Ail and PagC-Related Proteins in the Entomopathogenic Bacteria of Photorhabdus Genus

Abstract

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1 _Pl and ail2 _Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1 _Pl expression was directly controlled by PhoPQ and low external Mg²⁺ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2 _Pl and pagC _Pl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

Introduction

Various surface-exposed components present in the outer leaflet of the outer membrane play a crucial role in Gram-negative bacteria survival. Surface components have a dual role in virulent bacteria, first as factors maintaining the proper architecture of the outer membrane and as virulence factors [1]. About 50% of the outer membrane mass of Gram-negative bacteria consists of proteins, either lipoproteins that are anchoring the outer membrane to the underlying peptidoglycan or, integral membrane proteins [2]. The hallmark of integral outer membrane proteins is the folding into anti-parallel β-barrels [2], [3]. The most abundant integral membrane proteins of the bacterial outer membrane are porins, which are essentially trimeric β-barrels (16 or 18 β-strands) forming channels with various grades of selectivity [3]. Other barrel proteins having monomeric structure and fewer strands have been investigated, most displaying a specific function not related to the diffusion of hydrophilic molecules [3].

The family of related outer membrane proteins Ail/OmpX/PagC belongs to the latter category and was initially described in organisms like Yersinia enterocolitica and Yersinia pseudotuberculosis (Ail), Salmonella Typhimurium (Rck, PagC), Escherichia coli (OmpX, Lom) and Enterobacter (OmpX) [4]. These proteins display small size (from 15 to 18 kDa) and fold in eight β-barrels. Moreover, the Ail/OmpX/PagC proteins appear to be important for virulence by neutralizing host defense mechanisms. Ail from Y. enterocolitica promotes adhesion to and entry into eukaryotic tissue culture cells [5], [6]. PagC from S. Typhimurium is responsible for survival in macrophages [7], [8]. OmpX from Enterobacter aerogenes induces a β-lactam resistance mediated by a decrease in the porin production [9]. Lom from bacteriophage λ participates in E. coli adhesion to human buccal epithelial cells [10]. However, clear separation of functions between the different members of the Ail/OmpX/PagC family is not obvious. Indeed, Ail from Y. enterocolitica [5], [6], [11] and from Yersinia pestis [12], Rck from S. Typhimurium [6], PagC from S. enterica serovar Cholerasuis [13] are responsible for conferring resistance to complemented-mediated killing, but this property is not shared by PagC from S. Typhimurium, OmpX from E. cloacae or Lom from E. coli [6]. This discrepancy is probably due to gene annotation, which does not rely on an exhaustive phylogenetic analysis.

The expression of genes encoding outer membrane proteins has been found to be under a complex transcriptional regulation, acting as an adaptive response toward environmental physical attack of cell integrity. Modulation of expression of abundant outer membrane proteins such as porins are generally transcriptionally regulated by the two-component regulatory system OmpR-EnvZ and small RNAs [14]. Regulatory pathways controlling the expression of genes encoding proteins from the Ail/OmpX/PagC family have been elucidated in some Gram-negative bacteria, but there is no convergence towards a common pathway. For instance, in Y. enterocolitica 8081, ail is regulated by temperature [15]. By contrast, ail from Y. enterocolitica O: 9 is not subjected to thermoregulation but is under the control of the OmpR transcription factor [16]. ompX is regulated by a small RNA in S. Typhimurium [17]. The regulation of pagC is under the control of the two-component system PhoP-PhoQ through SlyA in S. Typhimurium [18]. This PhoPQ system has been extensively studied in bacteria and especially in S. Typhimurium, in which the response regulator PhoP regulates about 3% of Salmonella genes, subdivided into the PhoP-activated genes, pag, and the PhoP-repressing genes, prg [19]. In a variety of Gram-negative bacterial pathogens, numerous PhoP-regulated genes encode enzymes involved in LPS modifications [20]–[23], as well as several secreted and outer membrane proteins [24].

Photorhabdus (Enterobacteriaceae) is an insect pathogen living in a symbiotic association with entomopathogenic nematodes Heterorhabditis [25]. Heterorhabditis bacteriophora nematodes invade insect larvae and regurgitate bacteria from their gut directly into the hemolymph, the insect blood [26]. The bacteria overcome the insect immune system and colonize the insect body cavity leading to lethal septicemia [25]. Bacterial virulence factors and insecticidal toxins also participate to the insect death [27], [28]. Once the insect host is dead, bacteria bioconvert the tissues and the nematode partner feeds off the bacteria while nematode reproduction occurs through several generations [29]. Photorhabdus also successfully competes with saprophytic scavenging organisms. It produces antimicrobial factors in order to kill any invading and competing microbes [25]. Several rounds of nematode reproduction and bacterial replication lead to a new generation of infective juvenile (IJ) nematodes. Photorhabdus bacteria colonize specifically the posterior-intestinal cells of the maternal adult nematode before re-associating with the new IJ [30], [31]. The dual requirement for symbiosis and virulence makes Photorhabdus an excellent model organism for studying host-bacteria interactions. The genus Photorhabdus comprises four distinct species: Photorhabdus temperata, Photorhabdus luminescens, Photorhabdus heterorhabditis and Photorhabdus asymbiotica [32]. Although all four are highly pathogenic to insects, P. asymbiotica also causes infection in humans [33]–[35].

