Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae

English version

Autoři: Kimberly Filcek ^aff001; Katarina Vielfort ^aff002; Samada Muraleedharan ^aff002; Johan Henriksson ^aff002; Raphael H. Valdivia ^aff003; Patrik M. Bavoil ^aff001; Barbara S. Sixt ^aff002
Působiště autorů: Department of Microbial Pathogenesis, University of Maryland, School of Dentistry, Baltimore, MD, United States of America ^aff001; Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research, Department of Molecular Biology, Umeå University, Umeå, Sweden ^aff002; Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, United States of America ^aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0224324

Souhrn

The ability to introduce targeted genetic modifications in microbial genomes has revolutionized our ability to study the role and mode of action of individual bacterial virulence factors. Although the fastidious lifestyle of obligate intracellular bacterial pathogens poses a technical challenge to such manipulations, the last decade has produced significant advances in our ability to conduct molecular genetic analysis in Chlamydia trachomatis, a major bacterial agent of infertility and blindness. Similar approaches have not been established for the closely related veterinary Chlamydia spp., which cause significant economic damage, as well as rare but potentially life-threatening infections in humans. Here we demonstrate the feasibility of conducting site-specific mutagenesis for disrupting virulence genes in C. caviae, an agent of guinea pig inclusion conjunctivitis that was recently identified as a zoonotic agent in cases of severe community-acquired pneumonia. Using this approach, we generated C. caviae mutants deficient for the secreted effector proteins IncA and SinC. We demonstrate that C. caviae IncA plays a role in mediating fusion of the bacteria-containing vacuoles inhabited by C. caviae. Moreover, using a chicken embryo infection model, we provide first evidence for a role of SinC in C. caviae virulence in vivo.

Klíčová slova:

Chlamydia – Chlamydia infection – Introns – HeLa cells – Animal models of infection – Chicken models – Vero cells

Zdroje

1. Sachse K, Bavoil PM, Kaltenboeck B, Stephens RS, Kuo CC, Rossello-Mora R, et al. Emendation of the family Chlamydiaceae: proposal of a single genus, Chlamydia, to include all currently recognized species. Syst Appl Microbiol. 2015;38(2):99–103. Epub 2015/01/27. doi: 10.1016/j.syapm.2014.12.004 25618261.

2. Ward ME. The chlamydial developmental cycle. In: Barron AL, editor. Micobiology of Chlamydia. Boca Raton FL: CRC Press; 1988. p. 71–95.

3. Elwell C, Mirrashidi K, Engel J. Chlamydia cell biology and pathogenesis. Nat Rev Microbiol. 2016;14(6):385–400. doi: 10.1038/nrmicro.2016.30 27108705; PubMed Central PMCID: PMC4886739.

4. Wright HR, Turner A, Taylor HR. Trachoma. Lancet. 2008;371(9628):1945–54. Epub 2008/06/10. S0140-6736(08)60836-3 [pii] doi: 10.1016/S0140-6736(08)60836-3 18539226.

5. Newman L, Rowley J, Vander Hoorn S, Wijesooriya NS, Unemo M, Low N, et al. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One. 2015;10(12):e0143304. doi: 10.1371/journal.pone.0143304 26646541; PubMed Central PMCID: PMC4672879.

6. Burillo A, Bouza E. Chlamydophila pneumoniae. Infect Dis Clin North Am. 2010;24(1):61–71. Epub 2010/02/23. doi: 10.1016/j.idc.2009.10.002 20171546.

7. Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol. 2003;128(4):217–44. doi: 10.1053/jcpa.2002.0629 12834606.

8. Knittler MR, Sachse K. Chlamydia psittaci: update on an underestimated zoonotic agent. Pathogens and disease. 2015;73(1):1–15. Epub 2015/04/09. doi: 10.1093/femspd/ftu007 25853998.

