Advances and Obstacles in the Genetic Dissection of Chlamydial Virulence

  • Julie A. Brothwell
  • Matthew K. Muramatsu
  • Guangming Zhong
  • David E. NelsonEmail author
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 412)


Obligate intracellular pathogens in the family Chlamydiaceae infect taxonomically diverse eukaryotes ranging from amoebae to mammals. However, many fundamental aspects of chlamydial cell biology and pathogenesis remain poorly understood. Genetic dissection of chlamydial biology has historically been hampered by a lack of genetic tools. Exploitation of the ability of chlamydia to recombine genomic material by lateral gene transfer (LGT) ushered in a new era in chlamydia research. With methods to map mutations in place, genetic screens were able to assign functions and phenotypes to specific chlamydial genes. Development of an approach for stable transformation of chlamydia also provided a mechanism for gene delivery and platforms for disrupting chromosomal genes. Here, we explore how these and other tools have been used to test hypotheses concerning the functions of known chlamydial virulence factors and discover the functions of completely uncharacterized genes. Refinement and extension of the existing genetic tools to additional Chlamydia spp. will substantially advance understanding of the biology and pathogenesis of this important group of pathogens.


Chlamydial Virulence Lateral Gene Transfer (LGT) Chlamydial Biology Genus Chlamydia Targeting Induced Local Lesions In Genomes (TILLING) 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



DE Nelson was supported by grants AI099278 and AI116706, and G Zhong was supported by grants AI121989, AI105712, AI047997, from the United States National Institutes of Health, Division of Allery and Infectious Diseases. We would like Drs. Harlan Caldwell and Derek Fisher for discussion and insights regarding aspects of this manuscript. Finally, any oversights of relevant studies were not intentional and the authors would like to apologize for any instance of this in advance.


  1. Agaisse H, Derre I (2013) 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 8(2):e57090PubMedPubMedCentralCrossRefGoogle Scholar
  2. Albrecht M, Sharma CM, Reinhardt R, Vogel J, Rudel T (2010) Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome. Nucleic Acids Res 38(3):868–877PubMedCrossRefGoogle Scholar
  3. Azuma Y, Hirakawa H, Yamashita A, Cai Y, Rahman MA, Suzuki H, Mitaku S, Toh H, Goto S, Murakami T, Sugi K, Hayashi H, Fukushi H, Hattori M, Kuhara S, Shirai M (2006) Genome sequence of the cat pathogen, Chlamydophila felis. DNA Res 13(1):15–23PubMedCrossRefGoogle Scholar
  4. Barron AL, White HJ, Rank RG, Soloff BL, Moses EB (1981) A new animal model for the study of Chlamydia trachomatis genital infections: infection of mice with the agent of mouse pneumonitis. J Infect Dis 143(1):63–66PubMedCrossRefGoogle Scholar
  5. Bastidas RJ, Valdivia RH (2016) Emancipating Chlamydia: advances in the genetic manipulation of a recalcitrant intracellular pathogen. Microbiol Mol Biol Rev 80(2):411–427PubMedPubMedCentralCrossRefGoogle Scholar
  6. Bauler LD, Hackstadt T (2014) Expression and targeting of secreted proteins from Chlamydia trachomatis. J Bacteriol 196(7):1325–1334PubMedPubMedCentralCrossRefGoogle Scholar
  7. Binet R, Maurelli AT (2005) Fitness cost due to mutations in the 16S rRNA associated with spectinomycin resistance in Chlamydia psittaci 6BC. Antimicrob Agents Chemother 49(11):4455–4464PubMedPubMedCentralCrossRefGoogle Scholar
  8. Binet R, Maurelli AT (2007) Frequency of development and associated physiological cost of azithromycin resistance in Chlamydia psittaci 6BC and C. trachomatis L2. Antimicrob Agents Chemother 51(12):4267–4275PubMedPubMedCentralCrossRefGoogle Scholar
  9. Binet R, Maurelli AT (2009a) The chlamydial functional homolog of KsgA confers kasugamycin sensitivity to Chlamydia trachomatis and impacts bacterial fitness. Bmc Microbiology 9Google Scholar
  10. Binet R, Maurelli AT (2009b) Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation. Proc Natl Acad Sci USA 106(1):292–297PubMedCrossRefGoogle Scholar
  11. Binet R, Bowlin AK, Maurelli AT, Rank RG (2010) Impact of azithromycin resistance mutations on the virulence and fitness of Chlamydia caviae in guinea pigs. Antimicrob Agents Chemother 54(3):1094–1101PubMedPubMedCentralCrossRefGoogle Scholar