In an attempt to identify host-interacting bacterial proteins, we were interested in proteins from the Ail/OmpX/PagC family of Photorhabdus genus. Duchaud et al. [36] already described three Ail-like homologs in P. luminescens strain TT01. Thus, we exhaustively searched for proteins from the Ail/OmpX/PagC family encoded in the genomes of P. luminescens strain TT01 [36] and P. asymbiotica strain ATCC43949 [37]. Analysis of Ail/OmpX/PagC phylogeny supports a robust annotation showing that the Photorhabdus genus only encodes Ail and PagC orthologs. Then, we present the first detailed investigation into the role and the regulation of Ail and PagC proteins from Photorhabdus.

Materials and Methods

Bacterial strains, plasmids, primers and growth conditions

The strains and plasmids used in this study are listed in Table 1. All primers used in this study (Eurogentec) are listed in Supplemental Table 1. Photorhabdus and Escherichia coli strains were routinely grown at 28°C and 37°C, respectively, in Luria-Bertani (LB) broth or on LB solid medium. Photorhabdus was grown in M9 liquid medium with concentrations of MgSO₄ (10 µM and 10 mM) and supplemented with 0.1% casamino acids, 0.41 mM nicotinic acid, 9.1 mM sodium pyruvate, 0.2% glycerol and 0.1 mM CaCl₂. When required, antibiotics were used at the following final concentrations: kanamycin (Km) 20 µg/ml, gentamicin (Gm) 30 µg/ml, ampicillin (Ap) 100 µg/ml.

Table 1. Strains and plasmids used in this study.

Strain or plasmid	Genotype or relevant characteristics	Source or reference
P. luminescens strain
TT01	Strain isolated from the nematode Heterorhabditis bacteriophora THO1 in Trinidad; wild-type form	[75]
phoP	TT01 phoP::cat, phoP mutant	[21]
TT01/Pail1 Pl -gfp[AAV]	Conjugative strain, TT01 carrying Pail1 Pl -gfp[AAV] plasmid, KmR	this work
TT01/Pail2 Pl -gfp[AAV]	Conjugative strain, TT01 carrying Pail2 Pl -gfp[AAV] plasmid, KmR	this work
TT01/Plac -gfp[AAV]	Conjugative strain, TT01 carrying Plac -gfp[AAV] plasmid, KmR	[51]
phoP/Pail1 Pl -gfp[AAV]	Conjugative strain, phoP carrying Pail1 Pl -gfp[AAV] plasmid, KmR	this work
phoP/Pail2 Pl -gfp[AAV]	Conjugative strain, phoP carrying Pail2 Pl -gfp[AAV] plasmid, KmR	this work
E.coli strain
XL1Blue	F′ proAB lacI q ZΔM15 Tn10(TetR)	Laboratory stock
BL21 (DE3) pLysS	F− dcm ompT hsdS(rB −mB −) gal λ(DE3) (pLysS CamR)	Laboratory stock
WM3064	thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341::[erm pir (wt)]	[76]
Plasmid
pUC19	High copy number vector, ApR	Laboratory stock
pUC-ail Yp	0.65 kb PCR fragment obtained with y1324-PstI and y1324-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pUC-ail1 Pl	0.7 kb PCR fragment obtained with plu2480-PstI and plu2480-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pUC-ail2 Pl	0.7 kb PCR fragment obtained with plu2481-PstI and plu2481-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pUC-pagC Pl	0.8 kb PCR fragment obtained with plu1967-PstI and plu1967-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pUC-ail Pa	0.65 kb PCR fragment obtained with PAU_02047-PstI and PAU_02047-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pUC-pagC Pa	0.7 kb PCR fragment obtained with PAU_02601-PstI and PAU_02601-SacI primers and inserted between the PstI and SacI sites of pUC19	this work
pPROBE-gfp[AAV]	Plasmid (pBBR1 replicon) containing gfp[AAV] gene downstream from a multiple cloning site, KmR	[77]
Plac-gfp[AAV]	pPROBE with gfp[AAV] under the control of Plac promoter; KmR	[51]
Pail1 Pl -gfp[AAV]	pPROBE with gfp[AAV] under the control of ail1 (plu2480) gene promoter; KmR	this work
Pail2 Pl -gfp[AAV]	pPROBE with gfp[AAV] under the control of ail2 (plu2481) gene promoter; KmR	this work
pETPhos	pET28 replicon, ApR	[48]
PT7PhoP-His	pET28 producing PhoP(His-tag) in N-terminal under the control of T7 promoter; ApR	this work

Inference of the evolutionary relationships of Ail, PagC and OmpX-related proteins

Ail, PagC or OmpX annotated proteins in Photorhabdus luminescens TT01 (plu1967, plu2480, plu2481) and Photorhabdus asymbiotica ATCC43949 (PAU_02047 and PAU_02601) were identified and retrieved using the protein family analysis tool PipeAlign [38]. Outputs were pooled and resulting dataset were curated for protein multiple occurrences. The sequences were aligned using ClustalW [38] followed by manual curation. The sequence alignment was generated by Gblocks [39] and unambiguously aligned amino acid sites were retained for phylogeny inference using the maximum likelihood method implemented in PhyML [40]. Analyses were engendred under the LG model of amino acid replacement [41] with a gamma distribution of evolutionary rates across sites [42]. Internal branch supports were evaluated using the approximate Likelihood Ratio Test [43].