9. Beeckman DS, Vanrompay DC. Zoonotic Chlamydophila psittaci infections from a clinical perspective. Clin Microbiol Infect. 2009;15(1):11–7. Epub 2009/02/18. doi: 10.1111/j.1469-0691.2008.02669.x 19220335.

10. Murray ES. Guinea Pig Inclusion Conjunctivitis Virus. I. Isolation and Identification as a Member of the Psittacosis-Lymphogranuloma-Trachoma Group. J Infect Dis. 1964;114:1–12. Epub 1964/02/01. doi: 10.1093/infdis/114.1.1 14118043.

11. van Grootveld R, Bilsen MP, Boelsums TL, Heddema ER, Groeneveld GH, Gooskens J, et al. Chlamydia caviae causing community-acquired pneumonia: an emerging zoonosis. Vector Borne Zoonotic Dis. 2018. Epub 2018/07/10. doi: 10.1089/vbz.2018.2304 29985760.

12. Ramakers BP, Heijne M, Lie N, Le TN, van Vliet M, Claessen VPJ, et al. Zoonotic Chlamydia caviae presenting as community-acquired pneumonia. N Engl J Med. 2017;377(10):992–4. Epub 2017/09/07. doi: 10.1056/NEJMc1702983 28877022.

13. Azuma Y, Hirakawa H, Yamashita A, Cai Y, Rahman MA, Suzuki H, et al. Genome sequence of the cat pathogen, Chlamydophila felis. DNA Res. 2006;13(1):15–23. doi: 10.1093/dnares/dsi027 16766509.

14. Voigt A, Schofl G, Saluz HP. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens. PLoS One. 2012;7(4):e35097. Epub 2012/04/17. doi: 10.1371/journal.pone.0035097 22506068; PubMed Central PMCID: PMC3323650.

15. Thomson NR, Yeats C, Bell K, Holden MT, Bentley SD, Livingstone M, et al. The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation. Genome Res. 2005;15(5):629–40. doi: 10.1101/gr.3684805 15837807.

16. Read TD, Myers GS, Brunham RC, Nelson WC, Paulsen IT, Heidelberg J, et al. Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae. Nucleic Acids Res. 2003;31(8):2134–47. doi: 10.1093/nar/gkg321 12682364.

17. Peters J, Wilson DP, Myers G, Timms P, Bavoil PM. Type III secretion à la Chlamydia. Trends Microbiol. 2007;15(6):241–51. doi: 10.1016/j.tim.2007.04.005 17482820.

18. Mojica SA, Hovis KM, Frieman MB, Tran B, Hsia RC, Ravel J, et al. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors. Mol Biol Cell. 2015;26(10):1918–34. Epub 2015/03/20. doi: 10.1091/mbc.E14-11-1530 25788290; PubMed Central PMCID: PMC4436835.

19. Hower S, Wolf K, Fields KA. Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development. Mol Microbiol. 2009;72(6):1423–37. doi: 10.1111/j.1365-2958.2009.06732.x 19460098; PubMed Central PMCID: PMC2997736.

20. Bullock HD, Hower S, Fields KA. Domain analyses reveal that Chlamydia trachomatis CT694 protein belongs to the membrane-localized family of type III effector proteins. J Biol Chem. 2012;287(33):28078–86. Epub 2012/06/20. doi: 10.1074/jbc.M112.386904 22711538; PubMed Central PMCID: PMC3431695.

21. Sixt BS, Valdivia RH. Molecular genetic analysis of Chlamydia species. Annu Rev Microbiol. 2016;70:179–98. doi: 10.1146/annurev-micro-102215-095539 27607551.

22. Wang Y, Kahane S, Cutcliffe LT, Skilton RJ, Lambden PR, Clarke IN. Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog. 2011;7(9):e1002258. Epub 2011/10/04. doi: 10.1371/journal.ppat.1002258 21966270; PubMed Central PMCID: PMC3178582.