  12. Borges V, Ferreira R, Nunes A, Sousa-Uva M, Abreu M, Borrego MJ, Gomes JP (2013) Effect of long-term laboratory propagation on Chlamydia trachomatis genome dynamics. Infect Genet Evol 17:23–32PubMedCrossRefGoogle Scholar
  13. Brothwell JA, Muramatsu MK, Toh E, Rockey DD, Putman TE, Barta ML, Hefty PS, Suchland RJ, Nelson DE (2016) Interrogating genes that mediate Chlamydia trachomatis survival in cell culture using conditional mutants and recombination. J Bacteriol 198(15):2131–2139PubMedPubMedCentralCrossRefGoogle Scholar
  14. Brunham R, Yang C, Maclean I, Kimani J, Maitha G, Plummer F (1994) Chlamydia trachomatis from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the major outer membrane protein (omp1) gene. J Clin Invest 94(1):458–463PubMedPubMedCentralCrossRefGoogle Scholar
  15. Burall LS, Rodolakis A, Rekiki A, Myers GS, Bavoil PM (2009) Genomic analysis of an attenuated Chlamydia abortus live vaccine strain reveals defects in central metabolism and surface proteins. Infect Immun 77(9):4161–4167PubMedPubMedCentralCrossRefGoogle Scholar
  16. Caldwell HD, Wood H, Crane D, Bailey R, Jones RB, Mabey D, Maclean I, Mohammed Z, Peeling R, Roshick C, Schachter J, Solomon AW, Stamm WE, Suchland RJ, Taylor L, West SK, Quinn TC, Belland RJ, McClarty G (2003) Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates. J Clin Invest 111(11):1757–1769PubMedPubMedCentralCrossRefGoogle Scholar
  17. Carlson JH, Porcella SF, McClarty G, Caldwell HD (2005) Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains. Infect Immun 73(10):6407–6418PubMedPubMedCentralCrossRefGoogle Scholar
  18. Carlson JH, Whitmire WM, Crane DD, Wicke L, Virtaneva K, Sturdevant DE, Kupko JJ 3rd, Porcella SF, Martinez-Orengo N, Heinzen RA, Kari L, Caldwell HD (2008) The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor. Infect Immun 76(6):2273–2283PubMedPubMedCentralCrossRefGoogle Scholar
  19. Casson N, Medico N, Bille J, Greub G (2006) Parachlamydia acanthamoebae enters and multiplies within pneumocytes and lung fibroblasts. Microbes Infect 8(5):1294–1300PubMedCrossRefGoogle Scholar
  20. Casson N, Entenza JM, Borel N, Pospischil A, Greub G (2008) Murine model of pneumonia caused by Parachlamydia acanthamoebae. Microb Pathog 45(2):92–97PubMedCrossRefGoogle Scholar
  21. Chen C, Zhou Z, Conrad T, Yang Z, Dai J, Li Z, Wu Y, Zhong G (2015a) In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated pathogenicity in mouse upper genital tract. Infect Immun 83(5):1881–1892PubMedPubMedCentralCrossRefGoogle Scholar
  22. Chen J, Yang Z, Sun X, Tang L, Ding Y, Xue M, Zhou Z, Baseman J, Zhong G (2015b) Intrauterine infection with plasmid-free Chlamydia muridarum reveals a critical role of the plasmid in chlamydial ascension and establishes a model for evaluating plasmid-independent pathogenicity. Infect Immun 83(6):2583–2592PubMedPubMedCentralCrossRefGoogle Scholar
  23. Clifton DR, Fields KA, Grieshaber SS, Dooley CA, Fischer ER, Mead DJ, Carabeo RA, Hackstadt T (2004) A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin. Proc Natl Acad Sci USA 101(27):10166–10171PubMedPubMedCentralCrossRefGoogle Scholar
  24. Collingro A, Poppert S, Heinz E, Schmitz-Esser S, Essig A, Schweikert M, Wagner M, Horn M (2005) Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp. Microbiology 151(Pt 1):301–309PubMedCrossRefGoogle Scholar
  25. Collingro A, Tischler P, Weinmaier T, Penz T, Heinz E, Brunham RC, Read TD, Bavoil PM, Sachse K, Kahane S, Friedman MG, Rattei T, Myers GS, Horn M (2011) Unity in variety–the pan-genome of the Chlamydiae. Mol Biol Evol 28(12):3253–3270PubMedPubMedCentralCrossRefGoogle Scholar
  26. Conrad TA, Gong S, Yang Z, Matulich P, Keck J, Beltrami N, Chen C, Zhou Z, Dai J, Zhong G (2015) The chromosome-encoded hypothetical protein TC0668 Is an upper genital tract pathogenicity factor of Chlamydia muridarum. Infect Immun 84(2):467–479PubMedCrossRefGoogle Scholar
  27. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645PubMedPubMedCentralCrossRefGoogle Scholar
  28. Dean D, Schachter J, Dawson CR, Stephens RS (1992) Comparison of the major outer membrane protein variant sequence regions of B/Ba isolates: a molecular epidemiologic approach to Chlamydia trachomatis infections. J Infect Dis 166(2):383–392PubMedCrossRefGoogle Scholar
