Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2020 Jan 14;15(1):e0223033. doi: 10.1371/journal.pone.0223033

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus

Connor L Brown 1,2, Emily Garner 1,3, Guillaume Jospin 4, David A Coil 4, David O Schwake 5, Jonathan A Eisen 4,6, Biswarup Mukhopadhyay 2, Amy J Pruden 1,*
Editor: Daniel E Voth7
PMCID: PMC6959600  PMID: 31935215

Abstract

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1–3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.

Introduction

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and mediate the translocation of protein or peptide substrates with a broad range of function [1–4]. The core T1SS complex is composed of a dimerized inner membrane ATP-binding-cassette (ABC) transporter protein, a trimerized membrane fusion protein that spans the periplasm, and a trimerized outer membrane protein. The genes encoding the ABC transporter and membrane fusion protein are typically localized together in the genome [5], while the gene encoding the outer membrane protein is usually encoded elsewhere, reflecting the multifunctional nature of this family of proteins [6,7].

Three major classes of T1SSs can be described based on the N-terminal region of the ABC transporter [4,8] (Fig 1). The first class includes bacteriocin transporters, such as the colicin V system in Escherichia coli [10], which encode ABC transporter proteins with N-terminal C-39 peptidase domains that cleave N-terminal regions of nascent substrates during translocation [4] (Fig 1A). In another class, such as the HlyA secretion system in E. coli [11], the ABC transporters possess an N-terminal C-39 peptidase-like domain (CLD), which lacks the catalytic histidine [10] (Fig 1B). A third class of T1SSs are composed of ABC transporters that lack either the C-39 peptidase or CLD. These systems typically secrete smaller substrates, including epimerases and proteases in Azotobacter vinelandi and Pseudomonas aeruginosa, respectively [12,13] (Fig 1C).

Fig 1. Genomic organization and structure of three type 1 secretion systems with corresponding substrates below each system [3, 8, 9].

Fig 1

Open in a new tab

(A) The CvaC T1SS in E. coli. An N-terminal region of CvaC is cleaved during secretion by the C-39 peptidase motifs on CvaB (red diamonds). (B) The HlyA T1SS in E. coli. HlyB has the CLD domain (blue triangles). (C) The AprA T1SS. AprD lacks either the CLD or C-39 peptidase motif. (D) The LapA T1SS in P. fluorescens Pf01. LapA is secreted in a two-step fashion where the retention module (tan circle on the N-terminus of LapA) is cleaved by LapG before release into the extracellular environment.

In Legionella pneumophila, the prototypical T1SS is encoded at the lssXYZABD locus with lssB and lssD encoding the ABC transporter and membrane fusion protein, respectively [14,15]. This complex is responsible for secreting the virulence factor RtxA, which is associated with adherence, pore-formation, cytotoxicity, and entrance into host cells [16,17]. The RtxA T1SS belongs to a subset of the CLD-type T1SSs whose substrates possess an N-terminal retention module. The most well characterized system of this type is the adhesin LapA in Pseudomonas fluorescens strain Pf01 [3,18]. In P. fluorescens, LapA is secreted in a two-step fashion mediated by membrane fusion protein LapC, the ABC transporter LapB, outer membrane protein LapE, and a transglutaminase-like cysteine proteinase (BTLCP) LapG [3,18–20] (Fig 1D). However, no study has detected lssD (a lapE homolog) in any non-pneumophila genome since the discovery of the lssXYZABD locus in 2001 [15,17,21]. These observations have led to the assumption that RtxA is a key virulence determinant unique to L. pneumophila. In the present study, we report the broad distribution of the RtxA T1SS and four putative novel T1SSs throughout the Legionella genus and examine their occurrence among strains of disease-associated Legionella.

Results

Isolation and phylogenetic identification of a novel Legionella taurinensis strain containing four type 1 secretion systems

During a survey of municipal and well waters in Genesee County, Michigan, endemic L. pneumophila were targeted for isolation using standard culture methods, and isolates were subjected to whole genome sequencing [22]. Sequence and phylogenetic analysis revealed four isolates obtained from municipal water sourced from an aquifer to be novel Legionella taurinensis strains [23], a species that did not have a reference genome at that time (S1 File Table A). These genome sequences are deposited at DDBJ/ENA/GenBank under the accession PRJNA450138. While analyzing the genomes of the isolates for virulence factors, the strain was found to possess the lssXYZABD locus believed to be absent from non-pneumophila Legionella spp. [15,17,21,24] in addition to three novel T1SSs with low homology to the RtxA T1SS components.

Organization of the lssBD system in Legionella spp.

In contrast to previous reports [15,21], L. taurinensis was found to possess the entire lssXYZABD locus associated with RtxA secretion with two reading frames (ORFs) (472 base pair and 4.24 kilobase) between lssB and lssA, both of which encode hypothetical proteins (Fig 2A and 2B and S1 File Figs A and B). L. taurinensis and L. pneumophila LssB possess the LapB-type N-terminal CLD (S1 File Fig B, S1 File Table B). L. taurinensis possesses the lssXYZABD locus including a gene encoding an LssD homolog which is 60% identical to L. pneumophila LssD (S1 File Fig A).

Fig 2. Genomic organization of four type 1 secretion systems in the Legionella genus.

Fig 2

Open in a new tab

(A) Organization of the lssXYZABD locus in L. pneumophila. (B) The lssXYZABD locus in L. moravica DSM1924.(C) The lssXYZABD locus in L. taurinensis encodes homologs of all members of the lssXYZABD locus, but the operon has two inserted ORFs. The lssXYZABD locus in L. moravica DSM1924 (D) The lrpss T1SS found in L. taurinensis encodes a putative substrate at the LRPSS locus. (E and F) The lb1ss and lb2ss locus in L. taurinensis. White triangles are hypothetical proteins. (B-C) Percentages are percentage identity to L. pneumophila Philadelphia amino acid sequences. GenBank accession numbers are provided (S1 File Table B).