Molecular techniques and RNA preparation

DNA manipulations were carried out as previously described [44]. Plasmids were introduced into E. coli WM3064 (Table 1) by transformation and transferred to P. luminescens TT01 by filter mating [45]. All constructs were sequenced by Eurofins MWG Operon (Ebersberg, Germany). Total RNA was extracted with TRIzol reagent according to manufacturer's instructions (Invitrogen) and purified using RNeasy miniprep kit (Qiagen), including a DNase I treatment step. For each RNA preparation, we assessed DNA contamination by carrying out a control PCR. The quantity and quality of RNA were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific) and an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip kit (Agilent), respectively. Material for real-time quantitative polymerase chain reaction (RT-qPCR) analysis was prepared by extracting total RNA from the P. luminescens wild-type strain and the phoP mutant grown in Luria broth (OD₅₄₀ = 0.5–0.8). The gene expression level was evaluated during the growth phase.

Real-time quantitative polymerase chain reaction analysis

RT-qPCR was performed in two steps. First, the cDNA was synthesized from 500 ng of total RNA, with Super Script II Reverse Transcriptase (Invitrogen) and random hexamers (100 ng/µl) (Applied Biosystems). We then carried out qPCR in triplicate with the LightCycler 480 SYBR Green I Master kit from Roche Diagnostics, with 1 µl of cDNA synthesis mixture (diluted 1∶100) and 1 µM of specific primers for the genes studied (Supplemental Table 1). The enzyme was activated by heating for 10 min at 95°C. All qPCRs were performed in three technical replicates, with 45 cycles of 95°C for 5 seconds, 60°C for 5 seconds and 72°C for 10 seconds, and were monitored with the Light Cycler 480 system (Roche). Melting curves were analyzed for each reaction and each curve contained a single peak. The data for each sample were expressed relatively to the expression level of gyr, using REST software 2009 [46] as previously described [47]. This method provided a relative quantification of the target gene expression with respect to a reference gene (gyrB).

Overexpression and purification of PhoP recombinant protein

The entire coding region of phoP gene from TT01 strain was amplified by PCR and digested by NdeI and BamHI. The obtained PCR product was ligated into the same site of the expression vector, pETPhos [48] inserting a His-tag in the N-terminal part of PhoP thus generating the plasmid P_T7PhoP-His. The recombinant plasmid encoding a PhoP-His fusion protein was transformed into E. coli BL21 (DE3) pLysS cells. At an OD between 0.5–0.8, the expression of PhoP-His was induced by adding Isopropyl-beta-D-thiogalactoside at 0.5 mM. An overnight induction was then performed at 18°C. Bacterial culture was centrifuged at 7,000×g for 15 min at 4°C and washed twice in resuspension buffer (Tris 5 mM pH 7.5, NaCl 300 mM, glycerol 10%, Imidazole 10 mM). The pellet was frozen at −80°C for 30 min, then suspended in 5 ml resuspension buffer and lysed by sonication during 10 min at 4°C. Lysis products were centrifuged at 10,000×g during 30 min at 4°C. Five hundred µl of pre-equilibrated beads of Ni-NTA agarose (Qiagen) in wash buffer (Tris 5 mM pH 7.5, NaCl 300 mM, glycerol 10%, Imidazole 15 mM) were added to the supernatant fraction and incubated during 45 min with shaking at 4°C. The fraction was centrifuged at 500×g during 2 min at 4°C and wash 5 times with wash buffer. Protein was eluted twice in 1 ml elution buffer (Tris 5 mM pH 7.5, NaCl 300 mM, glycerol 10%, Imidazole 200 mM). Concentration of recombinant protein was assessed by Bradford assay and controlled by SDS-PAGE gel. Recombinant proteins were conserved at −80°C until use.

Electrophoretic mobility-shift assays

The promoter of ail1 _Pl was PCR-amplified from the genomic DNA of TT01 strain using specific primers (Supplemental Table 1) and purified using the High Pure PCR Product Purification kit (ROCHE). The 5′ ends of DNA fragment were labeled using [γ-³²P] ATP and T4 polynucleotide kinase (Promega). Radioactive DNA probe (2000 cpm/ml), 200 ng of poly(dI-dC)-poly(dI-dC) (SIGMA) and different amounts of PhoP-His were mixed with binding buffer (50 mM tris-HCl pH 8, 50 mM KCl, 50 µg/ml BSA) in a total 20 µl volume and incubated for 20 min at room temperature. The mixture was then loaded onto a native 6% (w/v) polyacrylamide TBE precast Gel (Invitrogen) and electrophoresed in 1% TBE (Tris-Borate-EDTA) buffer for 1 h at 100 V. Radioactive species were detected by autoradiography. PhoP-His was activated by in vitro phosphorylation with acetyl phosphate as previously described [49].