  29. DeMars R, Weinfurter J (2008) Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance. J Bacteriol 190(5):1605–1614PubMedCrossRefGoogle Scholar
  30. DeMars R, Weinfurter J, Guex E, Lin J, Potucek Y (2007) Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis. J Bacteriol 189(3):991–1003PubMedCrossRefGoogle Scholar
  31. Dessus-Babus S, Bebear CM, Charron A, Bebear C, de Barbeyrac B (1998) Sequencing of gyrase and topoisomerase IV quinolone-resistance-determining regions of Chlamydia trachomatis and characterization of quinolone-resistant mutants obtained In vitro. Antimicrob Agents Chemother 42(10):2474–2481PubMedPubMedCentralGoogle Scholar
  32. Ding H, Gong S, Tian Y, Yang Z, Brunham R, Zhong G (2013) Transformation of sexually transmitted infection-causing serovars of Chlamydia trachomatis using Blasticidin for selection. PLoS ONE 8(11):e80534PubMedPubMedCentralCrossRefGoogle Scholar
  33. Donati M, Huot-Creasy H, Humphrys M, Di Paolo M, Di Francesco A, Myers GS (2014) Genome Sequence of Chlamydia suis MD56, isolated from the conjunctiva of a weaned piglet. Genome Announc 2(3):e00425PubMedPubMedCentralCrossRefGoogle Scholar
  34. Draghi A 2nd, Popov VL, Kahl MM, Stanton JB, Brown CC, Tsongalis GJ, West AB, Frasca S Jr (2004) Characterization of “Candidatus piscichlamydia salmonis” (order Chlamydiales), a chlamydia-like bacterium associated with epitheliocystis in farmed Atlantic salmon (Salmo salar). J Clin Microbiol 42(11):5286–5297PubMedPubMedCentralCrossRefGoogle Scholar
  35. Dreses-Werringloer U, Padubrin I, Kohler L, Hudson AP (2003) Detection of nucleotide variability in rpoB in both rifampin-sensitive and rifampin-resistant strains of Chlamydia trachomatis. Antimicrob Agents Chemother 47(7):2316–2318PubMedPubMedCentralCrossRefGoogle Scholar
  36. Everett KD, Bush RM, Andersen AA (1999) Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49(Pt 2):415–440PubMedCrossRefGoogle Scholar
  37. Falkow S (1988) Molecular Koch’s postulates applied to microbial pathogenicity. Rev Infect Dis 10(Suppl 2):S274–276PubMedCrossRefGoogle Scholar
  38. Farencena A, Comanducci M, Donati M, Ratti G, Cevenini R (1997) Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma. Infect Immun 65(7):2965–2969PubMedPubMedCentralGoogle Scholar
  39. Fehlner-Gardiner C, Roshick C, Carlson JH, Hughes S, Belland RJ, Caldwell HD, McClarty G (2002) Molecular basis defining human Chlamydia trachomatis tissue tropism. A possible role for tryptophan synthase. J Biol Chem 277(30):26893–26903PubMedCrossRefGoogle Scholar
  40. Fischer A, Harrison KS, Ramirez Y, Auer D, Chowdhury BK, Prusty BK, Sauer F, Dimond Z, Kisker C, Scott Hefty P, Rudel T (2017) Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense. Elife 6Google Scholar
  41. Fitch WM, Peterson EM, de la Maza LM (1993) Phylogenetic analysis of the outer-membrane-protein genes of Chlamydiae, and its implication for vaccine development. Mol Biol Evol 10(4):892–913PubMedGoogle Scholar
  42. Gerard HC, Mishra MK, Mao G, Wang S, Hali M, Whittum-Hudson JA, Kannan RM, Hudson AP (2013) Dendrimer-enabled DNA delivery and transformation of Chlamydia pneumoniae. Nanomedicine 9(7):996–1008PubMedCrossRefGoogle Scholar
  43. Gomes JP, Bruno WJ, Borrego MJ, Dean D (2004) Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer. J Bacteriol 186(13):4295–4306PubMedPubMedCentralCrossRefGoogle Scholar
  44. Gong S, Yang Z, Lei L, Shen L, Zhong G (2013) Characterization of Chlamydia trachomatis plasmid-encoded open reading frames. J Bacteriol 195(17):3819–3826PubMedPubMedCentralCrossRefGoogle Scholar
  45. Greub G, Berger P, Papazian L, Raoult D (2003) Parachlamydiaceae as rare agents of pneumonia. Emerg Infect Dis 9(6):755–756PubMedPubMedCentralCrossRefGoogle Scholar
  46. Hadfield J, Harris SR, Seth-Smith HMB, Parmar S, Andersson P, Giffard PM, Schachter J, Moncada J, Ellison L, Vaulet MLG, Fermepin MR, Radebe F, Mendoza S, Ouburg S, Morre SA, Sachse K, Puolakkainen M, Korhonen SJ, Sonnex C, Wiggins R, Jalal H, Brunelli T, Casprini P, Pitt R, Ison C, Savicheva A, Shipitsyna E, Hadad R, Kari L, Burton MJ, Mabey D, Solomon AW, Lewis D, Marsh P, Unemo M, Clarke IN, Parkhill J, Thomson NR (2017) Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion. Genome Res 27(7):1220–1229PubMedPubMedCentralCrossRefGoogle Scholar
  47. Haider S, Wagner M, Schmid MC, Sixt BS, Christian JG, Hacker G, Pichler P, Mechtler K, Muller A, Baranyi C, Toenshoff ER, Montanaro J, Horn M (2010) Raman microspectroscopy reveals long-term extracellular activity of Chlamydiae. Mol Microbiol 77(3):687–700PubMedCrossRefGoogle Scholar