Following this observation, we examined 45 Legionella species whole genome sequences for the RtxA T1SS by comparing amino acid sequences of L. pneumophila LssB and LssD against the predicted proteomes of Legionella spp. using blastp (S1 File Table B). A species was considered to encode the RtxA T1SS if its genome encoded homologs of LssD and LssB with amino acid sequence ≥ 40% identity (S1 File Table B) and if the ABC transporter was monophyletic with L. pneumophila LssB (Fig 3, S1 File Figure C). These two proteins were chosen as they constitute two-thirds of the core components of a T1SS, the membrane fusion protein and ABC transporter. This analysis revealed that nearly half of species’ genomes examined (n = 19) encoded LssB and LssD homologs. Of these 19, several possessed the entire lssXYZABD locus, while others lacked some components of the locus or possessed additional ORFs within the cluster. In all in which the RtxA T1SS was detected, lssB and lssD were encoded adjacent to one another in the genome.

Fig 3. Unrooted maximum likelihood tree for trimmed sequences of 187 T1SS ABC transporters including the five Legionella systems.

Fig 3

Open in a new tab

Bootstrap values displayed as percentages. An un-modified tree used to classify the Legionella secretion systems, and the Legionella sequences, are provided (S1 File Fig E and Table B). Pink boxes indicate an association with disease.

Legionella moravica strain DSM19234 was found to possess the entire lssXYZABD locus with corresponding proteins 60–86% identical to those encoded by L. pneumophila Philadelphia 1 (Fig 2A and 2C, Table 1). This is noteworthy as a previous bioinformatic investigation reported the absence of this locus in L. moravica DSM19234 in its entirety [24]. This study reports using the tblastn algorithm (release 2.2.25) to compare the protein sequences of the locus with nucleotide sequences of Legionella spp. Repeating this approach using tblastn, L. moravica DSM19234 whole genome sequences (taxid: 1122165), were found to possess all genes of the lssXYZABD locus with identity values ranging from 56.28%-84.13% (Table 1). Therefore, whole genome sequences of L. moravica DSM19234 were found to possess genes encoding the RtxA T1SS. Last, we additionally confirmed the presence of an RtxA-like substrate in a subset of genomes, including those of L. taurinensis Genessee01 and L. moravica species, by identifying T1SS substrates with a putative N-terminal di-alanine retention module specific to LapA/RtxA class substrates (S1 File Figure D) [3,18] through tblastn searches against their whole genome sequences.

Table 1. L. moravica DSM19234 posesses the RtxA T1SS. Components were detected by tblastn. Identity values are adjusted for query cover.

Query Protein Accession ID Subject tblastn Identity
LssB CAD90959.1 L. moravica DSM19234 76.10%
LssD CAD90958.1 L. moravica DSM19234 84.13%
LssA CAD90960.1 L. moravica DSM19234 83.95%
LssZ CAD90961.1 L. moravica DSM19234 66.50%
LssY CAD90962.1 L. moravica DSM19234 56.28%
LssX CAD90963.1 L. moravica DSM19234 77.27%
Open in a new tab

Legionella longbeachae is the second most commonly reported causative agent of Legionnaires’ Disease, especially in New Zealand and Japan where reported cases of L. longbeachae infection occur about as often as cases of L. pneumophila infection [25]. Draft genome sequences of L. longbeachae strains F1157CHC and FDAARGOS-201 include lssB and lssD homologs with interrupting stop codons within the ORFs (S1 File Table B) indicating that these genes are non-functional or contain sequencing or annotation errors. A tblastn search with LssB and LssD from L. longbeachae strain FDAARGOS-201 as queries did not identify respective homologs in the genome of L. longbeachae strain NSW150 (E-value cut-off of 1E-5). Therefore, the only finished genome of L. longbeachae, strain NSW150, lacks homologs of both lssB and lssD. Further, all three genomes of L. longbeachae were examined for homologs of the L. pneumophila Philadelphia 1 RtxA (lpg0645) and LapE (lpg00827) using tblastn and neither were detected (E-value cut-off 1E-5).

L. taurinensis encodes three novel type 1 secretion systems

Analysis of the diversity of T1SSs across the Legionella genus has not been previously reported. Scanning the genome of L. taurinensis Genessee01 for additional lssB and lssD family genes revealed the presence of three putative novel T1SSs. One is composed of a 438 amino acid (aa) membrane fusion protein and 587 aa ABC transporter which were encoded adjacent to one another in the genome (Fig 2D). This ABC transporter lacks either the C-39 peptidase motif or CLD (S1 File Figs E and F) and a protein phylogeny of the ABC transporter indicated a relationship with T1SSs that secrete substrates of diverse function (Fig 3, S1 File Fig C). Additionally, the genomic locus in L. taurinensis that encodes the ABC transporter and membrane fusion protein also encodes a putative substrate with hemolysin type calcium binding motifs commonly associated with T1SS substrates. Because of this, the name Legionella repeat protein secretion system (LRPSS) is proposed for this T1SS.

L. taurinensis additionally encodes two putative bacteriocin transport T1SSs. One is a colicin V-like T1SS (Fig 2F, Fig 3), for which the name Legionella bacteriocin 1 secretion system (LB1SS) is proposed. The second putative bacteriocin transporter locus encodes two ABC transporter proteins at the same genomic locus, (Fig 2E and Fig 3, S1 File Fig E and S1 File Table B), with one of these (PUT41641.1) including a C-39 peptidase motif at the N-terminus. This ABC transporter was found to be phylogenetically related to a nitrile hydratase leader peptide (NHLP)-type bacteriocin ABC transporter in Nostoc sp. PCC 7120 [26] (Fig 3, S1 File Figs C and E). For this system, the name Legionella bacteriocin 2 secretion system (LB2SS) is proposed. Additionally, searching the genomes of the Legionella spp. for homologs of the LB2SS system genes revealed a similar NHLP-type bacteriocin T1SS. This was suggested by protein phylogeny (Fig 3, S1 File Fig C) to be a different T1SS or a highly diverged variant of the LB2SS. The name Legionella bacteriocin 3 secretion system (LB3SS) is proposed for this system.