Construction of plasmids expressing gfp[AAV] under the control of ail1 _Pl or ail2 _Pl promoters

Plasmids expressing the reporter gene gfp[AAV] under the control of ail1 _Pl, ail2 _Pl or lac gene promoters were constructed using a previously described method [50]. The construction of P_lac-gfp[AAV] has been described elsewhere [51]. Briefly, DNA fragments located upstream from ail1 _Pl (360 bp) and ail2 _Pl (358 bp) were amplified by PCR from P. luminescens TT01 genomic DNA with primers containing the EcoRI and BamHI restriction site (Supplemental Table 1). The PCR products were EcoRI- and BamHI-hydrolyzed and inserted into the corresponding sites of pPROBE-gfp[AAV]. Finally, P_lac-gfp[AAV], P_{ail1 Pl}-gfp[AAV], P_{ail2 Pl}-gfp[AAV] were transferred by filter bacterial mating [45] in P. luminescens TT01 wild type and phoP strains.

Quantification of ail1 _Pl and ail2 _Pl expression in bacterial populations grown in different media

Wild-type strains carrying either P_{ail1 Pl}-gfp[AAV], P_{ail2 Pl}-gfp[AAV] or P_lac-gfp[AAV] constructs were cultured in black-sided, clear-bottomed 96-well plates (Greiner). For each well, a 1∶20 dilution of an overnight culture was added to the M9 minimal medium supplemented with kanamycin and different concentrations of MgSO₄ or to LB medium supplemented with kanamycin. Then, the plates were incubated at 28°C for 45 h with shaking on an orbital shaker, in an Infinite M200 microplate reader (Tecan). Absorbance at 600 nm and GFP fluorescence intensity (excitation at 485±4.5 nm; emission at 520±10 nm) were measured every 30 min. Specific fluorescence was obtained by dividing fluorescence units (at the maximum level of expression) by the absorbance value.

Serum-killing assay

The serum-killing assay was performed as described previously [5] with overnight culture of Escherichia coli strain XL1Blue in LB with ampicillin 100 µg/ml at 37°C. For each bacterial strain, three independent assays were performed with human serum from human male AB sterile plasma (Sigma-Aldrich, reference: H4522) or with serum that was heat-treated to inactivate complement (56°C, 30 min). The number of viable bacteria after incubation with serum at 37°C for 60 min was calculated by serial dilution, plating on LB agar with ampicillin 100 µg/ml and counting the colony forming units (CFU). The degree of killing was calculated as follow: log kill  =  (log₁₀ CFU per milliliter of initially added bacteria) - (log₁₀ CFU per milliliter of bacteria surviving the incubation). The resistance was expressed as the difference in log kill between XL1-Blue harboring pUC19 incubated in 50% human serum and XL1Blue harboring the recombinant plasmid incubated either in 50% human serum or heat-inactivated serum.

Cell association assays

The cell invasion and association assays were performed as described previously [52]. Escherichia coli strain XL1Blue were grown at 37°C in LB broth supplemented with ampicillin 100 µg/ml for 2.5 hours (10⁸ CFU/ml; optical density at 540 nm of 0.5; xponential-growth phase). Chinese hamster ovary cells (CHO) were grown and maintained at 37°C in RPMI Medium 1460-glutamax I (Gibco) complemented with 10% foetal bovine serum (Lonza), and 1% PenStrep 5000 U/ml (Gibco). Spodoptera littoralis cells derived from hemocytes (Sl2b) were grown and maintained at 28°C in G3 Medium pH 6,2 (TC100 2% from Gibco modified with 0.037% α-ketoglutaric acid, 0.04% β-fructose, 0.005% fumaric acid, 0.067% malic acid, 0.006% succinic acid, 0.26% sucrose, 0.02% choline chloride, 0.02% β-alanine, 0.035% sodium bicarbonate, 0.33% lactalbumin hydrolysate, and complemented with 5% foetal bovine serum, 0.016% penicillin, 0.006% streptomycin).

Susceptibility to antimicrobial peptides

In vitro susceptibility to polymyxin B sulfate (Sigma), colistin methanesulfate (Sigma), cecropin A (Sigma), Spodoptera frugiperda cecropin B was evaluated by determination of minimal inhibition concentration as previously described [53].