  48. Hayes LJ, Yearsley P, Treharne JD, Ballard RA, Fehler GH, Ward ME (1994) Evidence for naturally occurring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Chlamydia trachomatis. Infect Immun 62(12):5659–5663PubMedPubMedCentralGoogle Scholar
  49. Holzer M, Laroucau K, Creasy HH, Ott S, Vorimore F, Bavoil PM, Marz M, Sachse K (2016) Whole-genome sequence of Chlamydia gallinacea type strain 08-1274/3. Genome Announc 4(4):e00708PubMedPubMedCentralCrossRefGoogle Scholar
  50. Hooppaw AJ, Fisher DJ (2016) A coming of age story: Chlamydia in the Post-Genetic Era. Infect Immun 84(3):612–621PubMedCentralCrossRefGoogle Scholar
  51. Horn M, Collingro A, Schmitz-Esser S, Beier CL, Purkhold U, Fartmann B, Brandt P, Nyakatura GJ, Droege M, Frishman D, Rattei T, Mewes HW, Wagner M (2004) Illuminating the evolutionary history of chlamydiae. Science 304(5671):728–730PubMedCrossRefGoogle Scholar
  52. Illingworth M, Hooppaw AJ, Ruan L, Fisher DJ, Chen L (2017) Biochemical and genetic analysis of the Chlamydia GroEL Chaperonins. J Bacteriol 199(12):e00844PubMedPubMedCentralCrossRefGoogle Scholar
  53. Jeffrey BM, Suchland RJ, Quinn KL, Davidson JR, Stamm WE, Rockey DD (2010) Genome sequencing of recent clinical Chlamydia trachomatis strains identifies loci associated with tissue tropism and regions of apparent recombination. Infect Immun 78(6):2544–2553PubMedPubMedCentralCrossRefGoogle Scholar
  54. Jeffrey BM, Suchland RJ, Eriksen SG, Sandoz KM, Rockey DD (2013) Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants. BMC Microbiol 13:142PubMedPubMedCentralCrossRefGoogle Scholar
  55. Johnson CM, Fisher DJ (2013) Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron. PLoS ONE 8(12):e83989PubMedPubMedCentralCrossRefGoogle Scholar
  56. Kalman S, Mitchell W, Marathe R, Lammel C, Fan J, Hyman RW, Olinger L, Grimwood J, Davis RW, Stephens RS (1999) Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nat Genet 21(4):385–389PubMedCrossRefGoogle Scholar
  57. Kannan RM, Gerard HC, Mishra MK, Mao G, Wang S, Hali M, Whittum-Hudson JA, Hudson AP (2013) Dendrimer-enabled transformation of Chlamydia trachomatis. Microb Pathog 65:29–35PubMedCrossRefGoogle Scholar
  58. Kari L, Whitmire WM, Carlson JH, Crane DD, Reveneau N, Nelson DE, Mabey DC, Bailey RL, Holland MJ, McClarty G, Caldwell HD (2008) Pathogenic diversity among Chlamydia trachomatis ocular strains in nonhuman primates is affected by subtle genomic variations. J Infect Dis 197(3):449–456PubMedCrossRefGoogle Scholar
  59. Kari L, Goheen MM, Randall LB, Taylor LD, Carlson JH, Whitmire WM, Virok D, Rajaram K, Endresz V, McClarty G, Nelson DE, Caldwell HD (2011) Generation of targeted Chlamydia trachomatis null mutants. Proc Natl Acad Sci USA 108(17):7189–7193PubMedPubMedCentralCrossRefGoogle Scholar
  60. Kari L, Southern TR, Downey CJ, Watkins HS, Randall LB, Taylor LD, Sturdevant GL, Whitmire WM, Caldwell HD (2014) Chlamydia trachomatis polymorphic membrane protein D is a virulence factor involved in early host-cell interactions. Infect Immun 82(7):2756–2762PubMedPubMedCentralCrossRefGoogle Scholar
  61. Karlsen M, Nylund A, Watanabe K, Helvik JV, Nylund S, Plarre H (2008) Characterization of ‘Candidatus Clavochlamydia salmonicola’: an intracellular bacterium infecting salmonid fish. Environ Microbiol 10(1):208–218PubMedGoogle Scholar
  62. Key CE, Fisher DJ (2017) Use of group II intron technology for targeted mutagenesis in Chlamydia trachomatis. Methods Mol Biol 1498:163–177PubMedCrossRefGoogle Scholar
  63. Kokes M, Dunn JD, Granek JA, Nguyen BD, Barker JR, Valdivia RH, Bastidas RJ (2015) Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17(5):716–725PubMedPubMedCentralCrossRefGoogle Scholar
  64. Lagkouvardos I, Jehl MA, Rattei T, Horn M (2014) Signature protein of the PVC superphylum. Appl Environ Microbiol 80(2):440–445PubMedPubMedCentralCrossRefGoogle Scholar
  65. Lampe MF, Suchland RJ, Stamm WE (1993) Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis. Infect Immun 61(1):213–219PubMedPubMedCentralGoogle Scholar
  66. Lei L, Chen J, Hou S, Ding Y, Yang Z, Zeng H, Baseman J, Zhong G (2014) Reduced live organism recovery and lack of hydrosalpinx in mice infected with plasmid-free Chlamydia muridarum. Infect Immun 82(3):983–992PubMedPubMedCentralCrossRefGoogle Scholar
