and its human host. Cell Microbiol. 2019:e13063 10.1111/cmi.13063 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Dos Santos Souza I, Maissa N, Ziveri J, Morand PC, Coureuil M, Nassif X, et al. Meningococcal disease: A paradigm of type-IV pilus dependent pathogenesis. Cell Microbiol. 2020;22(4):e13185 10.1111/cmi.13185 [DOI] [PubMed] [Google Scholar]
19.Sun YH, Bakshi S, Chalmers R, Tang CM. Functional genomics of Neisseria meningitidis pathogenesis. Nat Med. 2000;6(11):1269–73. 10.1038/81380 [DOI] [PubMed] [Google Scholar]
20.Exley RM, Sim R, Goodwin L, Winterbotham MS, M.C., Read RC, Tang CM. Identification of meningococcal genes necessary for colonization of human upper airway tissue. Infect Immun. 2009;77(1):45–51. 10.1128/IAI.00968-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Mendum TA, Newcombe J, Mannan AA, Kierzek AM, McFadden J. Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera. Genome Biol. 2011;12(12):R127 10.1186/gb-2011-12-12-r127 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Dietrich G, Kurz S, Hubner C, Aepinus C, Theiss S, Guckenberger M, et al. Transcriptome analysis of Neisseria meningitidis during infection. J Bacteriol. 2003;185(1):155–64. 10.1128/jb.185.1.155-164.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Hotopp JCD, Grifantini R, Kumar N, Tzeng YL, Fouts D, Frigimelica E, et al. Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes. Microbiology. 2006;152(Pt 12):3733–49. 10.1099/mic.0.29261-0 [DOI] [PubMed] [Google Scholar]
24.Joseph B, Schneiker-Bekel S, Blom J, Claus H, Linke B, Schwarz RF, et al. Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence. J Bacteriol. 2010;192(20):5363–77. 10.1128/JB.00883-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Joseph B, Frosch M, Schoen C, Schubert-Unkmeir A. Transcriptome analyses in the interaction of Neisseria meningitidis with mammalian host cells. Methods Mol Biol. 2012;799:267–93. 10.1007/978-1-61779-346-2_17 [DOI] [PubMed] [Google Scholar]
26.Hey A, Li MS, Hudson MJ, Langford PR, Kroll JS. Transcriptional profiling of Neisseria meningitidis interacting with human epithelial cells in a long-term in vitro colonization model. Infect Immun. 2013;81(11):4149–59. 10.1128/IAI.00397-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Echenique-Rivera H, Muzzi A, Del Tordello E, Seib K, Francois P, Rappuoli R, et al. Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival. PLoS Pathog. 2011;7(5):e1002027 10.1371/journal.ppat.1002027 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Hedman AK, Li MS, Langford PRF, Kroll JS. Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery. PLoS One. 2012;7(6):e39718 10.1371/journal.pone.0039718 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Bernardini G, Braconi D, Santucci A. The analysis of Neisseria meningitidis proteomes: Reference maps and their maps and their applications. Proteomics. 2007;7(16):2933–46. 10.1002/pmic.200700094 [DOI] [PubMed] [Google Scholar]
30.van Alen T, Claus H, Zahedi RP, Groh J, Blazyca H, Lappann M, et al. Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis. Proteomics. 2010;10(24):4512–21. 10.1002/pmic.201000267 [DOI] [PubMed] [Google Scholar]
31.Kanova E, Jimenez-Munguia I, Majerova P, Tkacova Z, Bhide K, Mertinkova P, et al. Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells. Front Microbiol. 2018;9:2294 10.3389/fmicb.2018.02294 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Siena E, Bodini M, Medini D. Interplay Between Virulence and Variability Factors as a Potential Driver of Invasive Meningococcal Disease. Comput Struct Biotechnol J. 2018;16:61–9. 10.1016/j.csbj.2018.02.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Takahashi H, Watanabe H. A broad-host-range vector of incompatibility group Q can work as a plasmid vector in Neisseria meningitidis: a new genetical tool. Microbiology. 2002;148(Pt 1):229–36. 10.1099/00221287-148-1-229 [DOI] [PubMed] [Google Scholar]
34.Soriani M. Unraveling Neisseria meningitidis pathogenesis: from functional genomics to experimental models. F1000Res. 2017;6:1228 10.12688/f1000research.11279.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Takahashi H, Kim KS, Watanabe H. Differential in vitro infectious abilities of two common Japan-specific sequence-type (ST) clones of disease-associated ST-2032 and carrier-associated ST-2046 Neisseria meningitidis strains in human endothelial and epithelial cell lines. FEMS Immunol Med Microbiol. 2008;52(1):36–46. 10.1111/j.1574-695X.2007.00342.x [DOI] [PubMed] [Google Scholar]
36.Takahashi H, Yanagisawa T, Kim KS, Yokoyama S, Ohnishi M. Meningococcal PilV potentiates Neisseria meningitidis type IV pilus-mediated internalization into human endothelial and epithelial cells. Infect Immun. 2012;80(12):4154–66. 10.1128/IAI.00423-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Takahashi H, Kim KS, Watanabe H. Meningococcal internalization into human endothelial and epithelial cells is triggered by the influx of extracellular L-glutamate via GltT L-glutamate ABC transporter in Neisseria meningitidis. Infect Immun. 2011;79(1):380–92. 10.1128/IAI.00497-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Takahashi H, Yanagisawa T, Kim KS, Yokoyama S, Ohnishi M. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM L-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells. Infect Immun. 2015;83(9):3555–67. 10.1128/IAI.00654-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE, Eisen JA, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science. 2000;287(5459):1809–15. 10.1126/science.287.5459.1809 [DOI] [PubMed] [Google Scholar]
40.Willis JCW, Chin JW. Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem. 2018;10(8):831–7. 10.1038/s41557-018-0052-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Wang L. Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts. Acc Chem Res. 2017;50(11):2767–75. 10.1021/acs.accounts.7b00376 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Holding AN. XL-MS: Protein cross-linking coupled with mass spectrometry. Methods. 2015;89:54–63. 10.1016/j.ymeth.2015.06.010 [DOI] [PubMed] [Google Scholar]
43.Dumas A, Lercher L, Spicer CD, Davis BG. Designing logical codon reassignment—Expanding the chemistry in biology. Chem Sci. 2015;6(1):50–69. 10.1039/c4sc01534g [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Chin JW, Martin AB, King DS, Wang L, Schultz PG. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A. 2002;99(17):11020–4. 10.1073/pnas.172226299 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Takahashi H, Watanabe H. Post-translational processing of Neisseria meningitidis g-glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space. FEMS Microbiol Lett. 2004;234(1):27–35. 10.1016/j.femsle.2004.03.003 [DOI] [PubMed] [Google Scholar]
46.Takahashi H, Watanabe H, Kim KS, Yokoyama S, Yanagisawa T. The Meningococcal Cysteine Transport System Plays a Crucial Role in Neisseria meningitidis survival in human brain microvascular endothelial cells. mBio. 2018;9(6):e02332–18. 10.1128/mBio.02332-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Takahashi H, Inada T, Postma P, Aiba H. CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA^(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli. Mol Gen Genet. 1998;259(3):317–26. 10.1007/s004380050818 [DOI] [PubMed] [Google Scholar]
48.Takahashi H, Hirose K, Watanabe H. Necessity of meningococcal gamma-glutamyl aminopeptidase for the Neisseria meningitidis growth in rat cerebrospinal fluid (CSF) and CSF mimicking medium. J Bacteriol. 2004;186(1):244–7. 10.1128/jb.186.1.244-247.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode. Chem Biol. 2008;15(11):1187–97. 10.1016/j.chembiol.2008.10.004 [DOI] [PubMed] [Google Scholar]
50.Lacey VK, Kouie GV, Noel JP, Wang L. Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase. Chembiochem. 2013;14(16):2100–5. 10.1002/cbic.201300400 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Stark MJ. Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene. 1987;51(2–3):255–67. 10.1016/0378-1119(87)90314-3 [DOI] [PubMed] [Google Scholar]
52.Schmidt TG, Skerra A. The Strep-tag system for one-step purification and high-affinity detection or. Capturing of proteins. Nat Protoc. 2007;2(6):1528–35. 10.1038/nprot.2007.209 [DOI] [PubMed] [Google Scholar]
53.Yanagisawa T, Takahashi M, Mukai T, Sato S, Wakamori M, Shirouzu M, et al. Multiple site-specific installations of Nepsilon-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis. Chembiochem. 2014;15(12):1830–8. 10.1002/cbic.201402291 [DOI] [PubMed] [Google Scholar]
54.Masuda A, Dohmae N. Examination of an absolute quantity of less than a hundred nanograms of proteins by amino acid analysis. Anal Bioanal Chem. 2013;405(25):8073–81. 10.1007/s00216-013-7056-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Takahashi H, Carlson RW, Muszynski A, Choudhury B, Kim KS, Stephens DS, et al. Modification of lipooligosaccharide with phosphoethanolamine by LptA in Neisseria meningitidis enhances meningococcal adhesion to human endothelial and epithelial cells. Infect Immun. 2008;76(12):5777–89. 10.1128/IAI.00676-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Ramsey ME, Hackett KT, Kotha C, Dillard JP. New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol. 2012;78(9):3068–78. 10.1128/AEM.07871-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Boyle-Vavra S, Seifert HS. Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae. Gene. 1995;155(1):101–6. 10.1016/0378-1119(94)00890-5 [DOI] [PubMed] [Google Scholar]
58.Yamaguchi A, Matsuda T, Ohtake K, Yanagisawa T, Yokoyama S, Fujiwara Y, et al. Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates. Bioconjug Chem. 2016;27(1):198–206. 10.1021/acs.bioconjchem.5b00602 [DOI] [PubMed] [Google Scholar]
59.Young TS, Ahmad I, Yin JA, Schultz PG. An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol. 2010;395(2):361–74. 10.1016/j.jmb.2009.10.030 [DOI] [PubMed] [Google Scholar]
60.Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc. 2002;124(31):9026–7. 10.1021/ja027007w [DOI] [PubMed] [Google Scholar]
61.Fabrini R, De Luca A, Stella L, Mei G, Orioni B, Ciccone S, et al. Monomer-dimer equilibrium in glutathione transferases: a critical re-examination. Biochemistry. 2009;48(43):10473–82. 10.1021/bi901238t [DOI] [PubMed] [Google Scholar]
62.Yanagisawa T, Kuratani M, Seki E, Hino N, Sakamoto K, Yokoyama S. Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase. Cell Chem Biol. 2019;26(7):936–49. 10.1016/j.chembiol.2019.03.008 [DOI] [PubMed] [Google Scholar]
63.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845–58. 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Zhang Z, Tan M, Xie Z, Dai LF, Y. C, Zhao Y. Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol. 2011;7(1):58–63. 10.1038/nchembio.495 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS One. 2012;7(7):e40410 10.1371/journal.pone.0040410 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Korndorfer IP, Skerra A. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site. Protein Sci. 2002;11(4):883–93. 10.1110/ps.4150102 [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Carbonnelle E, Helaine S, Nassif X, Pelicic V. A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol Microbiol. 2006;61(6):1510–22. 10.1111/j.1365-2958.2006.05341.x [DOI] [PubMed] [Google Scholar]
68.Kolappan S, Coureuil M, Yu X, Nassif X, Egelman EH, Craig L. Structure of the Neisseria meningitidis Type IV pilus. Nat Commun. 2016;7:13015 10.1038/ncomms13015 [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Carbonnelle E, Hill DJ, Morand P, Griffiths NJ, Bourdoulous S, Murillo I, et al. Meningococcal interactions with the host. Vaccine. 2009;27 Suppl 2:B78–89. 10.1016/j.vaccine.2009.04.069 [DOI] [PubMed] [Google Scholar]
70.Brown DR, Helaine S, Carbonnelle E, Pelicic V. Systematic functional analysis reveals that a set of seven genes is involved in Fine-Tuning of the Multiple Functions Mediated by Type IV Pili in Neisseria meningitidis. Infect Immun. 2010;78(7):3053–63. 10.1128/IAI.00099-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Imhaus AF, Dumenil G. The number of Neisseria meningitidis type IV pili determines host cell. EMBO J. 2014;33(16):1767–83. 10.15252/embj.201488031 [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Carbonnelle E, Hélaine S, Prouvensier L, Nassif X, Pelicic V. Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step. Mol Microbiol. 2005;55(1):54–64. 10.1111/j.1365-2958.2004.04364.x [DOI] [PubMed] [Google Scholar]
73.Pelicic V. Type IV pili: e pluribus unum? Mol Microbiol. 2008;68(4):827–37. 10.1111/j.1365-2958.2008.06197.x [DOI] [PubMed] [Google Scholar]
74.Hospenthal MK, Costa TRD, Waksman G. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol. 2017;15(6):365–79. 10.1038/nrmicro.2017.40 [DOI] [PubMed] [Google Scholar]
75.Wormann ME, Horien CL, Bennett JS, Jolley KA, Maiden MC, Tang CM, et al. Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences. BMC Genomics. 2014;15:253 10.1186/1471-2164-15-253 [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Helaine S, Carbonnelle E, Prouvensier L, Beretti JL, Nassif X, Pelicic V. PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol Microbiol. 2005;55(1):65–77. 10.1111/j.1365-2958.2004.04372.x [DOI] [PubMed] [Google Scholar]
77.Brissac T, Mikaty G, Dumenil G, Coureuil M, Nassif X. The meningococcal minor pilin PilX is responsible for type IV pilus conformational changes associated with signaling to endothelial cells. Infect Immun. 2012;80(9):3297–306. 10.1128/IAI.00369-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Mikaty G, Soyer M, Mairey E, Henry N, Dyer D, Forest KT, et al. Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress. PLoS Pathog. 2009;5(2):e1000314 10.1371/journal.ppat.1000314 [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Wolfgang M, van Putten JP, Hayes SF, Koomey M. The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol Microbiol. 1999;31(5):1345–57. 10.1046/j.1365-2958.1999.01269.x [DOI] [PubMed] [Google Scholar]
80.Hill DJ, Griffiths NJ, Borodina E, Virji M. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clin Sci (Lond). 2010;118(9):547–64. 10.1042/CS20090513 [DOI] [PMC free article] [PubMed] [Google Scholar]
81.Georgiadou M, Castagnini M, Karimova G, Ladant D, Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly. Mol Microbiol. 2012;84(5):857–73. 10.1111/j.1365-2958.2012.08062.x [DOI] [PubMed] [Google Scholar]
82.Lin S, Zhang Z, Xu H, Li L, Chen S, Li J, et al. Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens. J Am Chem Soc. 2011;133(50):20581–7. 10.1021/ja209008w [DOI] [PubMed] [Google Scholar]
83.Gan Q, Lehman BP, Bobik TA, Fan C. Expanding the genetic code of Salmonella with non-canonical amino acids. Sci Rep. 2016;6:39920 10.1038/srep39920 [DOI] [PMC free article] [PubMed] [Google Scholar]
84.Wang F, R S., Guo J, Shen W, Shen W, Schultz PG. Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis. PLoS One. 2010;5(2):e9354 10.1371/journal.pone.0009354 [DOI] [PMC free article] [PubMed] [Google Scholar]
85.Luo X, Zambaldo C, Liu T, Zhang Y, Xuan W, Wang C, et al. Recombinant thiopeptides containing noncanonical amino acids. Proc Natl Acad Sci U S A. 2016;113(13):3615–20. 10.1073/pnas.1602733113 [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Zhang M, Lin S, Song X, Liu J, Fu Y, Ge X, et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat Chem Biol. 2011;7(10):671–7. 10.1038/nchembio.644 [DOI] [PubMed] [Google Scholar]
87.O' Dwyer CA, Langford PR, Kroll JS. A novel neisserial shuttle plasmid: a useful new tool for meningococcal research. FEMS Microbiol Lett. 2005;251(1):143–7. 10.1016/j.femsle.2005.07.036 [DOI] [PubMed] [Google Scholar]
88.Spence JM, Wright L, Clark VL. Laboratory maintenance of Neisseria gonorrhoeae. Curr Protoc Microbiol. 2008;Chapter 4:Unit 4A.1 10.1002/9780471729259.mc04a01s8 [DOI] [PubMed] [Google Scholar]
89.Guibourdenche M, Popoff MY, Riou JY. Deoxyribonucleic acid relatedness among Neisseria gonorrhoeae, N. meningitidis. Ann Inst Pasteur Microbiol. 1986;137B(2):177–85. 10.1016/s0769-2609(86)80106-5 [DOI] [PubMed] [Google Scholar]
90.Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane. Bioinformatics. 1998;14(4):378–9. 10.1093/bioinformatics/14.4.378 [DOI] [PubMed] [Google Scholar]
91.Craig L, Volkmann N, Arvai AS, Pique ME, Yeager M, Egelman EH, et al. Type IV pilus structure by cryo-electron microscopy and crystallography. Mol Cell. 2006;23(5):651–62. 10.1016/j.molcel.2006.07.004 [DOI] [PubMed] [Google Scholar]
92.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35(6):1547–9. 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
93.Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–25. 10.1093/oxfordjournals.molbev.a040454 [DOI] [PubMed] [Google Scholar]
94.Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining. Proc Natl Acad Sci U S A. 2004;101(30):11030–5. 10.1073/pnas.0404206101 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0237883.r001