Results

Phylogeny of Ail, PagC and OmpX-related proteins among Bacteria

Ail, PagC and OmpX homologs were searched in genome databases (see Material and Methods). Phylogenetic analysis (Fig. 1A) clearly separated the 89 proteins from 53 bacterial species into three well-supported groups. The Ail group recovered the y1324 canonical Ail protein described in Y. pestis KIM [4]. The PagC group contained the STM3031 PagC protein from Salmonella Typhimurum [54]. Finally, the OmpX group was characterized by several proteins annotated OmpX, such as Ent638_1301 from Enterobacter sp. 638. Interestingly, the protein y1682 of Y. pestis KIM usually named Ail clusters with the OmpX group [4]. Our phylogenetic analysis clearly indicates that all the Photorhabdus proteins clustered within the Ail group (plu2480 and plu2481 from P. luminescens and PAU_02047 from P. asymbiotica) and the PagC group (plu1967 from P. luminescens and PAU_02601 from P. asymbiotica).

Figure 1. The Photorhabdus genus harbors ail and pagC genes.

A. Evolutionary relationships of Ail, PagC and OmpX-related proteins. Stars indicate branch supports higher than 0.85 (used as significance threshold). The scale bar corresponds to the number of substitutions per amino acid residue site. B. Conserved genomic context of the ail (up) and pagC (bottom) genes in Photorhabdus luminescens TT01 (Pl) and Photorhabdus asymbiotica ATCC43949 (Pa) genomes. The boxes above and below the axis represent ORFs in the forward and reverse orientations, respectively. The names of some genes are indicated. The names of genomic islands (GI) previously described in the P. luminescens and P. asymbiotica genomes [55] are given.

Genomic organization of ail and pagC genes in Photorhabdus genomes

Analysis of the P. asymbiotica ATCC43949 and P. luminescens TT01 genome sequences [36], [37] revealed that ail and pagC genes were located in conserved regions in the two Photorhabdus species, previously described as genomic islands in P. luminescens TT01 (Fig. 1B) [55]. Both insertion points and contents in the coding sequences were similar. The genomic islands from P. luminescens genomes were composed of additional genes encoding proteins potentially involved in infectious process. The toxBCD operon in the vicinity of the ail genes (GI_59) is involved in toxoflavin biosynthesis of Burkholderia glumae [56]. The phaxA1B1 genes in the vicinity of the pagC (GI_47) encodes the XaxAB-like binary toxin with insecticidal and cytotoxic activity [57], [58].

The two ail genes from P. luminescens, referred to as ail1_Pl and ail2_Pl hereafter, shared 71% of nucleotidic identity and 66% of aminoacids identity and were separated by 517 nucleotides. This adjacent position (Fig. 1B) together with their closed clustering inside the Ail group (Fig. 1A) suggests a recent tandem duplication. By contrast, the ail gene from P. asymbiotica (ail _Pa), the pagC genes of P. luminescens and P. asymbiotica (pagC _Pl and pagC _Pa, respectively) were present in single copy.

PhoP is directly regulating the expression of ail1 _Pl

Data on expression of genes encoding outer membrane proteins of the Ail/OmpX/PagC family remains limited except for PagC. Indeed, pagC gene in S. Typhimurium is a PhoP-activated gene [7]. A phoP mutant has been previously constructed and described for P. luminescens TT01 [21]. We therefore performed RT-qPCR to measure levels of pagC _Pl, ail1 _Pl and ail2 _Pl mRNA and to calculate their ratio of values in phoP and wild type backgrounds of P. luminescens TT01 (Fig. 2A). Only the level of ail1 _Pl transcript was lower in the phoP mutant than in the wild type strain, indicating that the ail1 _Pl gene expression requires PhoP. Contrarily, ail2 _Pl and pagC _Pl expression was not PhoP-dependent. The differential regulation of ail paralogs were independently confirmed by measuring expression of P_{ail1 Pl}-gfp[AAV] and P_{ail2 Pl}-gfp[AAV] fusions in wild-type and phoP backgrounds (Fig. 2B). Next, the possible direct binding of PhoP protein upstream of ail1 _Pl was investigated. Electrophoretic mobility shift assays (EMSAs) were carried out to compare the interaction profiles of different amounts of PhoP protein on the 360-bp ail1 _Pl promoter region (Fig. 2C). A recombinant N-terminal His-tag PhoP protein (PhoP-His) was first produced from P_T7PhoP-His vector (Table 1). The PhoP-His protein was purified and phosphorylated in vitro by incubation with acetyl phosphate. Phosphorylation efficiency and dimer formation were assessed by migration on precast Phos-tag gel (Fig. S1). Then, different amounts of phosphorylated and unphosphorylated PhoP-His were mixed with radiolabeled ail1 _Pl promoter. A gel shift pattern was observed when 1.5 µM of phosphorylated PhoP-His was added (Fig. 2C). No shifted bands were observed upon incubation with unphosphorylated PhoP-His. Therefore, PhoP-His protein can specifically bind to the promoter region of ail1 _Pl and the active form of PhoP corresponds to the phosphorylated isoform.

Figure 2. Only ail1 _Pl is directly regulated by PhoP.