23. Agaisse H, Derre I. A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter. PLoS One. 2013;8(2):e57090. doi: 10.1371/journal.pone.0057090 23441233; PubMed Central PMCID: PMC3575495.

24. Johnson CM, Fisher DJ. Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron. PLoS One. 2013;8(12):e83989. doi: 10.1371/journal.pone.0083989 24391860; PubMed Central PMCID: PMC3877132.

25. Mueller KE, Wolf K, Fields KA. Gene deletion by fluorescence-reported allelic exchange mutagenesis in Chlamydia trachomatis. mBio. 2016;7(1):e01817–15. doi: 10.1128/mBio.01817-15 26787828; PubMed Central PMCID: PMC4725004.

26. Lowden NM, Yeruva L, Johnson CM, Bowlin AK, Fisher DJ. Use of aminoglycoside 3' adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability. BMC research notes. 2015;8(1):570. doi: 10.1186/s13104-015-1542-9 26471806; PubMed Central PMCID: PMC4606545.

27. Sixt BS, Bastidas RJ, Finethy R, Baxter RM, Carpenter VK, Kroemer G, et al. The Chlamydia trachomatis inclusion membrane protein CpoS counteracts STING-mediated cellular surveillance and suicide programs Cell Host Microbe. 2017;21(1):113–21.

28. Schachter J, Wyrick PB. Culture and isolation of Chlamydia trachomatis. Methods in Enzymology. 236: Academic Press; 1994. p. 377–90. doi: 10.1016/0076-6879(94)36028-6 7968623

29. Tan C, Hsia RC, Shou H, Carrasco JA, Rank RG, Bavoil PM. Variable expression of surface-exposed polymorphic membrane proteins in in vitro-grown Chlamydia trachomatis. Cell Microbiol. 2010;12(2):174–87. Epub 2009/10/09. doi: 10.1111/j.1462-5822.2009.01389.x 19811502; PubMed Central PMCID: PMC3073146.

30. Alzhanov D, Barnes J, Hruby DE, Rockey DD. Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA. BMC microbiology. 2004;4:24. Epub 2004/07/03. doi: 10.1186/1471-2180-4-24 15230981; PubMed Central PMCID: PMC459217.

31. Chen YS, Bastidas RJ, Saka HA, Carpenter VK, Richards KL, Plano GV, et al. The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling. PLoS Pathog. 2014;10(2):e1003954. doi: 10.1371/journal.ppat.1003954 24586162; PubMed Central PMCID: PMC3930595.

32. Andersson C, Gripenland J, Johansson J. Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections. Nat Protoc. 2015;10(8):1155–64. Epub 2015/07/03. doi: 10.1038/nprot.2015.073 26134955.

33. Banks J, Eddie B, Schachter J, Meyer KF. Plaque formation by Chlamydia in L cells. Infect Immun. 1970;1(3):259–62. Epub 1970/03/01. 16557725; PubMed Central PMCID: PMC415889.

34. Suchland RJ, Rockey DD, Bannantine JP, Stamm WE. Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun. 2000;68(1):360–7. doi: 10.1128/iai.68.1.360-367.2000 10603409.

35. Rockey DD, Fischer ER, Hackstadt T. Temporal analysis of the developing Chlamydia psittaci inclusion by use of fluorescence and electron microscopy. Infect Immun. 1996;64(10):4269–78. Epub 1996/10/01. 8926099; PubMed Central PMCID: PMC174367.

36. Lutz-Wohlgroth L, Becker A, Brugnera E, Huat ZL, Zimmermann D, Grimm F, et al. Chlamydiales in guinea-pigs and their zoonotic potential. Journal of veterinary medicine. 2006;53(4):185–93. doi: 10.1111/j.1439-0442.2006.00819.x 16629952.

37. Rockey DD, Heinzen RA, Hackstadt T. Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Mol Microbiol. 1995;15(4):617–26. doi: 10.1111/j.1365-2958.1995.tb02371.x 7783634.