Distribution of T1SSs in the Legionella genus and their association with disease

We found that 19 of 45 (42%) Legionella spp. examined encode the RtxA T1SS (Fig 4). Of these 19, 7 (37%) are known pathogens [27]. Relatively few (10 of 45, 22%) encode the LRPSS system, while 16 of the 45 species (35%) encode the LB1SS; 9 of 45 species (20%) encode the LB2SS, and 11 of 45 species (24%) encode the LB3SS. Collectively, only eight species lacked any of the T1SSs described in this paper. We performed significance testing because previous studies have qualitatively evaluated differences in the distribution of T1SSs in relation to perceived virulence or propensity for intracellular growth. Two proportion Z-tests with continuity correction were performed to compare the prevalence of the RtxA T1SS within strains who have never been isolated from patients and those which have (Fig 4). No significant differences were detected (p = 0.4), but the sample sizes are small (n = 19). Thus, there was no detected difference in the proportions of disease and non-disease associated species that possessed any of the T1SSs to the extent of our current knowledge of pathogenicity in these Legionella species. However, our observation brings up the possibility that some of the Legionella species that thus far have not been isolated from patients are inefficient colonizers of built environments that are common exposure routes and, therefore, remain classified as non-pathogenic. Additionally, increasing the number of genomes analyzed could impact the results of the present analysis.

Fig 4. Nucleotide whole genome sequence FastTree tree predicted using a core set of marker genes predicted by PhyloSift of Legionella spp. overlaid with the distribution of T1SSs.

Fig 4

Open in a new tab

Pink color indicates species that have been associated with human infection [27].

Discussion

Multiple investigations report the restriction of the lssBD/RtxA system to L. pneumophila. Two studies did not detect lssD in non-pneumophila Legionella using Southern blotting and DNA macroarrays, respectively [15,21], while one study detected lssB in all non-pneumophila Legionella tested (n = 10) [15]. Incidentally, the presence of rtxA (determined by Southern blotting) in Legionella feeleii has been reported, but the study did not test for the presence of the T1SS components [17]. In retrospect, it may be unsurprising that several early studies did not detect the presence of lssD in non-pneumophila Legionella spp. given their reliance on DNA-DNA hybridization methods [15,16,21]. This component may be more variable due to the nature of its interactions with a rapidly evolving substrate, and therefore methods relying on the nucleotide sequence of L. pneumophila lssD as probe could plausibly cause false-negatives of this nature. On the other hand, Qin et al 2017 bioinformatically examined non-pneumophila Legionella genomes (n = 21) for the lssXYZABD components [24] and reported the absence of the lssXYZABD locus from all strains tested, including L. moravica strain DSM19234. Further, this study reported that Legionella lacking the lssXYZABD locus displayed reduced intracellular multiplication relative to L. pneumophila strains which possess the T1SS [24]. Thus, the emergent consensus model has been that the RtxA system is an important conserved genetic virulence determinant unique to L. pneumophila, despite the absence of additional experimental investigations of its function in animal models since the discovery of the enhanced entry locus in 2001 [15]. In contrast, we found bioinformatic evidence that homologs of the RtxA T1SS and the four novel T1SSs are prevalent throughout the genus, even among species not presently known to cause disease.

The 2014–2015 Center for Disease Control (CDC) Legionnaires’ Disease Surveillance Summary Report [28] documents the species isolated in culture confirmed Legionnaires’ Disease cases in the United States. Of 307 culture-confirmed cases in 2014–2015, 186 (60.6%) were caused by L. pneumophila, 3 (1%) by L. longbeachae, 4 (1.3%) by L. micdadei, 1 (0.3%) by L. bozemanii and 3 (1%) by L. feeleii. One hundred and eight (35%) were due to unreported species of Legionella. Of these, only L. feeleii and possibly some strains of L. longbeachae possess the RtxA T1SS. Additionally, Gomez-Valero et al 2019 measured replicative capacity of different Legionella spp. in THP-1 macrophages and found that L. jordanis, L. taurinensis, L. jamestowniensis, L parisiensis, L brunensis, and L. bozemanii displayed replicative capacity similar or superior to that of L. pneumophila [29]. Of these species, L. taurinensis, L. brunensis, and L. jamestowniensis, (three of six (50%)) encode the RtxA T1SS. Therefore, while many Legionella spp. possess the T1SS responsible for RtxA translocation, our results indicate that the system alone does not predict propensity for disease association or intracellular replication in macrophages.

In conclusion, we report that the RtxA T1SS and four novel T1SSs discovered in L. taurinensis are broadly distributed among Legionella species and provide the first extensive survey and classification of T1SSs in the Legionella genus. Sequence and phylogenetic analysis indicate that Legionella spp. encode T1SSs that facilitate diverse functions, including bacteriocin transport and protein secretion. Importantly, no relationship was detected between the possession of the RtxA T1SS with disease association, drawing into question the nature of RtxA and its T1SS as a genetic virulence determinant.

Methods

Determining epidemiological features of Legionella spp.

We considered a Legionella species to be “disease-associated” based on whether not any strain of the species has ever been isolated from a patient. To determine this, we referenced the online resource at https://www.specialpathogenslab.com/legionella-species.php (accessed late 2018—mid 2019) [27] and performed a literature review on PubMed Central to confirm accuracy.