Decision Letter 0

Nediljko Budisa

18 May 2020

PONE-D-20-12310

Successful genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: The new method shed light into the physiological function of a function-unknown NMB1345 protein in Neisseria meningitidis.

PLOS ONE

Dear Dr. Takahashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

After studying your manuscript, this academic editor found it confusing, poorly presented and not properly structured with lot of typos, style and grammatical errors. The both reviewers (independently) shared my view that the manuscript is in part difficult to comprehend and whole subject still requires to be substantially worked out (matured). Although one reviewer can even envisage a publication of the manuscript with the "minor revision"– the second reviewer opted for the "major revision" and expects a significant improvement in both data presentation and rewriting of the manuscript. After balancing their comments and the literature-check, it is difficult to escape the fact that the work still needs substantial further improvements. Thus, we feel that it has merit, but is not suitable for publication as it currently stands. Therefore, my decision is "Major Revision." We hope that criticism of the experts will help to improve the quality of your revised manuscript.

We would appreciate receiving your revised manuscript by Jul 02 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Ned Budisa

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Additional Editor Comments:

I asked highly experted reviewers and observers in the field for a fair, unbiased and balanced review on your paper. Now their comments are available to you. While the referee 2 justifiably voiced general concerns about the (a) scientific style (manuscript structure and focus), (b) practical relevance and (c) good scientific presentation in general, the referee 1 had many highly relevant technical issues which urgently needed to be addressed. All these concerns must be addressed correctly (point-to-point) in the revised manuscript (only when these conditions are fully met, I can handle the manuscript again as an academic editor). Obviously, the work still needs substantial improvements. I feel that it has merit, but is not suitable for publication as it currently stands. Therefore, my decision is again "Major Revision" and I invite you to submit a revised version of the manuscript that addresses the points raised as elaborated above.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has applied the genetic code expansion tools to reveal interaction of a hypothetical protein, NMB1345, to the PilE, a major component of meningococcal pili, in Neisseria meningiditis. Introduction of a new methodology to identify possible function of unknown factors in pathogenic bacteria is an interesting subject for further application of the orthogonal translation systems and create a new area of research. The manuscript has been prepared and written in scientific manner and just some minor issues has to be remained which mentioned below:

1- The quality of figures are very low and should be improved.

2- The number of figures is too high. Some of them could be moved in “Supporting information” part. For example, Fig. 4 and Fig. 7. Moreover, figures 5 and 6 should be combined.

3- There is some mistyping in the text. For example: line 86, “intarected!!”; line 110, “intection!!”; line 149 in Table 1, readjusting needed; line 635, double typing or moving after ref.; line 737, “seemd!!”

4- Line 122: “L plates”? Clarify.

5- Line 803: “proteins”; only one protein highlighted in red, but in the text two proteins mentioned. Delete “s” or highlight the second one (NMEN93004-1215).

6- In Fig. 3B: Western blotting would not be able to confirm proper incorporation of ncAA. The MS analysis should be done. Please clarify this issue in corresponding part.

7- The last but not least, please explain how chemically a protein like MBP-�N-PamA K278(pBPa) crosslinked to the endogenous proteins like Gln6-�N-PilE? Self-crosslinking speculation is possible but crosslinking with the accompanying proteins in vivo needs to be clarified in the text. A schematic illustration would be helpful.

Reviewer #2: Abstract:

“…Advances have been achieved in many technologies, and one of the most developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/ tRNA Pyl pair from Methanosarcina species;…”

What does most developed methods mean? This could just mean something with a reference system, otherwise it contains no information.

“… In the present study, we developed the new method to genetically incorporate ncAAs into N. meningitides…”

How can one introduce ncAAs into an organism? Maybe into a protein sequence?

“…In the present study, we developed the new method to genetically incorporate ncAAs into N. meningitidis and elucidate the biological function(s) of the NMB1345 protein in N. meningitidis by using ncAA-encoded photocrosslinking probes…”

Why extend the sentence unnecessarily? Say that your ncAAs are photcrosslinker. For example: In the present study, we developed the new method to genetically incorporate ncAA photocrosslinking probes into N. meningitidis and elucidate the biological function(s) of the NMB1345 protein in N. meningitidis

Introduction:

Line 42-45: “However, N. meningitidis exhibits the ability to cross the epithelial layer of the upper

43 respiratory tract, infiltrate the bloodstream, evade the defenses of the human immune

44 system, adhere to the endothelial layers of peripheral and brain vessels, cross the brain45

blood barrier, and replicate in cerebrospinal fluid.”

Citation is missing.

Line 84-86: “We previously attempted using already existing methods, such as the two-hybrid system or a pull-down assay, to identify the proteins intarected with NMB1345 protein, but were unsuccessful.”

Interacted is written wrong, also what does proteins interacted with NMB 1345 mean? Maybe protein-protein interaction?

Line 93: E. coli and C. elegans need to be written in full name. No full name before. Also C. elegans is written wrong.

Line 88-94: Needs rephrasing. The sentence is way to long. In addition, genetic code expansion with the Pyl system is anything but recently developed.

Line 97 99: No citation for the last claim in the sentence

Line 103-104: “... most useful …” this statement makes just sense if you say in what regard it is most useful. Nothing can be useful without stating the purpose.

Line 110: intection?

Line 111-112: If you state what PamA means, then do it the first time you use it, that would be in line 110.

Line 122: What are L plates or L broth? Please define or state the company.

Line 551: This sentence structure makes no sense. Your use of “and” implies that you genetically characterized the gene product. What does this mean? You did fluorescence.

Line 554: This is an interpretation of the data, normally in the text. In the figure caption one should describe what method was used and what was measured.

Line 563: using beneath makes no sense. Maybe you meant amongst or between strains?

Line 573-577: Sentence structure makes it almost incomprehensible.

Line 610-611: Your description is the opposite what the figure is showing, which is it?

Line 614: You say you used 123 amino acids but in figure 2B it is written that you used 113. Which is it? Figure 2B and the caption for that is confusing. Please clear up which parts you used and describe it with the correct length. Adapt the main text accordingly.

Line 616-618: This part is written extremely confusing. Where does strain 2215 and 2217 come from? Was not mentioned before or clearly described. Also Figure 2C does not annotate any of theses strains. Please adapt this.

Line 619-620: Please elaborate your conclusion.

Line 620-625: These claims are not evaluable since the Figure D and E are not consistent described. Please correct your Figure as mention above.

Line 630-632: “…while no one examined whether the orthogonality of the tRNAPyl-PylRS pair is specific or compatible with the meningococcal translational system.”

This should be put into the introduction, not results.

Line 642: The general description for Fig. 3does not match what is shown. You don’t show just the successful introduction of ncAAs into proteins.

Figure 3E: The gray arrow an text is not easy visible

Line 680: I guess you mean steric and not stereological. But since you have no further information why the suppression does not work I would omit this speculation. It could be also context effects, mRNA secondary structure effects, etc.

Line 684-685: “Moreover, when N. meningitidis was irradiated with UV light, a more dimer form of GST was detected with longer irradiation,…” Please rewrite, makes no sense

Line 705-707: What does this mean? Why mention HdeA from E.coli? Please elaborate the analogy or omit the sentence.

Line 709: “There were 40 Lys residues in the PamA protein...” wrong tense. Doesn’t PamA still has 40 Lys?

Line 720: You meant whole cell irradiation? Intact strains sounds weird.

Line 737: seemed not seemd

742-745: This sentence makes no sense, rewrite.

Line 747: The general description is not correct. Also boldening is not correct. Also no time of irradiation is mentioned.

Line 750: Portion?

Line 751: What was suppressed? How can an amino acid suppress something? Used several times. Please be more precise in the use of your language.

Line 759: If the contain other proteins then these are protein complexes.

Line 765-771: Super long sentence. Rephrase for clarity.

Line 789:-791 Why is there two times referred to A and one time to B. Just one time A.

Line 805-808: Phrasing makes no sense. Rephrase.

General remarks:

Check all abbreviations of consistency. Sometime you use IM for inner membrane; sometimes you use it for inner membrane-enriched fraction.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hamid Reza Karbalaei-Heidari

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 31;15(8):e0237883. doi: 10.1371/journal.pone.0237883.r002

Author response to Decision Letter 0

26 Jun 2020

Jun 26, 2020

Dr. Nediijko Budisa,

PLOS ONE

Academic editor

Dear Dr. Budisa,

Thank you very much for your favorable review of our manuscript.