  67. Liu Y, Chen C, Gong S, Hou S, Qi M, Liu Q, Baseman J, Zhong G (2014) Transformation of Chlamydia muridarum reveals a role for Pgp5 in suppression of plasmid-dependent gene expression. J Bacteriol 196(5):989–998PubMedPubMedCentralCrossRefGoogle Scholar
  68. Lohr M, Prohl A, Ostermann C, Liebler-Tenorio E, Schroedl W, Aeby S, Greub G, Reinhold P (2015) A bovine model of a respiratory Parachlamydia acanthamoebae infection. Pathog Dis 73(1):1–14PubMedGoogle Scholar
  69. Lowden NM, Yeruva L, Johnson CM, Bowlin AK, Fisher DJ (2015) Use of aminoglycoside 3’ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability. BMC Res Notes 8:570PubMedPubMedCentralCrossRefGoogle Scholar
  70. Luo ML, Leenay RT, Beisel CL (2016) Current and future prospects for CRISPR-based tools in bacteria. Biotechnol Bioeng 113(5):930–943PubMedCrossRefGoogle Scholar
  71. Matsumoto A, Izutsu H, Miyashita N, Ohuchi M (1998) Plaque formation by and plaque cloning of Chlamydia trachomatis biovar trachoma. J Clin Microbiol 36(10):3013–3019PubMedPubMedCentralGoogle Scholar
  72. McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations. Nat Biotechnol 18(4):455–457PubMedCrossRefGoogle Scholar
  73. McCoy AJ, Sandlin RC, Maurelli AT (2003) In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J Bacteriol 185(4):1218–1228PubMedPubMedCentralCrossRefGoogle Scholar
  74. Mishra MK, Gerard HC, Whittum-Hudson JA, Hudson AP, Kannan RM (2012) Dendrimer-enabled modulation of gene expression in Chlamydia trachomatis. Mol Pharm 9(3):413–421PubMedCrossRefGoogle Scholar
  75. Misiurina O, Shipitsina EV, Finashutina Iu P, Lazarev VN, Akopian TA, Savicheva AM, Govorun VM (2004) Analysis of point mutations in the ygeD, gyrA and parC genes in fluoroquinolones resistant clinical isolates of Chlamydia trachomatis. Mol Gen Mikrobiol Virusol 3:3–7Google Scholar
  76. Mojica S, Huot Creasy H, Daugherty S, Read TD, Kim T, Kaltenboeck B, Bavoil P, Myers GS (2011) Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58. J Bacteriol 193(14):3690PubMedPubMedCentralCrossRefGoogle Scholar
  77. Morrison RP, Caldwell HD (2002) Immunity to murine chlamydial genital infection. Infect Immun 70(6):2741–2751PubMedPubMedCentralCrossRefGoogle Scholar
  78. Moulder JW (1985) Comparative biology of intracellular parasitism. Microbiol Rev 49(3):298–337PubMedPubMedCentralGoogle Scholar
  79. Mueller KE, Wolf K, Fields KA (2016) Gene deletion by fluorescence-reported allelic exchange mutagenesis in Chlamydia trachomatis. MBio 7(1):e01817–01815PubMedPubMedCentralCrossRefGoogle Scholar
  80. Mueller KE, Wolf K, Fields KA (2017) Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis. Curr Protoc Microbiol 45: 11A 13 11–11A 13 15Google Scholar
  81. Muramatsu MK, Brothwell JA, Stein BD, Putman TE, Rockey DD, Nelson DE (2016) Beyond tryptophan synthase: identification of genes that contribute to Chlamydia trachomatis survival during IFN-gamma induced persistence and reactivation. Infect ImmunGoogle Scholar
  82. Nelson DE, Taylor LD, Shannon JG, Whitmire WM, Crane DD, McClarty G, Su H, Kari L, Caldwell HD (2007) Phenotypic rescue of Chlamydia trachomatis growth in IFN-gamma treated mouse cells by irradiated Chlamydia muridarum. Cell Microbiol 9(9):2289–2298PubMedCrossRefGoogle Scholar
  83. Nguyen BD, Valdivia RH (2012) Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches. Proc Natl Acad Sci USA 109(4):1263–1268PubMedPubMedCentralCrossRefGoogle Scholar
  84. Nguyen BD, Valdivia RH (2013) Forward genetic approaches in Chlamydia trachomatis. J Vis Exp 80:e50636Google Scholar
  85. Nunes A, Gomes JP (2014) Evolution, phylogeny, and molecular epidemiology of Chlamydia. Infect Genet Evol 23:49–64PubMedCrossRefGoogle Scholar
  86. O’Connell CM, Nicks KM (2006) A plasmid-cured Chlamydia muridarum strain displays altered plaque morphology and reduced infectivity in cell culture. Microbiology 152(Pt 6):1601–1607PubMedCrossRefGoogle Scholar
  87. O’Connell CM, Ingalls RR, Andrews CW Jr, Scurlock AM, Darville T (2007) Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease. J Immunol 179(6):4027–4034PubMedCrossRefGoogle Scholar
  88. Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA (2009) Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci USA 106(11):4430–4434PubMedPubMedCentralCrossRefGoogle Scholar