Sampling and culture methods

Two one-liter samples of water were collected from one tap of a school located in Genesee County serviced by a groundwater well in March of 2016 into sterile polypropylene bottles (Nalgene, Rochester, NY) with 24 mg of sodium thiosulfate per liter added to quench chlorine for preservation prior to microbial analysis. Samples were collected with the permission of the community school board supervisor. Within 24 hours, samples were filter-concentrated onto a sterile 0.22 μm pore size mixed-cellulose ester membrane (Millipore, Billerica, MA) and resuspended in 5 mL sterile tap water prior to culturing according to International Standards of Organization (ISO) methods [30] for the recovery of L. pneumophila on highly selective buffered charcoal yeast extract media with supplemented glycine, polymyxin B sulfate, cycloheximide and vancomycin.

Whole genome sequencing, assembly, and annotation

DNA was extracted using FastDNA SPIN Kit (MP Biomedicals, Solon, OH) according to manufacturer instructions. Purified DNA was quantified via a Qubit 2.0 Fluorometer (Thermo Fisher, Waltham, MA) and analyzed via gel electrophoresis to verify DNA integrity. Sequencing was conducted by MicrobesNG (Birmingham, United Kingdom) on a MiSeq platform (Illumina, San Diego, CA) with 2 x 250 bp paired-end reads. Libraries were constructed using a modified Nextera DNA library preparation kit (Illumina, San Diego, CA). Reads were trimmed using Trimmomatic [31] and de novo assemblies were generated using SPAdes [32]. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PRJNA453403. The version described in this paper is version PRJNA453403.

Sequence and phylogenetic analysis

The initial bioinformatic analysis that detected the T1SSs was performed using the integrated microbial genomes database and comparative analysis system (IMG) from the Joint Genome Institute [33].

To detect the RtxA T1SS components, L. pneumophila LssB and LssD amino acid sequences were compared with the protein sequences of Legionella spp. using blastp. For the four novel T1SSs, L. taurinensis amino acid sequences were used as query. A BLAST result was used to support a gene name when the amino acid sequence had overall identity of ≥40% (adjusted for incomplete query cover) with one of the query sequences. This criterion was used for all genes, except lb1ssD, for which many species displayed <40% amino acid homology relative to L. taurinensis lb1ssD (S1 File Table B). Despite this, these genes were consistently found to be co-localized with lb1ssB homologs with ≥40% homology to L. taurinensis (for instance, WP_012979428.1/WP_012979429.1 in L. longbeachae strain NSW150). The 40% identity cut-off is used by the Enzyme Commission to establish functional conservation between similar proteins [34,35]. Further, Qin et al. 2017 used the 40% identity cut-off previously to investigate the distribution of secretion systems in Legionella species [24]. Last, protein phylogeny of the ABC transporters was inferred to validate the results suggested by the blastp results. This analysis resulted in the renaming of several T1SSs which were near 40% homologous (S1 File Table B). Legionella T1SS sequences with suggested names based on the results of the protein phylogeny and the sequence identity analysis are compiled (S1 File Table B).

For the maximum likelihood (ML) tree, 187 amino acid sequences of T1SS ABC transporters were used. The sequences of previously described non-Legionella T1SS ABC transporters were chosen based on a review of the literature, especially [3,4,26]. These sequences were then compared with the non-redundant protein database [36] using blastp and the ten best hits for each non-Legionella T1SS ABC transporter were collected. These sequences and the Legionella sequences were then aligned using MUSCLE v3.8.31 [37] with default settings. The aligned sequences were then trimmed using the heuristic method for trimAl, which resulted in a 482 amino acid aligned region (available in online materials) [38]. Maximum likelihood trees for the trimmed sequence alignments were created using the RAxML webserver [39] with default settings including the GAMMA model of rate heterogeneity, the LG amino acid substitution matrix, and automatic bootstrapping (bootstopping cut-off = 0.03). The most likely tree was overlaid with bipartition values of 200 bootstrap replicates at the command line using RAxMLHPC v8.2.4. The tree constructed from the trimmed sequences is displayed in Fig 3.

Whole genome sequence tree

Reference genomes from 45 Legionella species were downloaded from NCBI (full list and sequences available at https://doi.org/10.6084/m9.figshare.9162152.v1) A set of core marker genes were identified using PhyloSift [40] and hmmer [41] to build a multiple sequence alignment. This alignment was used by FastTree [42] to generate a phylogenetic tree.

Availability of data and material

All data including all Legionella sequences identified in the present study are provided in the paper and its supplementary materials, and may be accessed online through FigShare at doi:10.6084/m9.figshare.9162161.v1, doi:10.6084/m9.figshare.9162146.v1, doi:10.6084/m9.figshare.9162152.v1, doi:10.6084/m9.figshare.9162161.v1. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PRJNA450138. The version described in this paper is version PRJNA450138.

Supporting information

S1 File. Supplementary information.

Supplementary figures and tables. PONE-D-19-25375R1_Supplementary_Information.

(DOCX)

Click here for additional data file. (6MB, docx)

Acknowledgments

The authors acknowledge and appreciate the time and support provided by Drs. William Rhoads and Marc Edwards.

Data Availability

All data including all Legionella sequences identified in the present study are provided in the paper and its supplementary materials, and may be accessed online through FigShare at doi:10.6084/m9.figshare.9162161.v1, doi:10.6084/m9.figshare.9162146.v1, doi:10.6084/m9.figshare.9162152.v1, doi:10.6084/m9.figshare.9162161.v1. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PRJNA450138. The version described in this paper is version PRJNA450138.