We have revised our manuscript according to the comments of the academic editor and two reviewers. In addition, we carefully rewrote the entire manuscript to improve the English and asked a native speaker to correct the English.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We revised the first page of manuscript to present on title, authors, affiliations, and corresponding author according to the PLOS ONE style.

We prepared the original image files according to these instructions. The original blots and gel images-files are as a PDF file in Supporting information, named as “Original blots and gel images”.

We removed all of the descriptions mentioned as “data not shown”.

Review Comments to the Author

1- The quality of figures are very low and should be improved.

We revised all of the figures according to the PLOS ONE style, and hope the present quality of the figures is acceptable.

2- The number of figures is too high. Some of them could be moved in “Supporting information” part. For example, Fig. 4 and Fig. 7. Moreover, figures 5 and 6 should be combined.

Since we consider Fig 4B to be essential to comprehend the content of this study, we retained Fig 4B as Fig 4 and moved the previous Fig 4A to the supporting information as the new S3 Fig.

According to reviewer 1’s suggestion, the previous Figures 5 and 6 were combined into one figure as the new Fig 5, and Fig 7 was moved to the supporting information as the new S7-9 Figs.

The typographic errors in lines 86,110 and 737 were corrected.

Table 1 was arranged as follows: since Fig 4A was moved to the supporting information as S3 Fig, the description of the meningococcal plasmids in the supporting information (new S3 Fig) was moved to the new S4 Table.

Double typing at line 635 was corrected.

4- Line 122: “L plates”? Clarify.

We corrected the description “L broth” and “L plate” to Luria-Bertani (LB) broth and LB plate (LB liquid medium containing 1.5% agarose).

5- Line 803: “proteins”; only one protein highlighted in red, but in the text two proteins mentioned. Delete “s” or highlight the second one (NMEN93004-1215).

We deleted “s”.

6- In Fig. 3B: Western blotting would not be able to confirm proper incorporation of ncAA. The MS analysis should be done. Please clarify this issue in corresponding part.

As reviewer 1 commented, from the physicochemical viewpoint, as MS analysis could confirm the proper incorporation of ncAA in OpaD K95amb, GST E51amb and GST F52amb.

On the other hand, purification of the OpaD and GST proteins from N. meningitidis strains was truly impossible since these proteins lacked tags and were expressed at comparable levels to endogenously produced proteins that could only be detected by Western blotting, as in higher organisms such as mammalian cells, Caenorhabditis elegans (ACS Chem Biol 20:1292-1302, 2012), neural cells (Stem cells 29:1231-1240, 2011) and zebrafish (Cell Research 27:294-297, 2017).

Considering of the technical difficulties, the purification of OpaD K95(mAzZLys) and OpaD K95(pBPa) from the N. meningitidis strain was impossible, and the results using OpaD K95amb shown in the former Fig 3BC were deleted in the revised manuscript.

As for GST E51(mAzZLys), GST E51(pBPa) and GST F52(pBPa), we constructed the new plasmids harboring gst genes fused to Strep2His6 tag for N. meningitidis. However, some materials required to introduce the plasmids into N. meningitidis were out of stock and we would have to wait for a month for their delivery. Moreover, even if we were able to obtain the N. meningitidis transformants, the purification of GST proteins followed by MS analysis will take approximately two months, since this is not an overproduction system. From these reasons, we could not purify GST E51(mAzZLys), GST E51(pBPa) and GST F52(pBPa) from N. meningitidis during this short period and could not prove the incorporation by MS analysis in the revised manuscript.

However, we purified the PamA K278(pBPa) protein from an N. meningitidis strain grown on approximately 200 plates (Fig 5A lane 6). We subjected the PamA K278(pBPa) protein to MS analysis and confirmed the selective incorporation of pBPa in N. meningitidis in the revised manuscript (S5 Fig).

We corrected the words “prove” or “confirm” to show that the proper incorporation of ncAA in GST E51amb and GST P52amb also occurred in N. meningitidis.

We hope our revised results sufficiently answer to the reviewer 1’s comments.

In our experiments, both the MBP-�N-PamA K278(pBPa) and Gln6-�N-PilE proteins were overproduced and crosslinked in E. coli. Our poor explanation probably led to reviewer 1’s confusion.

We revised the description to clearly show that the crosslinking between MBP-�N-PamA K278(pBPa) and Gln6-�N-PilE proteins was performed in E. coli. Moreover, a schematic illustration of these crosslinked proteins was added in S6D Fig.

We hope our revisions are suitable to answer for reviewer 1’s comments.

Reviewer #2:

Abstract:

What does most developed methods mean? This could just mean something with a reference system, otherwise it contains no information.

We revised the sentence by adding the following phrase “Among many biological technologies to examine transient protein-protein interactions in vivo” (Lines 24-25).

“… In the present study, we developed the new method to genetically incorporate ncAAs into N. meningitides…”

How can one introduce ncAAs into an organism? Maybe into a protein sequence?

We revised the sentence by adding the following phrase: ”encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair” (Lines 29-31).

We revised the sentence according to reviewer’s 2 suggestion as ”we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidate the biological function(s) of the NMB1345 protein” (Lines 29-31).

Introduction:

Line 42-45: “However, N. meningitidis exhibits the ability to cross the epithelial layer of the upper

43 respiratory tract, infiltrate the bloodstream, evade the defenses of the human immune

44 system, adhere to the endothelial layers of peripheral and brain vessels, cross the brain45

blood barrier, and replicate in cerebrospinal fluid.”

Citation is missing.

This citation was added in the revised manuscript (Line 44).

Interacted is written wrong, also what does proteins interacted with NMB 1345 mean? Maybe protein-protein interaction?

We revised the following phrase: “We previously attempted to examine protein-protein interaction using existing methods, such as the two-hybrid system and a pull-down assay, to find some cues on the function the NMB1345 protein” (Lines 82-85).

Line 93: E. coli and C. elegans need to be written in full name. No full name before. Also C. elegans is written wrong.

E. coli and C. elegans were rewritten as Escherichia coli and Caenorhabditis elegans, respectively (Lines 92-93).

Line 88-94: Needs rephrasing. The sentence is way to long. In addition, genetic code expansion with the Pyl system is anything but recently developed.

The sentence was divided into two parts. In addition, we removed the phrase “most recently” in the revised manuscript (Lines 87-94).

Line 97 99: No citation for the last claim in the sentence

The citation was added in the revised manuscript (Line 98).

Line 103-104: “... most useful …” this statement makes just sense if you say in what regard it is most useful. Nothing can be useful without stating the purpose.

We changed the phrase “most useful” to “most widely used” in the revised manuscript (Line 105).

Line 110: intection?

We corrected the mistyping.

Line 111-112: If you state what PamA means, then do it the first time you use it, that would be in line 110.

We mentioned the designation of NMB1345 as PamA in lines 109-110 in the revised manuscript.

Line 122: What are L plates or L broth? Please define or state the company.

Since both reviewers pointed out the same problems, we corrected the description “L broth” and “L plate” as Luria-Bertani (LB) broth and LB plate (LB liquid medium containing 1.5% agarose) (Lines 122-123).

Line 551: This sentence structure makes no sense. Your use of “and” implies that you genetically characterized the gene product. What does this mean? You did fluorescence.

We changed the title of Fig 1 to “Characterization of �pamA N. meningitidis” (Line 540).

Line 554: This is an interpretation of the data, normally in the text. In the figure caption one should describe what method was used and what was measured.

We changed the title of Fig 1B to “Effect of �pamA mutation on N. meningitidis infection of HBMEC” (Line 543).

Line 563: using beneath makes no sense. Maybe you meant amongst or between strains?

It is well known that bacterial infection elicits alternations of the signaling pathways concomitant with cytoskeletal reorganization in the host cells, and that ezrin is one of the key molecules controlling both cell shape and signaling. N. meningitidis reportedly induce ERM accumulation beneath the bacterial colony, and this accumulation is required for bacterial internalization into host cells (Takahashi et al, . IAI 2011, 2012 and 2015). This explanation was described in lines 560 to 562. In addition, since ezrin is the host cell’s factor, we considered that “beneath” would be an appropriate word to show the part about the condensation of ezrin under the meningococcal colonies, and this word was often used in our previous manuscripts (Takahashi et al, IAI 2011, 2012 and 2015).

We hope the term “beneath” is acceptable for reviewer 2.

Line 573-577: Sentence structure makes it almost incomprehensible.

We revised the sentence for clarification (Lines 562-564).