38. Rockey DD, Scidmore MA, Bannantine JP, Brown WJ. Proteins in the chlamydial inclusion membrane. Microbes Infect. 2002;4(3 SU -):333–40. 11909744

39. Hackstadt T, Scidmore-Carlson MA, Shaw EI, Fischer ER. The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cell Microbiol. 1999;1(2):119–30. 11207546.

40. Delevoye C, Nilges M, Dautry-Varsat A, Subtil A. Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes. J Biol Chem. 2004;279(45):46896–906. doi: 10.1074/jbc.M407227200 15316015.

41. Spears P, Storz J. Biotyping of Chlamydia psittaci based on inclusion morphology and response to diethylaminoethyl-dextran and cycloheximide. Infect Immun. 1979;24(1):224–32. Epub 1979/04/01. 457272; PubMed Central PMCID: PMC414287.

42. Hsia R, Ohayon H, Gounon P, Dautry-Varsat A, Bavoil PM. Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis. Microbes Infect. 2000;2(7):761–72. 10955956.

43. Geisler WM, Suchland RJ, Rockey DD, Stamm WE. Epidemiology and clinical manifestations of unique Chlamydia trachomatis isolates that occupy nonfusogenic inclusions. J Infect Dis. 2001;184(7):879–84. Epub 2001/08/31. doi: 10.1086/323340 11528595.

44. Suchland RJ, Jeffrey BM, Xia M, Bhatia A, Chu HG, Rockey DD, et al. Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations. Infect Immun. 2008;76(12):5438–46. Epub 2008/10/15. doi: 10.1128/IAI.00984-08 18852248; PubMed Central PMCID: PMC2583591.

45. Weber MM, Noriea NF, Bauler LD, Lam JL, Sager J, Wesolowski J, et al. A functional core of IncA is required for Chlamydia trachomatis inclusion fusion. J Bacteriol. 2016;198(8):1347–55. doi: 10.1128/JB.00933-15 26883826.

46. Alnassan AA, Shehata AA, Kotsch M, Lendner M, Daugschies A, Bangoura B. Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens. Parasitol Res. 2013;112(6):2299–306. Epub 2013/03/22. doi: 10.1007/s00436-013-3392-5 23515571.

47. Polakowska K, Lis MW, Helbin WM, Dubin G, Dubin A, Niedziolka JW, et al. The virulence of Staphylococcus aureus correlates with strain genotype in a chicken embryo model but not a nematode model. Microbes Infect. 2012;14(14):1352–62. Epub 2012/10/09. doi: 10.1016/j.micinf.2012.09.006 23041460.

48. Oh JY, Kang MS, Yoon H, Choi HW, An BK, Shin EG, et al. The embryo lethality of Escherichia coli isolates and its relationship to the presence of virulence-associated genes. Poult Sci. 2012;91(2):370–5. Epub 2012/01/19. doi: 10.3382/ps.2011-01807 22252350.

49. Horzempa J, O'Dee DM, Shanks RM, Nau GJ. Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo. Infect Immun. 2010;78(6):2607–19. Epub 2010/04/14. doi: 10.1128/IAI.00134-10 20385757; PubMed Central PMCID: PMC2876533.

50. Braukmann M, Sachse K, Jacobsen ID, Westermann M, Menge C, Saluz HP, et al. Distinct intensity of host-pathogen interactions in Chlamydia psittaci- and Chlamydia abortus-infected chicken embryos. Infect Immun. 2012;80(9):2976–88. Epub 2012/06/13. doi: 10.1128/IAI.00437-12 22689815; PubMed Central PMCID: PMC3418749.

51. Rank RG. In vivo chlamydial infection. In: Tan M, Bavoil P, editors. Intracellular Pathogens I: Chlamydiales. Washington, DC: ASM Press; 2012. p. 285–310.

Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae

Souhrn

Klíčová slova:

Zdroje

PLOS One

Aktuální možnosti diagnostiky a léčby litiáz