A. RT-qPCR: ail1 _Pl expression is PhoP-dependent. Total RNA from phoP mutant or TT01 wild-type strain of Photorhabdus luminescens was used for RT-qPCR analysis with internal primers specific for the indicated genes. mRNA levels were normalized against those of a reference gene (gyrB). Data are presented as a ratio of values for phoP mutant and TT01 wild-type strain. A ratio of 1 indicates no difference in expression level between both strains. The bars indicate standard errors calculated using Taylor's series. Significant differences (p-value <0.05) are indicated by asterisks (*). The relative quantification results were obtained from three independent experiments with the REST 2009 program. B. Gene transcription monitored by GFP quantification: ail1 _Pl promoter region is positively regulated by PhoP. The dynamic expression of ail1 _Pl and ail2 _Pl promoter in TT01 and phoP backgrounds was monitored over time after growth in LB medium. Each histogram represents the specific fluorescence at the peak of expression for each condition. One experiment representative of more than three independent experiments is shown. Standard deviations represent technical replicates. C. PhoP-His binds the promoter region of ail1 _Pl. Electrophoretic mobility shift assay was carried out to test the binding of PhoP-His protein activated in vitro with ACP 10 mM (P-PhoP-His) or non activated PhoP-His (PhoP-His) on ail1 _Pl promoter. The PhoP-His concentrations indicated are in micromolar. To ensure that the fixation is specific, we used BSA proteins and poly(dI-dC) in the binding buffer. D. ail1 _Pl expression is higher at low MgSO₄ concentrations. We evaluated the impact of low and high MgSO₄ concentrations on ail1 _Pl and ail2 _Pl expression. Cultures diluted at 1/200 were grown in M9 minimal medium supplemented with 10 µM or 10 mM MgSO₄. Each histogram represents specific fluorescence at the peak of expression for each condition. Experiments were realized at least three times.

ail1 _Pl expression is high at low MgSO₄ concentrations

It has been shown that low concentrations of Mg²⁺ activate the expression of PhoP-dependent genes in Salmonella whereas high Mg²⁺ concentrations repress the system (for review see [59], [60]. In order to evaluate the role of Mg²⁺ concentration on ail1 _Pl or ail2 _Pl expression, wild-type strain containing P_{ail1 Pl}-gfp[AAV], P_{ail2 Pl}-gfp[AAV] or P_lac-gfp[AAV] were grown in minimal medium M9 supplemented with 10 µM (activating concentration) or 10 mM (repressing concentration) of MgSO₄ and gene expression was monitored by recording GFP fluorescence (Fig. 2D). We observed a 5-fold decrease of ail1 _Pl expression at 10 mM MgSO₄, whereas ail2 _Pl expression was not dependent on the concentration of MgSO₄. As observed for PhoP-activated genes in S. Typhimurium [19], low MgSO4 concentration increases ail1 _Pl expression in Photorhabdus.

ail2 _Pl and pagC _Pl genes confer human serum resistance but no eukaryotic cell association phenotype

When introduced into Escherichia coli, individual members of the Ail/PagC/OmpX-related protein family have the property to confer resistance to human serum complement and to associate with eukaryotic cell [5], [6], [11], [13]. In order to test if the ail and pagC genes from P. luminescens and P. asymbiotica have similar phenotypes, we introduced ail1 _Pl, ail2 _Pl, pagC _Pl, ail _Pa and pagC _Pa under the control of the constitutive promoter P_lac into the high copy number pUC19 plasmid and transformed these pUC19 derivatives into an E. coli XL1blue strain. Active transcription of corresponding cloned genes in E. coli XL1Blue was controlled by real-time PCR (data not shown). We first evaluated the human serum resistance of the E. coli XL1Blue strains harboring the different derivative plasmids (Fig. 3). The positive control, E. coli XL1Blue expressing the ail gene from Y. pestis strain KIM, y1324 (ail _Yp), showed an elevated serum resistance, similar to the one observed in all strains when the complement in the serum was heated-inactivated. Moreover, we observed intermediate serum resistance with the ail2 _Pl and pagC _Pl genes whereas the resistance with the ail1 _Pl, ail _Pa and pagC _Pa genes was weak.

Figure 3. Human serum resistance of Escherichia coli XL1Blue strains carrying the plasmid pUC19 and its derivatives harboring ail or pagC genes.

Overnight grown bacteria were tested for viability at 37°C in 50% serum (black histograms) or heat-inactivated serum (hatched histograms). The resistance was expressed as the difference in log kill between XL1-Blue harboring pUC19 incubated in 50% human serum and XL1Blue harboring the recombinant plasmid incubated either in 50% human serum or heat-inactivated serum. Means and standard errors of results from triplicate experiments are shown.