Line 610-611: Your description is the opposite what the figure is showing, which is it?

As reviewer 2 pointed out, the description was opposite and corrected in the revised manuscript.

We mistyped 23 as “123” in the previous version. PamA consists of the first 3 amino acids (aa) residues, the putative membrane-spanning region (20aa) and the hydrophilic region (493aa) as shown in Fig 2B. On the other hand, in the experiments with lacZ or phoA fusion, the first 136 aa were fused to the lacZ or phoA gene to genetically examine the orientation of PamA in N. meningitidis. We corrected the number.

The explanations of HT2215 and HT2217 are described in the text. To prevent confusion, the two strains are referred to in Table 2 and the strain names are also shown in Fig 2B.

Line 619-620: Please elaborate your conclusion.

Genetical methods are widely used to determine the orientation of a protein relative to the inner membrane in bacteria by expressing LacZ and PhoA fusion proteins, since the alkaline phosphatase activity of PhoA is active only in the periplasmic space (Boyle-Vavra et al, 1995). The explanation is added in Lines 608-609.

Line 620-625: These claims are not evaluable since the Figure D and E are not consistent described. Please correct your Figure as mention above.

As answered above, the descriptions of Fig 2 D and E were opposite in the text and corrected in the revised manuscript.

Line 630-632: “…while no one examined whether the orthogonality of the tRNAPyl-PylRS pair is specific or compatible with the meningococcal translational system.”

This should be put into the introduction, not results.

The sentence “while no one examined whether the orthogonality of the tRNAPyl-PylRS pair is specific or compatible with the meningococcal translational system” was moved into the Introduction (Lines 98-100) in the revised manuscript.

Line 642: The general description for Fig. 3does not match what is shown. You don’t show just the successful introduction of ncAAs into proteins.

Figure 3E: The gray arrow an text is not easy visible

As reviewer 2 pointed out, the title of Fig 3 was not suitable for the content. We corrected the tile as “Incorporation of ncAAs into N. meningitidis proteins monitored by Western blotting” (Line 630).

As reviewer 2 pointed out, there are some reasons for the failure of mAzZLys incorporation at GST F52amb. However, we do not know the reason for the failure. To eliminate unnecessary confusion, the description of the steric hinderance was removed in the revised manuscript (Line 646).

Line 684-685: “Moreover, when N. meningitidis was irradiated with UV light, a more dimer form of GST was detected with longer irradiation,…” Please rewrite, makes no sense

We rewrote the sentence as “longer UV irradiation increased the amount of the dimer form of GST” (Line 658).

Line 705-707: What does this mean? Why mention HdeA from E.coli? Please elaborate the analogy or omit the sentence.

As reviewer 2 suggested the description of the E. coli HdeA protein was deleted in the revised manuscript.

Line 709: “There were 40 Lys residues in the PamA protein...” wrong tense. Doesn’t PamA still has 40 Lys?

As shown in S1 Fig, there are 40 Lys residues (shown in red) in the deduced amino acid sequence of the PamA protein.

Line 720: You meant whole cell irradiation? Intact strains sounds weird.

We rewrote the phrase “intact” as “whole bacterial cells” (Line 690).

Line 737: seemed not seemd

We corrected the mistyping.

742-745: This sentence makes no sense, rewrite.

We rewrote the sentence as “In the present study, we focused only on the proteins crosslinked to PamA K278(pBPa) because the three bands with the highest intensities were found in the PamA K278(pBPa) sample”.

Line 747: The general description is not correct. Also boldening is not correct. Also no time of irradiation is mentioned.

Since we revised Fig 4 according to the reviewer 1’s suggestion, the title for the new Fig 4 was rewritten in the revised manuscript.

This is the title of Fig 4, which is now shown in bold according to the guidelines.

PamA K208(pBPa), PamA K278(pBPa), PamA K314(pBPa), PamA K382(pBPa) and PamA K395(pBPa) proteins crosslinked to unidentified endogenous proteins in N. meningitidis. (Line 715-717)

While the irradiation time is described in the Materials and Methods, the irradiation time is also described in the figure citation (Line 720).

Line 750: Portion?

We rewrote the word as “aliquot” in the revised manuscript (Line 721)

Line 751: What was suppressed? How can an amino acid suppress something? Used several times. Please be more precise in the use of your language.

We meant to use the phrase “suppressed” in the genetic meaning. However, to exclude confusion, all phrases with “suppressed” in the old manuscript were rewritten as “expressed by pyrrolysine-based amber suppression with pBPa” in the revised manuscript.

Line 759: If the contain other proteins then these are protein complexes.

According to reviewer’s 2 suggestion, we rewrote the phrase “proteins” as “protein complexes” (Line 726).

Line 765-771: Super long sentence. Rephrase for clarity.

Since the explanation of Twin-strep-tag was cited in the text (Korndorfer and Skerra, 2002), we shortened the sentence by deleting the explanation (Lines 727-728).

Line 789:-791 Why is there two times referred to A and one time to B. Just one time A.

According to reviewer 1’s suggestion, we combined the previous Figs 4 and 5 into the new Fig 5. The revision changed the figure citations of the new Fig 5.

We corrected the reference to (A) in the figure citation of the new Fig 5.

Line 805-808: Phrasing makes no sense. Rephrase.

We rewrote the long sentences as follows: We then characterized the 85-kDa band, which could contain approximately 20 kDa endogenous protein crosslinked to PamA K278(pBPa). LC-MS/MS followed by MASCOT analyses revealed that two possible candidates, a fimbrial protein (17 kDa) and the hypothetical protein NMEN93004_1215 (18 kDa) (Table 3)(Lines 786-789).

General remarks:

Check all abbreviations of consistency. Sometime you use IM for inner membrane; sometimes you use it for inner membrane-enriched fraction.

We checked all of the abbreviations used in this study. As for the “IM”, we intended IM to mean Inner membrane, but not the Inner-membrane-rich fraction, and corrected it for clarity.

All figures were converted by PACE.

We appreciate your kind consideration of this revised manuscript and are looking forward to hearing a favorable response from you.

Sincerely yours,

Hideyuki Takahashi, Ph.D.

Department of Bacteriology I

National Institute of Infectious Diseases

Toyama 1-23-1, Shinjuku

Tokyo 162-8640, Japan

PLoS One. doi: 10.1371/journal.pone.0237883.r003

Decision Letter 1

Nediljko Budisa

5 Aug 2020

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein.

PONE-D-20-12310R1

Dear Dr. Takahashi,

The paper is critically evaluated by two highly experted reviewers in the field that are capable of a fair, unbiased and balanced review. As you can see from their comments, both reviewers agreed that the data in the manuscript are now better presented and manuscript is acceptable for publication with minor improvements.We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nediljko Budisa

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hamid Reza Karbalaei-Heidari

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0237883.r004

Acceptance letter

Nediljko Budisa

7 Aug 2020

PONE-D-20-12310R1

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein.

Dear Dr. Takahashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. Nediljko Budisa

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Nucleotide sequence and deduced amino acid sequence of the pamA gene in N. meningitidis HT1125 strain.

(PDF)

Click here for additional data file.^{(427.8KB, pdf)}

S2 Fig. Photocrosslinking of PamA K148(mAzZLys), K273(mAzZLys), and K388(mAzZLys) in N. meningitidis.

(PDF)

Click here for additional data file.^{(500.1KB, pdf)}

S3 Fig. PamA K278(pBPa) protein crosslinks with endogenous proteins in N. meningitidis.

(PDF)

Click here for additional data file.^{(1.3MB, pdf)}

S4 Fig. Schematic structure of the C-terminal region of PamA K278-Strep₂-His₆.

(PDF)

Click here for additional data file.^{(32.5KB, pdf)}

S5 Fig. Mass spectrometry analysis of PamA K278amb expressed by the pyrrolysine-based amber suppression with pBPa in N. meningitidis.

(PDF)

Click here for additional data file.^{(83.1KB, pdf)}

S6 Fig. Examination of purified MBP-ΔN-PamA K278(pBPa) and Gln₆-ΔN-PilE recombinant proteins from E. coli strains for LC-MS/MS analysis.

(PDF)

Click here for additional data file.^{(661.3KB, pdf)}

S7 Fig. Analysis of amino acid residues (region) in PilE crosslinked to PamA K278 (pBPa) by LC-MS/MS.

(PDF)

Click here for additional data file.^{(68.8KB, pdf)}

S8 Fig. MS/MS spectrum of the peptide IEVGK(pBPa)LAFSTK from MBP-ΔN-PamA K278(pBPa).

The sequence was read from the annotated b and y ion series; b3, y4, y5, y6, y7, y8, y9, and y10 ions were observed.

