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

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.

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

(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.

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

(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).

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

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.

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.

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