In order to assess if the association of E. coli XL1Blue to eukaryotic cells was affected by expression of Photorhabdus ail or pagC genes, association assays of mammals cells (CHO) and insect cells (Sl2b) were performed. E. coli XL1Blue expressing ail _Yp displayed association with CHO cells (% of association of 30.43±17.06) in regards to E. coli XL1Blue harboring the pUC19 plasmid (% of association of 0.57±0.05), which is consistent with previously published data [52]. None of the Photorhabdus over-expressed genes, ail1 _Pl, ail2 _Pl, pagC _Pl, ail _Pa and pagC _Pa, was able to confer to E. coli XL1Blue strain an improved association with Sl2b cells to E. coli XL1Blue strain (average % of association of 10.60±6.80).

ail1 _Pl, ail2 _Pl and pagC _Pl do not confer antimicrobial peptide resistance

Since P. luminescens TT01 and P. asymbiotica ATCC43949 are pathogenic towards insect, the role of ail and pagC genes was studied by cultivating recombinant E. coli in insect hemolymph, which is analogous to the mammalian blood regarding the immune system. The growth of the E. coli XL1Blue derivatives was not affected when cultivated in sterile hemolymph of Spodoptera littoralis fifth-instar larvae (data not shown). Then, we compared the susceptibility to different antimicrobial peptides of the negative control E. coli XL1Blue/pUC19 and the E. coli XL1Blue harboring the different derivative plasmids. Similar minimal inhibitory concentrations were observed towards colistin (1.5–3 µg/ml), polymyxin B (0.1–0.2 µg/ml), cecropin A (6–12.5 µg/ml) or cecropin B from the Lepidoptera Spodoptera frugiperda (1.5–3 µg/ml).

Discussion

Photorhabdus genomes only harbor ail and pagC gene homologs

Our analysis of 89 proteins from 53 bacterial species belonging to the Ail/PagC/OmpX family clearly distinguished three sub-families. In the two whole-assembled genomes of Photorhabdus, both Ail and PagC proteins are encoded. Interestingly, in P. luminescens TT01, we identified two intra-genome homologs ail1 and ail2. Such homologs can arise through duplication, where both gene copies are named paralogs, or by acquiring similar genes from outside sources through horizontal gene transfer, where both gene copies are named xenologs [61]. The adjacent position of ail1 _Pl and ail2 _Pl genes suggests recent tandem duplication. Duplication is an important hallmark of the genome plasticity of P. luminescens both in short-term adaptation and long-term evolution. Several dozen of duplicated genes have already been described in P. luminescens TT01 [36]. Interestingly, another gene encoding an outer membrane protein, the ompF-like gene, probably underwent a tandem duplication [62]. Characteristic features of an ancient whole-genome duplication were also detected in the P. luminescens TT01 genome [63]. In addition, a 275-kilobase single block duplication, with cryptic phenotypic consequences, was observed in phenotypic variants of P. luminescens [64].

What are the factors regulating ail/pagC gene expression in Photorhabdus?

No data about transcription factors involved in the control of ail/pagC expression in the Photorhabdus genus are currently available. P. asymbiotica is considered an emerging human pathogen [34]. In an attempt to find host-interacting proteins that are relevant to either human or insect infections is interesting, Wilkinson et al. identified a thermoregulation of the secretion for its Ail-like homolog in P. asymbiotica ATCC43949 (Ail_Pa in our study) as Ail_Pa is secreted at 37°C but not at 30°C [37]. In our study, we decided to use the available phoP mutant of P. luminescens TT01 [21] to compare relative expression of ail1 _Pl, ail2 _Pl and pagC _Pl in wild-type and phoP backgrounds. Only P. luminescens ail1 expression is PhoP-dependent by contrast with ail2 _Pl and pagC _Pl. The pagC gene from S. Typhimurium is regulated by PhoP in a indirect way via SlyA [18]. Surprisingly, our electromobility shift assays showed that the His-tagged PhoP protein from P. luminescens directly binds to the promoter region of ail1. This is the first time that ail has been described to rely on the direct control of PhoP in Gram-negative bacteria. In addition, we monitored the kinetic of ail1 expression in different culture conditions and determined that ail1 expression was reduced in the presence of high concentrations of MgSO₄. The environmental deprivation of Mg²⁺ as a signal activating the PhoP/PhoQ signal transduction cascade was first described in Salmonella [19]. In Photorhabdus, Derzelle et al. [21] showed that PhoP-dependent expression of the first gene of the pbgPE operon, involved in LPS modifications, relies on the Mg²⁺ concentrations in the culture medium. Like pbgPE, expression ail1 _Pl is higher at low Mg²⁺ concentrations than at high concentrations. In Salmonella and P. luminescens, both the phoP gene and the pbgPE operon are involved in virulence in the mouse and the insect models, respectively [7], [21], [65], [66]. In P. luminescens, it is likely that the deficience of PhoP-dependent pbgPE expression is responsible for the avirulence of phoP mutant rather than the one of ail1 _Pl.

What are the functions of Ail and PagC proteins in the life cycle of Photorhabdus?