  89. Omsland A, Sager J, Nair V, Sturdevant DE, Hackstadt T (2012) Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium. Proc Natl Acad Sci USA 109(48):19781–19785PubMedPubMedCentralCrossRefGoogle Scholar
  90. Omsland A, Sixt BS, Horn M, Hackstadt T (2014) Chlamydial metabolism revisited: interspecies metabolic variability and developmental stage-specific physiologic activities. FEMS Microbiol Rev 38(4):779–801PubMedPubMedCentralCrossRefGoogle Scholar
  91. Palmer L, Falkow S (1986) A common plasmid of Chlamydia trachomatis. Plasmid 16(1):52–62PubMedCrossRefGoogle Scholar
  92. Peterson EM, Markoff BA, Schachter J, de la Maza LM (1990) The 7.5-kb plasmid present in Chlamydia trachomatis is not essential for the growth of this microorganism. Plasmid 23(2):144–148PubMedCrossRefGoogle Scholar
  93. Pickett MA, Everson JS, Pead PJ, Clarke IN (2005) The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents. Microbiology-Sgm 151:893–903CrossRefGoogle Scholar
  94. Qin B, McClarty G (1992) Effect of 6-thioguanine on Chlamydia trachomatis growth in wild-type and hypoxanthine-guanine phosphoribosyltransferase-deficient cells. J Bacteriol 174(9):2865–2873PubMedPubMedCentralCrossRefGoogle Scholar
  95. Rajaram K, Giebel AM, Toh E, Hu S, Newman JH, Morrison SG, Kari L, Morrison RP, Nelson DE (2015) Mutational analysis of the Chlamydia muridarum plasticity zone. Infect Immun 83(7):2870–2881PubMedPubMedCentralCrossRefGoogle Scholar
  96. Ramsey KH, Sigar IM, Schripsema JH, Denman CJ, Bowlin AK, Myers GA, Rank RG (2009) Strain and virulence diversity in the mouse pathogen Chlamydia muridarum. Infect Immun 77(8):3284–3293PubMedPubMedCentralCrossRefGoogle Scholar
  97. Rank RG, Yeruva L (2014) Hidden in plain sight: chlamydial gastrointestinal infection and its relevance to persistence in human genital infection. Infect Immun 82(4):1362–1371PubMedPubMedCentralCrossRefGoogle Scholar
  98. Rank RG, Bowlin AK, Reed RL, Darville T (2003) Characterization of chlamydial genital infection resulting from sexual transmission from male to female guinea pigs and determination of infectious dose. Infect Immun 71(11):6148–6154PubMedPubMedCentralCrossRefGoogle Scholar
  99. Read TD, Brunham RC, Shen C, Gill SR, Heidelberg JF, White O, Hickey EK, Peterson J, Utterback T, Berry K, Bass S, Linher K, Weidman J, Khouri H, Craven B, Bowman C, Dodson R, Gwinn M, Nelson W, DeBoy R, Kolonay J, McClarty G, Salzberg SL, Eisen J, Fraser CM (2000) Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res 28(6):1397–1406PubMedPubMedCentralCrossRefGoogle Scholar
  100. Read TD, Myers GS, Brunham RC, Nelson WC, Paulsen IT, Heidelberg J, Holtzapple E, Khouri H, Federova NB, Carty HA, Umayam LA, Haft DH, Peterson J, Beanan MJ, White O, Salzberg SL, Hsia RC, McClarty G, Rank RG, Bavoil PM, Fraser CM (2003) Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae. Nucleic Acids Res 31(8):2134–2147PubMedPubMedCentralCrossRefGoogle Scholar
  101. Rodolakis A (1983) In vitro and in vivo properties of chemically induced temperature-sensitive mutants of Chlamydia psittaci var. ovis: screening in a murine model. Infect Immun 42(2):525–530PubMedPubMedCentralGoogle Scholar
  102. Rodolakis A, Souriau A (1983) Response of ewes to temperature-sensitive mutants of Chlamydia psittaci (var ovis) obtained by NTG mutagenesis. Ann Rech Vet 14(2):155–161PubMedGoogle Scholar
  103. Rodolakis A, Souriau A (1986) Response of goats to vaccination with temperature-sensitive mutants of Chlamydia psittaci obtained by nitrosoguanidine mutagenesis. Am J Vet Res 47(12):2627–2631PubMedGoogle Scholar
  104. Russell M, Darville T, Chandra-Kuntal K, Smith B, Andrews CW Jr, O’Connell CM (2011) Infectivity acts as in vivo selection for maintenance of the chlamydial cryptic plasmid. Infect Immun 79(1):98–107PubMedCrossRefGoogle Scholar
  105. Sachse K, Laroucau K (2015) Two more species of Chlamydia-does it make a difference? Pathog Dis 73(1):1–3PubMedCrossRefGoogle Scholar
  106. Sachse K, Laroucau K, Riege K, Wehner S, Dilcher M, Creasy HH, Weidmann M, Myers G, Vorimore F, Vicari N, Magnino S, Liebler-Tenorio E, Ruettger A, Bavoil PM, Hufert FT, Rossello-Mora R, Marz M (2014) Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst Appl Microbiol 37(2):79–88PubMedCrossRefGoogle Scholar
  107. Schofl G, Voigt A, Litsche K, Sachse K, Saluz HP (2011) Complete genome sequences of four mammalian isolates of Chlamydophila psittaci. J Bacteriol 193(16):4258PubMedPubMedCentralCrossRefGoogle Scholar