Funding Statement

This study was supported by the US National Science Foundation via RAPID Award no. 1556258: Recipient: A.P.; a Graduate Research Fellowship Program Grant (DGE 0822220): Recipient: E.G.; Supplementary funding associated with award #1336650: Recipient: A.P. Additional support was provided by the Alfred P. Sloan Foundation Microbiology of the Built Environment program (https://sloan.org/programs/research/microbiology-of-the-built-environment#apply): Recipient: E.G.; the American Water Works Association Abel Wolman Fellowship: Recipient: E.G.; and the Institute for Critical Technology and Applied Science and Engineering of the Exposome Center (https://ictas.vt.edu/) at Virginia Polytechnic Institute and State University: Recipient: A.P. In addition to the funding sources noted, colleague Marc A. Edwards contributed discretionary research funding to support the sequencing study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Thomas S, Holland IB, Schmitt L. The Type 1 secretion pathway—The hemolysin system and beyond. Biochim Biophys Acta—Mol Cell Res. 2014;1843: 1629–1641. 10.1016/J.BBAMCR.2013.09.017 [DOI] [PubMed] [Google Scholar]
  • 2.Holland IB, Schmitt L, Young J. Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review). Mol Membr Biol. 22: 29–39. Available: http://www.ncbi.nlm.nih.gov/pubmed/16092522 10.1080/09687860500042013 [DOI] [PubMed] [Google Scholar]
  • 3.Smith TJ, Sondermann H, O’Toole GA. Type 1 Does the Two-Step: Type 1 Secretion Substrates with a Functional Periplasmic Intermediate. J Bacteriol. 2018;200: JB.00168–18. 10.1128/JB.00168-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kanonenberg K, Schwarz CKW, Schmitt L. Type I secretion systems–a story of appendices. Res Microbiol. 2013;164: 596–604. 10.1016/j.resmic.2013.03.011 [DOI] [PubMed] [Google Scholar]
  • 5.Abby SS, Cury J, Guglielmini J, Néron B, Touchon M, Rocha EPC. Identification of protein secretion systems in bacterial genomes. Sci Rep. 2016;6: 23080 10.1038/srep23080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ferhat M, Atlan D, Vianney A, Lazzaroni J-C, Doublet P, Gilbert C. The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion. Bereswill S, editor. PLoS One. 2009;4: e7732 10.1371/journal.pone.0007732 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Koronakis V, Eswaran J, Hughes C. Structure and Function of TolC: The Bacterial Exit Duct for Proteins and Drugs. Annu Rev Biochem. 2004;73: 467–489. 10.1146/annurev.biochem.73.011303.074104 [DOI] [PubMed] [Google Scholar]
  • 8.Thomas S, Holland IB, Schmitt L. The Type 1 secretion pathway—The hemolysin system and beyond. Biochim Biophys Acta—Mol Cell Res. 2014;1843: 1629–1641. 10.1016/J.BBAMCR.2013.09.017 [DOI] [PubMed] [Google Scholar]
  • 9.Guzzo J, Pages J-M, Duong F, Lazdunski A, Murgierl M. Pseudomonas aeruginosa Alkaline Protease: Evidence for Secretion Genes and Study of Secretion Mechanism. J Bacteriol. 1991. Available: http://jb.asm.org/ [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lecher J, Schwarz CKW, Stoldt M, Smits SHJ, Willbold D, Schmitt L. An RTX Transporter Tethers Its Unfolded Substrate during Secretion via a Unique N-Terminal Domain. Structure. 2012;20: 1778–1787. 10.1016/j.str.2012.08.005 [DOI] [PubMed] [Google Scholar]
  • 11.Bohach GA, Snyder IS. Chemical and immunological analysis of the complex structure of Escherichia coli alpha-hemolysin. J Bacteriol. 1985;164: 1071–80. Available: http://www.ncbi.nlm.nih.gov/pubmed/3905764 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Czjzek M, Arnoux P, Haser R, Izadi N, Lecroisey A, Delepierre M, et al. The crystal structure of HasA, a hemophore secreted by Serratia marcescens. Nat Struct Biol. 1999;6: 516–520. 10.1038/9281 [DOI] [PubMed] [Google Scholar]
  • 13.Gimmestad M, Steigedal M, Ertesvag H, Moreno S, Christensen BE, Espin G, et al. Identification and Characterization of an Azotobacter vinelandii Type I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases. J Bacteriol. 2006;188: 5551–5560. 10.1128/JB.00236-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fuche F, Vianney A, Andrea C, Doublet P, Gilbert C. Functional Type 1 Secretion System Involved in Legionella pneumophila Virulence. Parkinson JS, editor. J Bacteriol. 2015;197: 563–571. 10.1128/JB.02164-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Jacobi S, Heuner K. Description of a putative type I secretion system in Legionella pneumophila. Int J Med Microbiol. 2003;293: 349–358. 10.1078/1438-4221-00276 [DOI] [PubMed] [Google Scholar]
  • 16.Cirillo SLG, Bermudez LE, El-Etr SH, Duhamel GE, Cirillo JD. Legionella pneumophila Entry Gene rtxA Is Involved in Virulence. Infect Immun. 2001;69: 508–517. 10.1128/IAI.69.1.508-517.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Littman M, Cirillo JD, Yan L, Samrakandi MM, Cirillo SLG. Role of the Legionella pneumophila rtxA gene in amoebae. Microbiology. 2002;148: 1667–1677. 10.1099/00221287-148-6-1667 [DOI] [PubMed] [Google Scholar]
  • 18.Smith TJ, Font ME, Kelly CM, Sondermann H, O’toole GA 4. An N-terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas 1 fluorescens at the Cell Surface: A Novel Sub-family of Type I Secretion Systems 2 3 Downloaded from. J Bacteriol. 2018. [cited 13 Nov 2018]. 10.1128/JB.00734-17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Chatterjee D, Boyd CD, O’Toole GA, Sondermann H. Structural characterization of a conserved, calcium-dependent periplasmic protease from Legionella pneumophila. J Bacteriol. 2012;194: 4415–25. 10.1128/JB.00640-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ginalski K, Kinch L, Rychlewski L, Grishin N V. BTLCP proteins: a novel family of bacterial transglutaminase-like cysteine proteinases. Trends Biochem Sci. 2004;29: 392–395. 10.1016/j.tibs.2004.06.001 [DOI] [PubMed] [Google Scholar]
  • 21.Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Etienne J, et al. Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. Genome Res. 2008;18: 431–41. 10.1101/gr.7229808 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Garner E, Brown C, Schwake DO, Rhoads WJ, Arango-Argoty G, Zhang L, et al. Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016. Emerg Infect Dis. 2019;25 10.3201/eid2511.181032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lo Presti F, Riffard S, Meugnier H, Reyrolle M, Lasne Y, Grimont PAD, et al. Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis. Int J Syst Bacteriol. 1999;49: 397–403. 10.1099/00207713-49-2-397 [DOI] [PubMed] [Google Scholar]
  • 24.Qin T, Zhou H, Ren H, Liu W. Distribution of Secretion Systems in the Genus Legionella and Its Correlation with Pathogenicity. Front Microbiol. 2017;8: 388 10.3389/fmicb.2017.00388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Whiley H, Bentham R. Legionella longbeachae and legionellosis. Emerg Infect Dis. 2011;17: 579–83. 10.3201/eid1704.100446 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Haft DH, Basu MK, Mitchell DA. Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family. BMC Biol. 2010;8: 70 10.1186/1741-7007-8-70 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Special Pathogens Legionella Species Index. Available: https://www.specialpathogenslab.com/legionella-species.php
  • 28.Shah Albert Barskey Alison Binder Chris Edens Sooji Lee Jessica Smith Stephanie Schrag Cynthia Whitney Laura Cooley P. Legionnaires’ Disease Surveillance Summary Report, United States-2014-2015. 2014. Available: https://www.cdc.gov/legionella/health-depts/surv-reporting/2014-15-surv-report-508.pdf [Google Scholar]
  • 29.Gomez-Valero L, Rusniok C, Carson D, Mondino S, Pérez-Cobas AE, Rolando M, et al. More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells. Proc Natl Acad Sci U S A. 2019;116: 2265–2273. 10.1073/pnas.1808016116 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.ISO 11731:2017(en), Water quality—Enumeration of Legionella. [cited 12 Sep 2018]. Available: https://www.iso.org/obp/ui/#iso:std:iso:11731:ed-2:v1:en
  • 31.Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30: 2114–2120. 10.1093/bioinformatics/btu170 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012;19: 455–477. 10.1089/cmb.2012.0021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Grechkin Y, et al. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 2012;40: D115–22. 10.1093/nar/gkr1044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wilson CA, Kreychman J, Gerstein M. Assessing annotation transfer for genomics: Quantifying the relations between protein sequence, structure and function through traditional and probabilistic scores. J Mol Biol. 2000;297: 233–249. 10.1006/jmbi.2000.3550 [DOI] [PubMed] [Google Scholar]
  • 35.Todd AE, Orengo CA, Thornton JM. Evolution of function in protein superfamilies, from a structural perspective. J Mol Biol. 2001;307: 1113–43. 10.1006/jmbi.2001.4513 [DOI] [PubMed] [Google Scholar]
  • 36.Pruitt KD, Tatusova T, Maglott DR. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005;33: D501–4. 10.1093/nar/gki025 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32: 1792–1797. 10.1093/nar/gkh340 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25: 1972–3. 10.1093/bioinformatics/btp348 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kozlov A, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. bioRxiv. 2018; 447110 10.1101/447110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Darling AE, Jospin G, Lowe E, Matsen FA, Bik HM, Eisen JA. PhyloSift: phylogenetic analysis of genomes and metagenomes. PeerJ. 2014;2: e243 10.7717/peerj.243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14: 755–763. 10.1093/bioinformatics/14.9.755 [DOI] [PubMed] [Google Scholar]
  • 42.Price MN, Dehal PS, Arkin AP. FastTree 2 –Approximately Maximum-Likelihood Trees for Large Alignments. Poon AFY, editor. PLoS One. 2010;5: e9490 10.1371/journal.pone.0009490 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Daniel E Voth

