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

Autoři: Connor L. Brown aff001;  Emily Garner aff001;  Guillaume Jospin aff004;  David A. Coil aff004;  David O. Schwake aff005;  Jonathan A. Eisen aff004;  Biswarup Mukhopadhyay aff002;  Amy J. Pruden aff001
Působiště autorů: Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, United States of America aff001;  Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States of America aff002;  Department of Civil and Environmental Engineering, West Virginia University, Morgantown, WV, United States of America aff003;  Genome Center, University of California, Davis, CA, United States of America aff004;  Department of Natural Sciences, Middle Georgia State University, Macon, GA, United States of America aff005;  Evolution and Ecology, Medical Microbiology and Immunology, University of California, Davis, CA, United States of America aff006
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223033


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.

Klíčová slova:

Genetic loci – Legionella – Outer membrane proteins – Phylogenetic analysis – Proteases – Secretion systems – Sequence alignment – Legionella pneumophila


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. doi: 10.1016/J.BBAMCR.2013.09.017 24129268

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 doi: 10.1080/09687860500042013 16092522

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. doi: 10.1128/JB.00168-18 29866808

4. Kanonenberg K, Schwarz CKW, Schmitt L. Type I secretion systems–a story of appendices. Res Microbiol. 2013;164: 596–604. doi: 10.1016/j.resmic.2013.03.011 23541474

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. doi: 10.1038/srep23080 26979785

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. doi: 10.1371/journal.pone.0007732 19888467

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. doi: 10.1146/annurev.biochem.73.011303.074104 15189150

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. doi: 10.1016/J.BBAMCR.2013.09.017 24129268

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/

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. doi: 10.1016/j.str.2012.08.005 22959622

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 3905764

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. doi: 10.1038/9281 10360351

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. doi: 10.1128/JB.00236-06 16855245

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. doi: 10.1128/JB.02164-14 25422301

15. Jacobi S, Heuner K. Description of a putative type I secretion system in Legionella pneumophila. Int J Med Microbiol. 2003;293: 349–358. doi: 10.1078/1438-4221-00276 14695063

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. doi: 10.1128/IAI.69.1.508-517.2001 11119544

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. doi: 10.1099/00221287-148-6-1667 12055287

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]. doi: 10.1128/JB.00734-17 29437852

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. doi: 10.1128/JB.00640-12 22707706

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. doi: 10.1016/j.tibs.2004.06.001 15288868

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. doi: 10.1101/gr.7229808 18256241

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. doi: 10.3201/eid2511.181032 31625848

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. doi: 10.1099/00207713-49-2-397 10319460

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. doi: 10.3389/fmicb.2017.00388 28352254

25. Whiley H, Bentham R. Legionella longbeachae and legionellosis. Emerg Infect Dis. 2011;17: 579–83. doi: 10.3201/eid1704.100446 21470444

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. doi: 10.1186/1741-7007-8-70 20500830

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

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. doi: 10.1073/pnas.1808016116 30659146

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. doi: 10.1093/bioinformatics/btu170 24695404

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. doi: 10.1089/cmb.2012.0021 22506599

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. doi: 10.1093/nar/gkr1044 22194640

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. doi: 10.1006/jmbi.2000.3550 10704319

35. Todd AE, Orengo CA, Thornton JM. Evolution of function in protein superfamilies, from a structural perspective. J Mol Biol. 2001;307: 1113–43. doi: 10.1006/jmbi.2001.4513 11286560

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. doi: 10.1093/nar/gki025 15608248

37. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32: 1792–1797. doi: 10.1093/nar/gkh340 15034147

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. doi: 10.1093/bioinformatics/btp348 19505945

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. doi: 10.1101/447110

40. Darling AE, Jospin G, Lowe E, Matsen FA, Bik HM, Eisen JA. PhyloSift: phylogenetic analysis of genomes and metagenomes. PeerJ. 2014;2: e243. doi: 10.7717/peerj.243 24482762

41. Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14: 755–763. doi: 10.1093/bioinformatics/14.9.755 9918945

42. Price MN, Dehal PS, Arkin AP. FastTree 2 –Approximately Maximum-Likelihood Trees for Large Alignments. Poon AFY, editor. PLoS One. 2010;5: e9490. doi: 10.1371/journal.pone.0009490 20224823

Článek vyšel v časopise


2020 Číslo 1