To answer this question, we used a well-established assay by expressing in E. coli ail and pagC homologs from the Photorhabdus strains. Such strategy was successfully used for characterizing the role of proteins from the Ail/PagC/OmpX family in Salmonella and Yersinia in human serum resistance and invasion/adherence to eukaryotic culture cells [4]. Recombinant Escherichia coli clones, expressing cosmids from Photorhabdus, were also used in assays to study gain of toxicity against insects, nematodes, amoeba, and mammalian macrophages [67] and to attribute biological function to several Photorhabdus potential virulence loci [68]–[70]. With this heterologous assay, we showed that none of the over-expressed genes displayed a role in adherence to the tested mammal (CHO) or insect (Sl2b) cells. Photorhabdus life cycle is mainly extracellular except a transient invasive stage during the symbiont transmission to the new generation of IJ before they exit the insect cadaver [30]. This transmission stage is dependent on the production of the bacterial Mad pili [71]. By contrast with Ail and PagC from Yersinia and Salmonella species [5]–[8], our results do not suggest a role of Ail1, Ail2 or PagC in cell invasion. While P. asymbiotica ATCC43949 is a clinical isolate from human wounds resistant to human serum at 30°C and 37°C [37], it is surprising that neither ail1 _Pa nor pagC _Pa participated in the human serum resistance. It is tempting to speculate that other P. asymbiotica proteins could be involved in serum resistance. Finally, when expressed in E. coli, only ail2 _Pl and pagC _Pl appear to play a role in human serum resistance whereas P. luminescens TT01 was not described as a clinical isolate. One hypothesis could be that Ail2_Pl and PagC_Pl could play a role in resistance toward components of the insect blood, the hemolymph. Thus, we tested the resistance of recombinant E. coli bacteria toward different AMPs, a key component of the insect humoral insect immunity, though without any success. In pathogenic Yersinia species, Ail proteins bind substrates such as the host cell extracellular matrix proteins, fibronectin and laminin, as well as the complement regulatory proteins C4bp and factor H [72]. Therefore, in insects, Ail2_Pl and PagC_Pl may interact with similar hemolymph components yet to be identified.

What is the evolutionary significance of the two Ail proteins in P. luminescens TT01, Ail1_Pl and Ail2_Pl?

In Y. pestis KIM, four genes encoding the proteins from the Ail/PagC/OmpX family were identified including one OmpX protein (y1682) and three Ail proteins (y2446, y2034 and y1324). These three Ail proteins are phylogenetically distant and their corresponding genes are not adjacent on the Y. pestis KIM genome [73]. In P. luminescens TT01, we propose that the two ail genes result from a recent tandem duplication (see above). The genomic redundancy in prokaryotes can be explained as a consequence of three selective processes, (i) elevated protein dosage (identical and duplicated genes), (ii) protein diversification (divergent paralogs) and (iii) adaptation to environmental variations (ecoparalogs of intermediate divergence) [74]. The intermediate aminoacids identity between Ail1_Pl and Ail2_Pl suggests a case of ecoparalogs. Three clues also argue in favour of this hypothesis. First, as already described with ecoparalogs predicted to be on the outer membrane or in the periplasmic space where the environment influence is important for protein function and stability [74], Ail1_Pl and Ail2_Pl differ by their isoelectric point values (respectively, 9.0 and 7.0). Second, a usual role identified in the Ail/PagC/OmpX family, the resistance to human serum, was only conserved for one protein, Ail2_Pl. Finally, regulation of the expression of ail1 _Pl and ail2 _Pl genes is obviously different and in the case of ail1 _Pl, we showed that the influence of external environment fluctuation by the way of magnesium concentration is important. All together, the existence of these two ecoparalogs is highly suggestive of an adaptation to multiple niches (at least, insect and nematode) in response to external fluctuation.

Supporting Information

Figure S1

Acetyl phosphate can phosphorylate PhoP-His in vitro. To evaluate the efficiency of PhoP-His phosphorylation by acetyl phosphate, precast 12.5% polyacrylamide Mn²⁺-Phos-tag gel (Wako Chemicals, Japon) was used. When present the Phos-tag and its associated divalent cation Mn²⁺ form a complex with phosphorylated forms and retard protein migration. Four micrograms of purified PhoP-His were incubated in vitro either with 50 mM acetyl phosphate (lane 2) or without acetyl phosphate (lane 1) using the buffer described for EMSA protocol. SDS-PAGE was performed using standard protocols and gel was run at 4°C and 150 V to avoid phosphate hydrolysis until 10 min after loading blue sorting. Thereafter, the gel was incubated during 10 min in the Cathode buffer (40 mM 6-amino caproic acid, 25 mM Tris, 20% methanol) supplemented with 1 mM EDTA in order to quench Mn²⁺ cations and 20 min in the cathode buffer without EDTA to remove excess of EDTA. The gel was stained with coomassie brilliant blue. In absence of acetyl phosphate, only unphosphorylated PhoP-His is found (lane 1) whereas phosphorylated PhoP-His and dimerization are observed in presence of acetyl phosphate (lane 2) showing that acetyl phosphate can phosphorylate PhoP-His in vitro.

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Table S1

Primers used in this study.

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Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by grant n ° 2011-1333-03 from National Institute of Agronomical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.