14 Oct 2019

PONE-D-19-25375

Whole genome sequence analysis reveals broad distribution of the RtxA type 1 secretion system and four novel type 1 secretion systems throughout the Legionella genus

PLOS ONE

Dear Dr. Pruden,

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.

We would appreciate receiving your revised manuscript by Nov 28 2019 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,

Daniel E. Voth, Ph.D.

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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. 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 (if provided):

[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: Yes

**********

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: Yes

**********

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: This is a well organized report on the prevalence of T1SS in isolates of Legionella. Genome sequence was generated from specimen isolated in Flint, Michigan groundwater. Four isolates were described with one novel genome revealed.

The emphasis of the study examines distribution of RtxA-like type 1 secretion systems, with the important conclusion that this system is not limited to distribution among human pathogenic strains of Legionella as previously proposed.

The majority of the paper reads like a comprehensive review of classes of T1SS, focusing on classifying new secretion systems in L.taurinensis.

The use of 'virulence factor' throughout to describe the RtxA locus in L.pneumophila is somewhat generous. When one examines the papers that this study cites, nearly all phenotypes said to be associated with the rtxA locus present as less than two-fold differences in most experiments. of the three enhanced entry loci described in reference 15, the rtxA-containing locus has not been pursued since these publications. Other enhanced entry (enh) have been explored.

The wording in the abstract (lines 41-42) 'conserved virulence factor in L.pneumophila' doesn't convey the message correctly. It should be changed to 'virulence factor exclusive to L.pneumophila'.