(PDF)

Click here for additional data file.^{(28.3KB, pdf)}

(PDF)

Click here for additional data file.^{(48.8KB, pdf)}

S10 Fig. Sequence alignment between neisserial PilE and crystal structure of full-length gonococcal PilE.

(PDF)

Click here for additional data file.^{(159.5KB, pdf)}

S11 Fig. Characterization of pili.

(PDF)

Click here for additional data file.^{(4.5MB, pdf)}

S12 Fig. Detection of homomeric crosslinking between two MBP-ΔN-PamA K278(pBPa).

(PDF)

Click here for additional data file.^{(282.5KB, pdf)}

S13 Fig. Phylogenetic tree showing the relation of pamA genes among neisserial species: N. meningitidis, N. gonorrhoeae and N. lactamica.

(PDF)

Click here for additional data file.^{(20.2KB, pdf)}

S1 Table. Oligonucleotides used in this study.

(DOCX)

Click here for additional data file.^{(69.8KB, docx)}

S2 Table. Plasmids to construct N. meningitidis mutants.

(DOCX)

Click here for additional data file.^{(22KB, docx)}

S3 Table. Plasmids for production of protein recombinants expressed in E. coli.

(DOCX)

Click here for additional data file.^{(16.2KB, docx)}

S4 Table. IncQ plasmids for N. meningitidis strains which results were shown in supporting information.

(DOCX)

Click here for additional data file.^{(16.4KB, docx)}

S5 Table. Analysis of Pili components by Tandem Mass Tag labeling.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S1 File

(PDF)