  108. Seth-Smith HM, Harris SR, Persson K, Marsh P, Barron A, Bignell A, Bjartling C, Clark L, Cutcliffe LT, Lambden PR, Lennard N, Lockey SJ, Quail MA, Salim O, Skilton RJ, Wang Y, Holland MJ, Parkhill J, Thomson NR, Clarke IN (2009) Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain. BMC Genom 10:239CrossRefGoogle Scholar
  109. Shao L, Melero J, Zhang N, Arulanandam B, Baseman J, Liu Q, Zhong G (2017) The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract. PLoS ONE 12(5):e0177691PubMedPubMedCentralCrossRefGoogle Scholar
  110. Sixt BS, Bastidas RJ, Finethy R, Baxter RM, Carpenter VK, Kroemer G, Coers J, Valdivia RH (2017) The Chlamydia trachomatis inclusion membrane protein CpoS counteracts STING-mediated cellular surveillance and suicide programs. Cell Host Microbe 21(1):113–121PubMedCrossRefGoogle Scholar
  111. Snavely EA, Kokes M, Dunn JD, Saka HA, Nguyen BD, Bastidas RJ, McCafferty DG, Valdivia RH (2014) Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis 71(3):336–351PubMedPubMedCentralCrossRefGoogle Scholar
  112. Song L, Carlson JH, Whitmire WM, Kari L, Virtaneva K, Sturdevant DE, Watkins H, Zhou B, Sturdevant GL, Porcella SF, McClarty G, Caldwell HD (2013) Chlamydia trachomatis plasmid-encoded Pgp4 is a transcriptional regulator of virulence-associated genes. Infect Immun 81(3):636–644PubMedPubMedCentralCrossRefGoogle Scholar
  113. Song L, Carlson JH, Zhou B, Virtaneva K, Whitmire WM, Sturdevant GL, Porcella SF, McClarty G, Caldwell HD (2014) Plasmid-mediated transformation tropism of chlamydial biovars. Pathog Dis 70(2):189–193PubMedCrossRefGoogle Scholar
  114. Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, Aravind L, Mitchell W, Olinger L, Tatusov RL, Zhao Q, Koonin EV, Davis RW (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282(5389):754–759PubMedCrossRefGoogle Scholar
  115. Stephens RS, Myers G, Eppinger M, Bavoil PM (2009) Divergence without difference: phylogenetics and taxonomy of Chlamydia resolved. FEMS Immunol Med Microbiol 55(2):115–119PubMedCrossRefGoogle Scholar
  116. Stothard DR, Williams JA, Van Der Pol B, Jones RB (1998) Identification of a Chlamydia trachomatis serovar E urogenital isolate which lacks the cryptic plasmid. Infect Immun 66(12):6010–6013PubMedPubMedCentralGoogle Scholar
  117. Stride MC, Polkinghorne A, Miller TL, Groff JM, Lapatra SE, Nowak BF (2013) Molecular characterization of “Candidatus Parilichlamydia carangidicola,” a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, “Candidatus Parilichlamydiaceae” fam. nov. (order Chlamydiales). Appl Environ Microbiol 79(5):1590–1597PubMedPubMedCentralCrossRefGoogle Scholar
  118. Sturdevant GL, Kari L, Gardner DJ, Olivares-Zavaleta N, Randall LB, Whitmire WM, Carlson JH, Goheen MM, Selleck EM, Martens C, Caldwell HD (2010) Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract. Infect Immun 78(9):3660–3668PubMedPubMedCentralCrossRefGoogle Scholar
  119. Suchland RJ, Rockey DD, Bannantine JP, Stamm WE (2000) Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun 68(1):360–367PubMedPubMedCentralCrossRefGoogle Scholar
  120. Suchland RJ, Sandoz KM, Jeffrey BM, Stamm WE, Rockey DD (2009) Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro. Antimicrob Agents Chemother 53(11):4604–4611PubMedPubMedCentralCrossRefGoogle Scholar
  121. Tam JE, Davis CH, Wyrick PB (1994) Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation. Can J Microbiol 40(7):583–591PubMedCrossRefGoogle Scholar
  122. Taylor-Brown A, Vaughan L, Greub G, Timms P, Polkinghorne A (2015) Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae. Pathog Dis 73(1):1–15PubMedCrossRefGoogle Scholar
  123. Thomas NS, Lusher M, Storey CC, Clarke IN (1997) Plasmid diversity in Chlamydia. Microbiology 143(Pt 6):1847–1854PubMedCrossRefGoogle Scholar
  124. Thomson NR, Yeats C, Bell K, Holden MT, Bentley SD, Livingstone M, Cerdeno-Tarraga AM, Harris B, Doggett J, Ormond D, Mungall K, Clarke K, Feltwell T, Hance Z, Sanders M, Quail MA, Price C, Barrell BG, Parkhill J, Longbottom D (2005) The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation. Genome Res 15(5):629–640PubMedPubMedCentralCrossRefGoogle Scholar