Reviewer #2: This manuscript describes an in silico analysis of predicted type I secretion system (T1SS) distribution in multiple Legionella species. Brown et al. describe isolation of four new strains of Legionella taurinensis from Genesee county Michigan. Upon genome sequencing of these strains, the authors identified four putative T1SSs, one with homology to the RtxA T1SS and three putative novel T1SSs. They also probed the genomes of 45 additional Legionella species for the presence of T1SSs using amino acid sequence homology as an indicator. They found predicted RtxA and novel LB2SS T1SS in previously sequenced Legionella species and determined that distribution of T1SS is not a predictor of virulence. This is an interesting study that re-evaluates the distribution of T1SS within Legionella.

Comments:

The title should be changed to ..”four putative novel type 1 secretion…”. There is no experimental evidence demonstrating that these are functional type 1 secretion systems.

Table 1 is missing from the submission files.

As there is a virulence defect observed for loss of the rtxA T1SS in L. pneumophila (Cirillo et al, 2001), the authors should rephrase the sentence in lines 56-57. The RtxA system could be a virulence determinant, it’s just not sufficient if other virulence factors are not present/active, etc. Another example is that the Dot/Icm secretion system is present in all sequences strains of Legionella but they're not all pathogenic to humans.

Line 96: add the word “potential” or “putative” in front of “novel T1SS”

Line 103: Remove “(Garner et al. in review)” unless the study has been published.

Figure 2: B and C are switched in the figure legend and text.

Out of curiosity, any idea what the hypothetical genes in the L. taurinensis lssXYZABD locus could be doing?

Line 130: How did you decide on a 40% identity cut-off? In your predictions, was the rtxA substrate homolog also identified?

Line 147: The blastp algorithm was used here, correct? If so, please specify.

Line 200: Please reference the sentence starting “Of these 19,…”.

Line 216: Please reference the statement ending in “…Legionella tested (n = 10)”.

Line 229: please change the language in the sentence starting with “In contrast,…” to emphasize that presence if these systems is predicted based on homology.

Figure 4 is of poor quality (pixelated), please improve the quality of this image to increase its readability. Also, what does the light pink color indicate?

Please also keep in mind that just because a Legionella species has not been isolated from a patient, it does not have potential to cause disease. It is likely that some species may be less able to colonize sources of human infection.

**********

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: No

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 Jan 14;15(1):e0223033. doi: 10.1371/journal.pone.0223033.r002

Author response to Decision Letter 0

10 Dec 2019

PONE-D-19-25375

Whole genome sequence analysis reveals broad distribution of the RtxA type 1 secretion system and four novel type 1 secretion systems throughout the Legionella genus

PLOS ONE

Dear Dr. Pruden,

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.

We would appreciate receiving your revised manuscript by Nov 28 2019 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,

Daniel E. Voth, Ph.D.

Academic Editor

PLOS ONE

[Note: Line references refer to the revised version of the manuscript, rather than the version showing tracked changes.]

Reviewers’ Comments to Authors:

Reviewer #1: This is a well-organized report on the prevalence of T1SS in isolates of Legionella. Genome sequence was generated from specimen isolated in Flint, Michigan groundwater. Four isolates were described with one novel genome revealed. The emphasis of the study examines distribution of RtxA-like type 1 secretion systems, with the important conclusion that this system is not limited to distribution among human pathogenic strains of Legionella as previously proposed. The majority of the paper reads like a comprehensive review of classes of T1SS, focusing on classifying new secretion systems in L.taurinensis.

1. The use of 'virulence factor' throughout to describe the RtxA locus in L. pneumophila is somewhat generous. When one examines the papers that this study cites, nearly all phenotypes said to be associated with the rtxA locus present as less than two-fold differences in most experiments. of the three enhanced entry loci described in reference 15, the rtxA-containing locus has not been pursued since these publications. Other enhanced entry (enh) have been explored.

We agree that the nature of RtxA as a virulence factor is uncertain. However, several experimental studies since Cirillo et al. 2001 have supported the role of RtxA and its type 1 secretion system (T1SS) as a possible virulence factor (Cirillo et al. 2002, Fuche et al 2015, Qin et al. 2017) and these observations have been reported in a substantial review (Smith et al. 2019). Of note, Cirillo et al. 2002 examined 20 non-pneumophila Legionella genomes and found that RtxA was restricted to L. pneumophila and L. feeleii; this led the authors to conclude:

“This observation, along with previous studies demonstrating that this gene plays an important role in virulence (Cirillo et al., 2001), has helped to fulfill ‘molecular Koch’s postulates’ for rtxA, supporting the conclusion that this gene plays a role in disease.”

Therefore, we argue that the use of “virulence factor” is justified to reflect the assumptions of the current available literature. However, as these studies were not conducted in animal models, we have updated the first paragraph of the discussion (lines 234-236) to reflect the spirit of the reviewer’s comment:

Thus, the emergent consensus model has been that the RtxA system is an important conserved genetic virulence determinant unique to L. pneumophila, despite the absence of additional experimental investigation of its function in animal models since the discovery of the enhanced entry locus in 2001 [15].

2. The wording in the abstract (lines 41-42) 'conserved virulence factor in L.pneumophila' doesn't convey the message correctly. It should be changed to 'virulence factor exclusive to L. pneumophila'.

The wording has been changed to reflect that it is a virulence factor exclusive to L. pneumophila rather than being conserved (lines 40-41).