Click here for additional data file.^{(18.6MB, pdf)}

Data Availability Statement

[pone.0237883.ref001] 1.Takahashi H, Haga M, Sunagawa T, Saitoh T, Kitahara T, Matsumoto S, et al. Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens. J Infect Chemother. 2016;22(7):501–4. 10.1016/j.jiac.2015.12.016 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref002] 2.Chang Q, Tzeng YL, Stephens DS. Meningococcal disease: changes in epidemiology and prevention. Clinical epidemiology. 2012;4:237–45. 10.2147/CLEP.S28410 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref003] 3.Coureuil M, Jamet A, Bille E, Lecuyer H, Bourdoulous S, Nassif X. Molecular interactions between Neisseria meningitidis and its human host. Cell Microbiol. 2019;21(11):e13063 10.1111/cmi.13063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref004] 4.Brouwer MC, Read RC, van de Beek D. Host genetics and outcome in meningococcal disease: a systematic review and meta-analysis. The Lancet Infectious diseases. 2010;10(4):262–74. 10.1016/S1473-3099(10)70045-1 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref005] 5.Codjoe SN, Nabie VA. Climate change and cerebrospinal meningitis in the Ghanaian meningitis belt. International Journal of Environmental Research and Public Health. 2014;11(7):6923–39. 10.3390/ijerph110706923 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref006] 6.Peak IR, Srikhanta Y, Dieckelmann M, Moxon ER, Jennings MP. Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis. FEMS Immunol Med Microbiol. 2000;28(4):329–34. 10.1111/j.1574-695X.2000.tb01494.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref007] 7.Virji M. Pathogenic neisseriae: surface modulation, pathogenesis and infection control. Nat Rev Microbiol. 2009;7(4):274–86. 10.1038/nrmicro2097 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref008] 8.Coureuil M, Lecuyer H, Bourdoulous S, Nassif X. A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers. Nat Rev Microbiol. 2017;15(3):149–59. 10.1038/nrmicro.2016.178 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref009] 9.Schubert-Unkmeir A. Molecular mechanisms involved in the interaction of Neisseria meningitidis with cells of the human blood-cerebrospinal fluid barrier. Pathog Dis. 2017;75(2). 10.1093/femspd/ftx023 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref010] 10.Capecchi B, Adu-Bobie J, Di Marcello F, Ciucchi L, Masignani V, Taddei A, et al. Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells. Mol Microbiol. 2005;55(3):687–98. 10.1111/j.1365-2958.2004.04423.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref011] 11.Serruto D, Adu-Bobie J, Scarselli M, Veggi D, Pizza M, Rappuoli R, et al. Neisseria meningitidis App, a new adhesin with autocatalytic serine protease activity. Mol Microbiol. 2003;48(2):323–34. 10.1046/j.1365-2958.2003.03420.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref012] 12.van Ulsen P, van Alphen L, ten Hove J, Fransen F, van der Ley P, Tommassen J. A Neisserial autotransporter NalP modulating the processing of other autotransporters. Mol Microbiol. 2003;50(3):1017–30. 10.1046/j.1365-2958.2003.03773.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref013] 13.Turner DP, Marietou AG, Johnston L, Ho KK, Rogers AJ, Wooldridge KG, et al. Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis. Infect Immun. 2006;74(5):2957–64. 10.1128/IAI.74.5.2957-2964.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref014] 14.Oldfield NJ, Bland SJ, Taraktsoglou M, Dos Ramos FJ, Robinson K, Wooldridge KG, et al. T-cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells. Cell Microbiol. 2007;9(2):463–78. 10.1111/j.1462-5822.2006.00803.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref015] 15.Hung MC, Heckels JE, Christodoulides M. The adhesin complex protein (ACP) of Neisseria meningitidis is a new adhesin with vaccine potential. MBio. 2013;4(2). 10.1128/mBio.00041-13 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref016] 16.Fletcher LD, Bernfield L, Barniak V, Farley JE, Howell A, Knauf M, et al. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun. 2004;72(4):2088–2100. 10.1128/iai.72.4.2088-2100.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref017] 17.Coureuil M, Jamet A, Bille E, Lecuyer H, Bourdoulous S, Nassif X. Molecular interactions between Neisseria meningitidis and its human host. Cell Microbiol. 2019:e13063 10.1111/cmi.13063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref018] 18.Dos Santos Souza I, Maissa N, Ziveri J, Morand PC, Coureuil M, Nassif X, et al. Meningococcal disease: A paradigm of type-IV pilus dependent pathogenesis. Cell Microbiol. 2020;22(4):e13185 10.1111/cmi.13185 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref019] 19.Sun YH, Bakshi S, Chalmers R, Tang CM. Functional genomics of Neisseria meningitidis pathogenesis. Nat Med. 2000;6(11):1269–73. 10.1038/81380 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref020] 20.Exley RM, Sim R, Goodwin L, Winterbotham MS, M.C., Read RC, Tang CM. Identification of meningococcal genes necessary for colonization of human upper airway tissue. Infect Immun. 2009;77(1):45–51. 10.1128/IAI.00968-08 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref021] 21.Mendum TA, Newcombe J, Mannan AA, Kierzek AM, McFadden J. Interrogation of global mutagenesis data with a genome scale model of Neisseria meningitidis to assess gene fitness in vitro and in sera. Genome Biol. 2011;12(12):R127 10.1186/gb-2011-12-12-r127 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref022] 22.Dietrich G, Kurz S, Hubner C, Aepinus C, Theiss S, Guckenberger M, et al. Transcriptome analysis of Neisseria meningitidis during infection. J Bacteriol. 2003;185(1):155–64. 10.1128/jb.185.1.155-164.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref023] 23.Hotopp JCD, Grifantini R, Kumar N, Tzeng YL, Fouts D, Frigimelica E, et al. Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes. Microbiology. 2006;152(Pt 12):3733–49. 10.1099/mic.0.29261-0 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref024] 24.Joseph B, Schneiker-Bekel S, Blom J, Claus H, Linke B, Schwarz RF, et al. Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence. J Bacteriol. 2010;192(20):5363–77. 10.1128/JB.00883-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref025] 25.Joseph B, Frosch M, Schoen C, Schubert-Unkmeir A. Transcriptome analyses in the interaction of Neisseria meningitidis with mammalian host cells. Methods Mol Biol. 2012;799:267–93. 10.1007/978-1-61779-346-2_17 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref026] 26.Hey A, Li MS, Hudson MJ, Langford PR, Kroll JS. Transcriptional profiling of Neisseria meningitidis interacting with human epithelial cells in a long-term in vitro colonization model. Infect Immun. 2013;81(11):4149–59. 10.1128/IAI.00397-13 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref027] 27.Echenique-Rivera H, Muzzi A, Del Tordello E, Seib K, Francois P, Rappuoli R, et al. Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival. PLoS Pathog. 2011;7(5):e1002027 10.1371/journal.ppat.1002027 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref028] 28.Hedman AK, Li MS, Langford PRF, Kroll JS. Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery. PLoS One. 2012;7(6):e39718 10.1371/journal.pone.0039718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref029] 29.Bernardini G, Braconi D, Santucci A. The analysis of Neisseria meningitidis proteomes: Reference maps and their maps and their applications. Proteomics. 2007;7(16):2933–46. 10.1002/pmic.200700094 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref030] 30.van Alen T, Claus H, Zahedi RP, Groh J, Blazyca H, Lappann M, et al. Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis. Proteomics. 2010;10(24):4512–21. 10.1002/pmic.201000267 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref031] 31.Kanova E, Jimenez-Munguia I, Majerova P, Tkacova Z, Bhide K, Mertinkova P, et al. Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells. Front Microbiol. 2018;9:2294 10.3389/fmicb.2018.02294 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref032] 32.Siena E, Bodini M, Medini D. Interplay Between Virulence and Variability Factors as a Potential Driver of Invasive Meningococcal Disease. Comput Struct Biotechnol J. 2018;16:61–9. 10.1016/j.csbj.2018.02.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref033] 33.Takahashi H, Watanabe H. A broad-host-range vector of incompatibility group Q can work as a plasmid vector in Neisseria meningitidis: a new genetical tool. Microbiology. 2002;148(Pt 1):229–36. 10.1099/00221287-148-1-229 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref034] 34.Soriani M. Unraveling Neisseria meningitidis pathogenesis: from functional genomics to experimental models. F1000Res. 2017;6:1228 10.12688/f1000research.11279.1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref035] 35.Takahashi H, Kim KS, Watanabe H. Differential in vitro infectious abilities of two common Japan-specific sequence-type (ST) clones of disease-associated ST-2032 and carrier-associated ST-2046 Neisseria meningitidis strains in human endothelial and epithelial cell lines. FEMS Immunol Med Microbiol. 2008;52(1):36–46. 10.1111/j.1574-695X.2007.00342.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref036] 36.Takahashi H, Yanagisawa T, Kim KS, Yokoyama S, Ohnishi M. Meningococcal PilV potentiates Neisseria meningitidis type IV pilus-mediated internalization into human endothelial and epithelial cells. Infect Immun. 2012;80(12):4154–66. 10.1128/IAI.00423-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref037] 37.Takahashi H, Kim KS, Watanabe H. Meningococcal internalization into human endothelial and epithelial cells is triggered by the influx of extracellular L-glutamate via GltT L-glutamate ABC transporter in Neisseria meningitidis. Infect Immun. 2011;79(1):380–92. 10.1128/IAI.00497-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref038] 38.Takahashi H, Yanagisawa T, Kim KS, Yokoyama S, Ohnishi M. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM L-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells. Infect Immun. 2015;83(9):3555–67. 10.1128/IAI.00654-15 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref039] 39.Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE, Eisen JA, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science. 2000;287(5459):1809–15. 10.1126/science.287.5459.1809 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref040] 40.Willis JCW, Chin JW. Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem. 2018;10(8):831–7. 10.1038/s41557-018-0052-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref041] 41.Wang L. Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts. Acc Chem Res. 2017;50(11):2767–75. 10.1021/acs.accounts.7b00376 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref042] 42.Holding AN. XL-MS: Protein cross-linking coupled with mass spectrometry. Methods. 2015;89:54–63. 10.1016/j.ymeth.2015.06.010 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref043] 43.Dumas A, Lercher L, Spicer CD, Davis BG. Designing logical codon reassignment—Expanding the chemistry in biology. Chem Sci. 2015;6(1):50–69. 10.1039/c4sc01534g [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref044] 44.Chin JW, Martin AB, King DS, Wang L, Schultz PG. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A. 2002;99(17):11020–4. 10.1073/pnas.172226299 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref045] 45.Takahashi H, Watanabe H. Post-translational processing of Neisseria meningitidis g-glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space. FEMS Microbiol Lett. 2004;234(1):27–35. 10.1016/j.femsle.2004.03.003 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref046] 46.Takahashi H, Watanabe H, Kim KS, Yokoyama S, Yanagisawa T. The Meningococcal Cysteine Transport System Plays a Crucial Role in Neisseria meningitidis survival in human brain microvascular endothelial cells. mBio. 2018;9(6):e02332–18. 10.1128/mBio.02332-18 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref047] 47.Takahashi H, Inada T, Postma P, Aiba H. CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA^(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli. Mol Gen Genet. 1998;259(3):317–26. 10.1007/s004380050818 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref048] 48.Takahashi H, Hirose K, Watanabe H. Necessity of meningococcal gamma-glutamyl aminopeptidase for the Neisseria meningitidis growth in rat cerebrospinal fluid (CSF) and CSF mimicking medium. J Bacteriol. 2004;186(1):244–7. 10.1128/jb.186.1.244-247.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref049] 49.Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode. Chem Biol. 2008;15(11):1187–97. 10.1016/j.chembiol.2008.10.004 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref050] 50.Lacey VK, Kouie GV, Noel JP, Wang L. Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase. Chembiochem. 2013;14(16):2100–5. 10.1002/cbic.201300400 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref051] 51.Stark MJ. Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene. 1987;51(2–3):255–67. 10.1016/0378-1119(87)90314-3 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref052] 52.Schmidt TG, Skerra A. The Strep-tag system for one-step purification and high-affinity detection or. Capturing of proteins. Nat Protoc. 2007;2(6):1528–35. 10.1038/nprot.2007.209 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref053] 53.Yanagisawa T, Takahashi M, Mukai T, Sato S, Wakamori M, Shirouzu M, et al. Multiple site-specific installations of Nepsilon-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis. Chembiochem. 2014;15(12):1830–8. 10.1002/cbic.201402291 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref054] 54.Masuda A, Dohmae N. Examination of an absolute quantity of less than a hundred nanograms of proteins by amino acid analysis. Anal Bioanal Chem. 2013;405(25):8073–81. 10.1007/s00216-013-7056-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref055] 55.Takahashi H, Carlson RW, Muszynski A, Choudhury B, Kim KS, Stephens DS, et al. Modification of lipooligosaccharide with phosphoethanolamine by LptA in Neisseria meningitidis enhances meningococcal adhesion to human endothelial and epithelial cells. Infect Immun. 2008;76(12):5777–89. 10.1128/IAI.00676-08 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref056] 56.Ramsey ME, Hackett KT, Kotha C, Dillard JP. New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol. 2012;78(9):3068–78. 10.1128/AEM.07871-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref057] 57.Boyle-Vavra S, Seifert HS. Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae. Gene. 1995;155(1):101–6. 10.1016/0378-1119(94)00890-5 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref058] 58.Yamaguchi A, Matsuda T, Ohtake K, Yanagisawa T, Yokoyama S, Fujiwara Y, et al. Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates. Bioconjug Chem. 2016;27(1):198–206. 10.1021/acs.bioconjchem.5b00602 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref059] 59.Young TS, Ahmad I, Yin JA, Schultz PG. An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol. 2010;395(2):361–74. 10.1016/j.jmb.2009.10.030 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref060] 60.Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc. 2002;124(31):9026–7. 10.1021/ja027007w [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref061] 61.Fabrini R, De Luca A, Stella L, Mei G, Orioni B, Ciccone S, et al. Monomer-dimer equilibrium in glutathione transferases: a critical re-examination. Biochemistry. 2009;48(43):10473–82. 10.1021/bi901238t [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref062] 62.Yanagisawa T, Kuratani M, Seki E, Hino N, Sakamoto K, Yokoyama S. Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase. Cell Chem Biol. 2019;26(7):936–49. 10.1016/j.chembiol.2019.03.008 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref063] 63.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10(6):845–58. 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref064] 64.Zhang Z, Tan M, Xie Z, Dai LF, Y. C, Zhao Y. Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol. 2011;7(1):58–63. 10.1038/nchembio.495 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref065] 65.Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS One. 2012;7(7):e40410 10.1371/journal.pone.0040410 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref066] 66.Korndorfer IP, Skerra A. Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site. Protein Sci. 2002;11(4):883–93. 10.1110/ps.4150102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref067] 67.Carbonnelle E, Helaine S, Nassif X, Pelicic V. A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol Microbiol. 2006;61(6):1510–22. 10.1111/j.1365-2958.2006.05341.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref068] 68.Kolappan S, Coureuil M, Yu X, Nassif X, Egelman EH, Craig L. Structure of the Neisseria meningitidis Type IV pilus. Nat Commun. 2016;7:13015 10.1038/ncomms13015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref069] 69.Carbonnelle E, Hill DJ, Morand P, Griffiths NJ, Bourdoulous S, Murillo I, et al. Meningococcal interactions with the host. Vaccine. 2009;27 Suppl 2:B78–89. 10.1016/j.vaccine.2009.04.069 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref070] 70.Brown DR, Helaine S, Carbonnelle E, Pelicic V. Systematic functional analysis reveals that a set of seven genes is involved in Fine-Tuning of the Multiple Functions Mediated by Type IV Pili in Neisseria meningitidis. Infect Immun. 2010;78(7):3053–63. 10.1128/IAI.00099-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref071] 71.Imhaus AF, Dumenil G. The number of Neisseria meningitidis type IV pili determines host cell. EMBO J. 2014;33(16):1767–83. 10.15252/embj.201488031 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref072] 72.Carbonnelle E, Hélaine S, Prouvensier L, Nassif X, Pelicic V. Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step. Mol Microbiol. 2005;55(1):54–64. 10.1111/j.1365-2958.2004.04364.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref073] 73.Pelicic V. Type IV pili: e pluribus unum? Mol Microbiol. 2008;68(4):827–37. 10.1111/j.1365-2958.2008.06197.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref074] 74.Hospenthal MK, Costa TRD, Waksman G. A comprehensive guide to pilus biogenesis in Gram-negative bacteria. Nat Rev Microbiol. 2017;15(6):365–79. 10.1038/nrmicro.2017.40 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref075] 75.Wormann ME, Horien CL, Bennett JS, Jolley KA, Maiden MC, Tang CM, et al. Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences. BMC Genomics. 2014;15:253 10.1186/1471-2164-15-253 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref076] 76.Helaine S, Carbonnelle E, Prouvensier L, Beretti JL, Nassif X, Pelicic V. PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol Microbiol. 2005;55(1):65–77. 10.1111/j.1365-2958.2004.04372.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref077] 77.Brissac T, Mikaty G, Dumenil G, Coureuil M, Nassif X. The meningococcal minor pilin PilX is responsible for type IV pilus conformational changes associated with signaling to endothelial cells. Infect Immun. 2012;80(9):3297–306. 10.1128/IAI.00369-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref078] 78.Mikaty G, Soyer M, Mairey E, Henry N, Dyer D, Forest KT, et al. Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress. PLoS Pathog. 2009;5(2):e1000314 10.1371/journal.ppat.1000314 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref079] 79.Wolfgang M, van Putten JP, Hayes SF, Koomey M. The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation. Mol Microbiol. 1999;31(5):1345–57. 10.1046/j.1365-2958.1999.01269.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref080] 80.Hill DJ, Griffiths NJ, Borodina E, Virji M. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clin Sci (Lond). 2010;118(9):547–64. 10.1042/CS20090513 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref081] 81.Georgiadou M, Castagnini M, Karimova G, Ladant D, Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly. Mol Microbiol. 2012;84(5):857–73. 10.1111/j.1365-2958.2012.08062.x [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref082] 82.Lin S, Zhang Z, Xu H, Li L, Chen S, Li J, et al. Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens. J Am Chem Soc. 2011;133(50):20581–7. 10.1021/ja209008w [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref083] 83.Gan Q, Lehman BP, Bobik TA, Fan C. Expanding the genetic code of Salmonella with non-canonical amino acids. Sci Rep. 2016;6:39920 10.1038/srep39920 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref084] 84.Wang F, R S., Guo J, Shen W, Shen W, Schultz PG. Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis. PLoS One. 2010;5(2):e9354 10.1371/journal.pone.0009354 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref085] 85.Luo X, Zambaldo C, Liu T, Zhang Y, Xuan W, Wang C, et al. Recombinant thiopeptides containing noncanonical amino acids. Proc Natl Acad Sci U S A. 2016;113(13):3615–20. 10.1073/pnas.1602733113 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref086] 86.Zhang M, Lin S, Song X, Liu J, Fu Y, Ge X, et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat Chem Biol. 2011;7(10):671–7. 10.1038/nchembio.644 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref087] 87.O' Dwyer CA, Langford PR, Kroll JS. A novel neisserial shuttle plasmid: a useful new tool for meningococcal research. FEMS Microbiol Lett. 2005;251(1):143–7. 10.1016/j.femsle.2005.07.036 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref088] 88.Spence JM, Wright L, Clark VL. Laboratory maintenance of Neisseria gonorrhoeae. Curr Protoc Microbiol. 2008;Chapter 4:Unit 4A.1 10.1002/9780471729259.mc04a01s8 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref089] 89.Guibourdenche M, Popoff MY, Riou JY. Deoxyribonucleic acid relatedness among Neisseria gonorrhoeae, N. meningitidis. Ann Inst Pasteur Microbiol. 1986;137B(2):177–85. 10.1016/s0769-2609(86)80106-5 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref090] 90.Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane. Bioinformatics. 1998;14(4):378–9. 10.1093/bioinformatics/14.4.378 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref091] 91.Craig L, Volkmann N, Arvai AS, Pique ME, Yeager M, Egelman EH, et al. Type IV pilus structure by cryo-electron microscopy and crystallography. Mol Cell. 2006;23(5):651–62. 10.1016/j.molcel.2006.07.004 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref092] 92.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35(6):1547–9. 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237883.ref093] 93.Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–25. 10.1093/oxfordjournals.molbev.a040454 [DOI] [PubMed] [Google Scholar]

[pone.0237883.ref094] 94.Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining. Proc Natl Acad Sci U S A. 2004;101(30):11030–5. 10.1073/pnas.0404206101 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein

Hideyuki Takahashi

Naoshi Dohmae

Kwang Sik Kim

Ken Shimuta

Makoto Ohnishi

Shigeyuki Yokoyama

Tatsuo Yanagisawa

Roles

Abstract

Introduction

Materials and methods

Bacterial growth conditions

Table 1. IncQ plasmids introduced into N. meningitidis strains which results were shown in this study.

Table 2. Strains used in this study.

Production of anti-PamA protein rabbit antiserum

Construction of meningococcal mutants

Fig 2. Characterization of the biochemical properties of the PamA protein in N. meningitidis.

Assessment of host cell-associated and internalized bacteria

Western blotting

Observations of meningococci and ezrin accumulation by immunofluorescence staining

Construction of IncQ plasmids for N. meningitidis

UV crosslinking of whole N. meningitidis cells expressing the PamA Lys-amber (pamA K-amb) mutants by pyrrolysine-based amber suppression with pBPa

Identification of endogenous proteins crosslinked with PamA K278(pBPa) in N. meningitidis

Identification of the counterpart of the amino acid residue of PilE crosslinked with PamA K278(pBPa)

Transmission Electron Microscopy (TEM)

Measurement of the ratio of pilus components to PilE in purified pili

Results

Genetic evidence that a hypothetical protein annotated as NMB1345 is important for N. meningitidis internalization into HBMEC

Fig 1. Characterization of ΔpamA N. meningitidis mutant.

Characterization of the biochemical properties of PamA in N. meningitidis

Incorporation of ncAAs into proteins in living N. meningitidis

Fig 3. Incorporation of ncAAs into N. meningitidis proteins monitored by Western blotting.

Incorporation of a genetically encoded photocrosslinking amino acid into the PamA protein to identify the accompanying protein in N. meningitidis

Fig 4. PamA K208(pBPa), PamA K278(pBPa), PamA K314(pBPa), PamA K382(pBPa) and PamA K395(pBPa) proteins crosslinked to unidentified endogenous proteins in N. meningitidis.

Fig 5. Purification and identification of an endogenous protein crosslinked to PamA K278(pBPa)-Strep2-His6.

Table 3. Summary of the protein candidates crosslinked to PamA in N. meningitidis identified by mass spectrometry.

Mapping the amino acid residue in PilE that crosslinks to the PamA protein

Genetic elucidation of the interaction between PamA and PilE in N. meningitidis and its important for meningococcal internalization into HBMEC

Fig 6. The pilE I12A N. meningitidis mutant also showed reduced internalization into HBMEC, the induction of cytoskeleton rearrangements, and interactions with PamA in N. meningitidis.

Characterization of pili in the ΔpamA N. meningitidis mutant

Fig 7. Disruption of the interaction between PamA and PilE leads to abnormal proportions of PilV and PilX relative to PilE in meningococcal pili.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nediljko Budisa

Roles

Author response to Decision Letter 0

Decision Letter 1

Nediljko Budisa

Roles

Acceptance letter

Nediljko Budisa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 5. Purification and identification of an endogenous protein crosslinked to PamA K278(pBPa)-Strep₂-His₆.