  125. Thomson NR, Holden MT, Carder C, Lennard N, Lockey SJ, Marsh P, Skipp P, O’Connor CD, Goodhead I, Norbertzcak H, Harris B, Ormond D, Rance R, Quail MA, Parkhill J, Stephens RS, Clarke IN (2008) Chlamydia trachomatis: genome sequence analysis of lymphogranuloma venereum isolates. Genome Res 18(1):161–171PubMedPubMedCentralCrossRefGoogle Scholar
  126. Tipples G, McClarty G (1991) Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure. J Bacteriol 173(16):4932–4940PubMedPubMedCentralCrossRefGoogle Scholar
  127. Treharne JD, Yearsley PJ, Ballard RC (1989) In vitro studies of Chlamydia trachomatis susceptibility and resistance to rifampin and rifabutin. Antimicrob Agents Chemother 33(8):1393–1394PubMedPubMedCentralCrossRefGoogle Scholar
  128. Wang Y, Nagarajan U, Hennings L, Bowlin AK, Rank RG (2010) Local host response to chlamydial urethral infection in male guinea pigs. Infect Immun 78(4):1670–1681PubMedPubMedCentralCrossRefGoogle Scholar
  129. Wang Y, Kahane S, Cutcliffe LT, Skilton RJ, Lambden PR, Clarke IN (2011) Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathog 7(9):e1002258PubMedPubMedCentralCrossRefGoogle Scholar
  130. Wang YB, Cutcliffe LT, Skilton RJ, Ramsey KH, Thomson NR, Clarke IN (2014) The genetic basis of plasmid tropism between Chlamydia trachomatis and Chlamydia muridarum. Pathogens and Disease 72(1):19–23PubMedPubMedCentralCrossRefGoogle Scholar
  131. Weber MM, Bauler LD, Lam J, Hackstadt T (2015) Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis. Infect Immun 83(12):4710–4718PubMedPubMedCentralCrossRefGoogle Scholar
  132. Weber MM, Lam JL, Dooley CA, Noriea NF, Hansen BT, Hoyt FH, Carmody AB, Sturdevant GL, Hackstadt T (2017) Absence of specific Chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death. Cell Rep 19(7):1406–1417PubMedPubMedCentralCrossRefGoogle Scholar
  133. Weisburg WG, Hatch TP, Woese CR (1986) Eubacterial origin of chlamydiae. J Bacteriol 167(2):570–574PubMedPubMedCentralCrossRefGoogle Scholar
  134. Wesolowski J, Weber MM, Nawrotek A, Dooley CA, Calderon M, St Croix CM, Hackstadt T, Cherfils J, Paumet F (2017) Chlamydia hijacks ARF GTPases to coordinate microtubule posttranslational modifications and golgi complex positioning. MBio 8(3):e02280PubMedPubMedCentralCrossRefGoogle Scholar
  135. Wickstrum J, Sammons LR, Restivo KN, Hefty PS (2013) Conditional gene expression in Chlamydia trachomatis using the tet system. PLoS ONE 8(10):e76743PubMedPubMedCentralCrossRefGoogle Scholar
  136. Wolner-Hanssen P, Patton DL, Holmes KK (1991) Protective immunity in pig-tailed macaques after cervical infection with Chlamydia trachomatis. Sex Transm Dis 18(1):21–25PubMedCrossRefGoogle Scholar
  137. Wylie JL, Wang LL, Tipples G, McClarty G (1996) A single point mutation in CTP synthetase of Chlamydia trachomatis confers resistance to cyclopentenyl cytosine. J Biol Chem 271(26):15393–15400PubMedCrossRefGoogle Scholar
  138. Yang C, Starr T, Song L, Carlson JH, Sturdevant GL, Beare PA, Whitmire WM, Caldwell HD (2015) Chlamydial lytic exit from host cells is plasmid regulated. MBio 6(6):e01648–01615PubMedPubMedCentralCrossRefGoogle Scholar
  139. Yang C, Kari L, Sturdevant GL, Song L, Patton MJ, Couch CE, Ilgenfritz JM, Southern TR, Whitmire WM, Briones M, Bonner C, Grant C, Hu P, McClarty G, Caldwell HD (2017) Chlamydia trachomatis ChxR is a transcriptional regulator of virulence factors that function in in vivo host-pathogen interactions. Pathog Dis 75(3):ftx035PubMedCentralCrossRefGoogle Scholar
  140. Yeruva L, Spencer N, Bowlin AK, Wang Y, Rank RG (2013) Chlamydial infection of the gastrointestinal tract: a reservoir for persistent infection. Pathog Dis 68(3):88–95PubMedPubMedCentralCrossRefGoogle Scholar
  141. Zhang H, Cheng QX, Liu AM, Zhao GP, Wang J (2017) A novel and efficient method for bacteria genome editing employing both CRISPR/Cas9 and an antibiotic resistance cassette. Front Microbiol 8:812PubMedPubMedCentralCrossRefGoogle Scholar
  142. Zhong G (2017) Chlamydial plasmid-dependent pathogenicity. Trends Microbiol 25(2):141–152PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Julie A. Brothwell
    • 1
  • Matthew K. Muramatsu
    • 1
  • Guangming Zhong
    • 2
  • David E. Nelson
    • 1
    Email author
  1. 1.Department of Microbiology and ImmunologyIndiana University School of MedicineIndianapolisUSA
  2. 2.Department of Microbiology, Immunology and Molecular GeneticsUniversity of Texas Health Science Center at San AntonioSan AntonioUSA

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