Reviewer #2: This manuscript describes an in-silico analysis of predicted type I secretion system (T1SS) distribution in multiple Legionella species. Brown et al. describe isolation of four new strains of Legionella taurinensis from Genesee county Michigan. Upon genome sequencing of these strains, the authors identified four putative T1SSs, one with homology to the RtxA T1SS and three putative novel T1SSs. They also probed the genomes of 45 additional Legionella species for the presence of T1SSs using amino acid sequence homology as an indicator. They found predicted RtxA and novel LB2SS T1SS in previously sequenced Legionella species and determined that distribution of T1SS is not a predictor of virulence. This is an interesting study that re-evaluates the distribution of T1SS within Legionella.

1. The title should be changed to ”four putative novel type 1 secretion…”. There is no experimental evidence demonstrating that these are functional type 1 secretion systems.

The title has been updated to reflect that these systems are putative.

2. Table 1 is missing from the submission files.

This table has been uploaded along with the resubmission.

3. As there is a virulence defect observed for loss of the rtxA T1SS in L. pneumophila (Cirillo et al, 2001), the authors should rephrase the sentence in lines 56-57. The RtxA system could be a virulence determinant, it’s just not sufficient if other virulence factors are not present/active, etc. Another example is that the Dot/Icm secretion system is present in all sequences strains of Legionella but they're not all pathogenic to humans.

We have updated the final line of the abstract (lines 56-57):

“These results draw into question the nature of RtxA and its T1SS as a singular genetic virulence determinant. Future investigations should focus on mechanistic explanations for the association of RtxA with virulence.”

4. Line 96: add the word “potential” or “putative” in front of “novel T1SS”

This line has been updated (line 96).

5. Line 103: Remove “(Garner et al. in review)” unless the study has been published.

This reference has been updated (lines 103).

6. Figure 2: B and C are switched in the figure legend and text.

This has been fixed (lines 111-117).

7. Out of curiosity, any idea what the hypothetical genes in the L. taurinensis lssXYZABD locus could be doing?

These proteins appear to be part of a zeta toxin/antitoxin system. The only homologs of these genes we could find were in Legionella species, and the colocalization of the two genes was preserved in all Legionella genomes we looked at. Analyzing the amino acid sequence of the larger protein, we found evidence of an NTP-ase p-loop domain associated with UDP-kinase/zeta toxin activity (domain IPR010488). On the Integrated Microbial Genomes repository, many of the homologs of the larger protein were annotated as putative zeta toxins.

8. Line 130: How did you decide on a 40% identity cut-off? In your predictions, was the rtxA substrate homolog also identified?

The 40% identity cut-off is used by the Enzyme Commission and, when comparing domains with the same fold, reflects specific functional conservation well (Wilson et al. 2000). Further, Qin et al. 2017 used this cut-off value to identify homologs of secretion system components in Legionella spp.

This text (and appropriate citations) was added to reflect this (lines 298-301):

“The 40% identity cut-off is used by the Enzyme Commission to establish functional conservation between similar proteins (33, 34) Further, Qin et al. 2017 used the 40% identity cut-off previously to investigate the distribution of secretion systems in Legionella species (23).”

9. Line 147: The blastp algorithm was used here, correct? If so, please specify.

This line was updated with the search algorithm used (tblastn) (lines 146-147).

10. Line 200: Please reference the sentence starting “Of these 19,…”.

A reference was added to this line (lines 202).

11. Line 216: Please reference the statement ending in “…Legionella tested (n = 10)”.

A reference was added to this line (line 222).

12. Line 229: please change the language in the sentence starting with “In contrast,…” to emphasize that presence if these systems is predicted based on homology.

This sentence was changed to (lines 236-238):

“In contrast, we found bioinformatic evidence that homologs of the RtxA T1SS and the four novel T1SSs are prevalent throughout the genus, even among species not presently known to cause disease.”

13. Figure 4 is of poor quality (pixelated), please improve the quality of this image to increase its readability. Also, what does the light pink color indicate?

The resolution of Figure 4 has been improved and a sentence was added to the figure legend of Figure 4 (line 197-198):

“Pink color indicates species that have been associated with human infections (27)”

Please note: the version of the figure within the PLOS submission PDF has less resolution than the actual TIF file. To see the actual figure, please use the download link in the top right corner of the submission document.

14. Please also keep in mind that just because a Legionella species has not been isolated from a patient, it does not have potential to cause disease. It is likely that some species may be less able to colonize sources of human infection.

We agree with the reviewer’s comment and have introduced the following changes to the end of the results section (lines 212-217):

“However, our observation brings up the possibility that some of the Legionella species that thus far have not been isolated from patients are inefficient colonizers of built environments that are common exposure routes and, therefore, remain classified as non-pathogenic.”

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (23KB, docx)

Decision Letter 1

Daniel E Voth

18 Dec 2019

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus

PONE-D-19-25375R1

Dear Dr. Pruden,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Daniel E. Voth, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Daniel E Voth

27 Dec 2019

PONE-D-19-25375R1

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus

Dear Dr. Pruden:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Daniel E. Voth

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 File. Supplementary information.

    Supplementary figures and tables. PONE-D-19-25375R1_Supplementary_Information.

    (DOCX)

    Click here for additional data file. (6MB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (23KB, docx)

    Data Availability Statement

    All data including all Legionella sequences identified in the present study are provided in the paper and its supplementary materials, and may be accessed online through FigShare at doi:10.6084/m9.figshare.9162161.v1, doi:10.6084/m9.figshare.9162146.v1, doi:10.6084/m9.figshare.9162152.v1, doi:10.6084/m9.figshare.9162161.v1. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PRJNA450138. The version described in this paper is version PRJNA450138.

    All data including all Legionella sequences identified in the present study are provided in the paper and its supplementary materials, and may be accessed online through FigShare at doi:10.6084/m9.figshare.9162161.v1, doi:10.6084/m9.figshare.9162146.v1, doi:10.6084/m9.figshare.9162152.v1, doi:10.6084/m9.figshare.9162161.v1. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PRJNA450138. The version described in this paper is version PRJNA450138.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES