Proteomic characterisation of the Chlamydia abortus outer membrane complex (COMC) using combined rapid monolithic column liquid chromatography and fast MS/MS scanning

Autoři: David Longbottom aff001;  Morag Livingstone aff001;  Kevin D. Aitchison aff001;  Lisa Imrie aff001;  Erin Manson aff001;  Nicholas Wheelhouse aff001;  Neil F. Inglis aff001
Působiště autorů: Moredun Research Institute, Pentlands Science Park, Edinburgh, United Kingdom aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)—the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.

Klíčová slova:

Gram negative bacteria – Chlamydia – Chlamydia infection – Integral membrane proteins – Membrane proteins – Outer membrane proteins – Molecular mass


1. Longbottom D., Coulter L.J. Animal chlamydioses and zoonotic implications. J. Comp. Pathol. (2003) 128: 217–244. doi: 10.1053/jcpa.2002.0629 12834606

2. Holmes K. K., Handsfield H. H., Wang S. P., Wentworth B. B., Turck M., Anderson J.B. et al. Etiology of non-gonococcal urethritis. N. Eng. J. Med. (1975) 292: 1199. doi: 10.1056/NEJM197506052922301 165407

3. Tang F.-F., Chang H.-L., Huang Y. Studies on the etiology of trachoma with special reference to isolation of the virus in chick embryo. Chin. Med J. (1957) 75: 429–447. 13461224

4. Grayston J.T., Kuo C., Wang S.P., Altman J. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N. Engl. J. Med. (1986) 315:161–168. doi: 10.1056/NEJM198607173150305 3724806

5. Harkinezhad T., Geens T, Vanrompay D. Chlamydophila psittaci infections in birds: A review with emphasis on zoonotic consequences. Vet. Microbiol. (2009) 135:68–77. doi: 10.1016/j.vetmic.2008.09.046 19054633

6. McKinlay A.W., White N., Buxton D., Inglis J.M., Johnson F.W., Kurtz J.B. et al. Severe Chlamydia psittaci sepsis in pregancy. Q. J. Med. (1985) 57: 689–696. 4080958

7. Johnson F.W., Matheson B.A., Williams H. Laing A.G., Jandial V., Davidson-Lamb R. et al. Abortion due to infection with Chlamydia psittaci in a sheep farmer's wife. Br. Med. J. (1985) 290: 5924. doi: 10.1136/bmj.290.6468.592 3918685

8. Sillis M. and Longbottom D. (2011). Chlamydiosis. In: Zoonoses ( Palmer S.R., Soulsby Lord, Torgerson P.R. and Brown D.W.G., eds.), second edition, Oxford University Press, pp.146–57.

9. Scidmore M. A., Fischer E. R., Hackstadt T. Restricted Fusion of Chlamydia trachomatis Vesicles with Endocytic Compartments during the Initial Stages of Infection. Infect. Immun. (2003) 71: 973–984. doi: 10.1128/IAI.71.2.973-984.2003 12540580

10. Caldwell H.D., Kromhout J., Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. (1981) 31: 1161–1176. 7228399

11. Tan T.W., Herring A.J., Anderson I.E., Jones G.E. Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein. Infect. Immun. (1990) 58: 3101–3108. 2387636

12. Batteiger B.E., Rank R.G., Bavoil P.M., Soderberg L.S. Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis. J. Gen. Microbiol. (1993) 12: 2965–2972. doi: 10.1099/00221287-139-12-2965 7510322

13. Sandbulte J., TerWee J., Wigington K., Sabara M. Evaluation of Chlamydia psittaci subfraction and subunit preparations for their proective capacities. Vet. Microbiol. (1996) 48: 269–282. doi: 10.1016/0378-1135(95)00166-2 9054123

14. Pal S., Theodor I., Peterson E.M., de la Maza L.M. Immunization with an acellular vaccine consisting of the outer membrane complex of Chlamydia trachomatis induces protection against a genital challenge. Infect. Immun. (1997) 8: 3361–3369. 9234798

15. Birkelund S., Morgan-Fisher M., Timmerman E., Gevaert K., Shaw A.C., Christiansen G. Analysis of proteins in Chlamydia trachomatis L2 outer membrane complex, COMC. FEMS Immunol. Med. Microbiol. (2009) 55: 187–195. doi: 10.1111/j.1574-695X.2009.00522.x 19187221

16. Liu X., Afrane M., Clemmer D.E., Zhong G., Nelson D.E. Identification of Chlamydia trachomatis outer membrane complex proteins by differential proteomics. J. Bacteriol. (2010) 192: 2852–2860. doi: 10.1128/JB.01628-09 20348250

17. Shevchenko A., Jensen O.N., Podtelejnikov A.V., Sagliocco F., Wilm M., Vorm O. et al. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. U.S.A. (1996) 93: 14440–14445. doi: 10.1073/pnas.93.25.14440 8962070

18. Henzel W. J., Billeci T. M., Stults J. T., Wong S. C., Grimley C., Watanabe C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Nat. Acad. Sci. (1993) 90: 5011–5015. doi: 10.1073/pnas.90.11.5011 8506346

19. Hughes V., Bannantine J.P., Denham S., Smith S., Garcia-Sanchez A., Sales J. et al. Immunogenicity of proteome-determined Mycobacterium avium subsp. paratuberculosis-specific proteins in sheep with paratuberculosis. Clin. Vaccine Immunol. (2008) 15: 1824–1833. doi: 10.1128/CVI.00099-08 18845834

20. Watson E., Alberdi M.P., Inglis N.F., Lainson A., Porter M.E., Manson E. et al. Proteomicanalysis of Lawsonia intracellularis reveals expression of outer embrane proteins during infection. Vet. Microbiol. (2014) 174: 448–455. doi: 10.1016/j.vetmic.2014.10.002 25457368

21. Batycka M., Inglis N.F., Cook K., Adam A, Fraser-Pitt D., Smith D.G.E. et al. Ultra-fast MS/MS scanning combined with monolithic column LC increases throughput in proteomic analysis. Rapid Commun. Mass Spectrom. (2006) 20: 2074–2080. doi: 10.1002/rcm.2563 16773668

22. Burgess K.E.V., Lainson A., Imrie L., Fraser-Pitt D., Yaga R., Smith D.G.E. et al. Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning. Proteomics (2009) 9: 1720–1726. doi: 10.1002/pmic.200800200 19242933

23. Anderson I.E. Comparison of the virulence in mice of some ovine isolates of Chlamydia psittaci. Vet. Microbiol. (1986) 12: 213–220. doi: 10.1016/0378-1135(86)90050-7 3776092

24. McClenaghan M., Herring A.J., Aitken I.D. Comparison of Chlamydia psittaci isolates by DNA restriction endonuclease analysis. Infect. Immun. (1984) 45: 384–389. 6086526

25. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. (1970) 227: 680–685. doi: 10.1038/227680a0 5432063

26. Taylor G.K., Goodlett D.R. Rules governing protein identification by mass spectrometry. Rapid Commun. Mass Spectrom. (2005) 19: 3420. doi: 10.1002/rcm.2225 16252315

27. Yu N.Y., Wagner J.R., Laird M.R., Melli G., Rey S., Lo R. et al. PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics (2010) 26: 1608–1615. doi: 10.1093/bioinformatics/btq249 20472543

28. Neilsen H. Predicting secretory sroteins with SignalP. In Kihara D (ed): Protein Function Prediction (Methods in Molecular Biology vol. 1611) pp. 59–73, Springer 2017. doi: 10.1007/978-1-4939-7015-5_6

29. Kelley L.A., Mezulis S., Yates C.M., Wass M.N., Sternberg M.J.E. The Phyre2 web portal for protein modeling, prediction and analysis. (2015) Nature Protocols (2015) 10: 845–858. doi: 10.1038/nprot.2015.053 25950237

30. Jones P., Binns D. Chang H.Y., Fraser M. Li W., McAnulla C. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. (2014) 30: 1236–1240. doi: 10.1093/bioinformatics/btu031 24451626

31. Skipp P., Robinson J., O'Connor C.D., Clarke I.N. Shotgun proteomic analysis of Chlamydia trachomatis. Proteomics (2005) 5: 1558–1573. doi: 10.1002/pmic.200401044 15838905

32. Wuppermann F.N., Molleken K., Julien M., Jantos C.A., Hegemann J.H. Chlamydia pneumoniae GroEL1 protein is cell surface associated and required for infection of HEp-2 cells. J. Bacteriol. (2008) 190: 3757–3767. doi: 10.1128/JB.01638-07 18310329

33. Koenigs A., Zipfel P.F., Kraiczy P. Translation elongation factor Tuf of Acinetobacter baumannii is a plasminogen-binding protein. PLoS One (2015) 31: 10(7): e0134418. doi: 10.1371/journal.pone.0134418 26230848

34. Kolberg J., Hammerschmidt S., Frank R., Jonak J., Sanderova H., Aase A. The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci. FEMS Immunol. Med. Microbiol. (2008) 53: 222–230. doi: 10.1111/j.1574-695X.2008.00419.x 18462389

35. Wyllie S., Ashley R.H., Longbottom D., Herring A.J. The major outer membrane protein of Chlamydia psittaci functions as a porin-like ion channel. Infect. Immun. (1998) 66: 5202–5207. 9784523

36. Confer A.W., Ayalew S. The OmpA family of proteins: Roles in bacterial pathogenesis and immunity. Vet. Microbiol. (2013) 163: 207–222. doi: 10.1016/j.vetmic.2012.08.019 22986056

37. Buzoni-Gatel D., Bernard F., Andersen A., Rodolakis A. Protective effect of polyclonal and monoclonal antibodies against abortion in mice infected by Chlamydia psittaci. Vaccine. (1990) 8: 342–346. doi: 10.1016/0264-410x(90)90092-z 2396474

38. de Sa C., Souriau A., Bernard F., Salinas J., Rodolakis A. An oligomer of the major outer membrane protein of Chlamydia psittaci is recognized by monoclonal antibodies which protect mice from abortion. Infect. Immun. (1995) 63: 4912–4916. 7591155

39. Sun G., Pal S., Weiland J., Peterson E.M., de la Maza L.M. Protection against an intranasal challenge by vaccines formulated with native and recombinant preparations of the Chlamydia trachomatis major outer membrane protein. Vaccine. (2009) 27: 5020–5025. doi: 10.1016/j.vaccine.2009.05.008 19446590

40. Kubo A., Stephens R.S. Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target. Mol. Microbiol. (2000) 38:772–780. doi: 10.1046/j.1365-2958.2000.02167.x 11115112

41. Kubo A., Stephens R.S. Substrate-specific diffusion of select dicarboxylates through Chlamydia trachomatis PorB. Microbiology-SGM (2001) 147: 3135–3140. doi: 10.1099/00221287-147-11-3135 11700364

42. Kawa D.E., Schachter J., Stephens R.S. Immune response to the Chlamydia trachomatis outer membrane protein PorB. Vaccine. (2004) 22: 4282–4286. doi: 10.1016/j.vaccine.2004.04.035 15474719

43. Ifere G.O., He Q., Igietseme J.U., Ananaba G.A., Lyn D., Lubitz W., et al. Immunogenicity and protection against genital Chlamydia infection and its complications by a multisubunit candidate vaccine. J. Microbiol. Immunol. Infect. (2007) 40: 188–200. 17639158

44. Henderson I.R., Lam A.C. Polymorphic proteins of Chlamydia spp.—autotransporters beyond the Proteobacteria, Trends. Microbiol. (2001) 9: 573–578. doi: 10.1016/s0966-842x(01)02234-x 11728862

45. Thomson N.R., Yeats C., Bell K., Holden M.T., Bentley S.D., Livingstone M., et al. The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation. Genome Res (2005) 15: 629–640. doi: 10.1101/gr.3684805 15837807

46. Stephens R.S., Kalman S, Lammel C., Fan J., Marathe R., Aravind L. et al. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. (1998) Science 282: 754–759. doi: 10.1126/science.282.5389.754 9784136

47. Read T.D., Myers G.S., Brunham R.C., Nelson W.C., Paulsen I.T., 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. (2003) Nucleic Acids Res. 15: 2134–2147. doi: 10.1093/nar/gkg321 12682364

48. Azuma Y., Hirakawa H., Yamashita A., Cai Y., Rahman M.A., Suzuki H. et al. Genome sequence of the cat pathogen, Chlamydophila felis. DNA Res. (2006) 28: 15–23. doi: 10.1093/dnares/dsi027 16766509

49. Voigt A.1., Schöfl G., Saluz H.P. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens. (2012) PLoS One.7(4):e35097. doi: 10.1371/journal.pone.0035097 22506068

50. Wheelhouse N., Aitchison K., Spalding L., Livingstone M., Longbottom D., Transcriptional analysis of in vitro expression patterns of Chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle, Vet. Res. (2009) 40:47– doi: 10.1051/vetres/2009030 19454212

51. Wheelhouse N., Sait M., Wilson K., Aitchison K., McLean K., Smith D.G., Longbottom D., Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle, Vet. Microbiol. (2012) 160: 525–529. doi: 10.1016/j.vetmic.2012.06.017 22776512

52. Longbottom D., Russell M., Jones G.E., Lainson F.A., Herring A.J. Identification of a multigene family coding for the 90 kda proteins of the ovine abortion subtype of Chlamydia psittaci, FEMS. Microbiol. Lett. (1996) 142: 277–281. doi: 10.1111/j.1574-6968.1996.tb08443.x 8810511

53. Longbottom D., Russell M., Dunbar S.M., Jones G.E., Herring A.J. Molecular cloning and characterization of the genes coding for the highly immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia psittaci subtype that causes abortion in sheep. Infect. Immun. (1998) 66:1317–1324. 9529048

54. Pedersen A.S., Christiansen G., Birkelund S. Differential expression of Pmp10 in cell culture infected with Chlamydia pneumoniae CWL029. FEMS. Microbiol. Lett. (2001) 203:153–159. doi: 10.1111/j.1574-6968.2001.tb10834.x 11583841

55. Wehrl W., Brinkmann V., Jungblut P.R., Meyer T.F., Szczepek A.J. From the inside out–processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells. (2004) Mol. Microbiol. 51: 319–334. doi: 10.1046/j.1365-2958.2003.03838.x 14756775

56. Kiselev A.O., Stamm W.E., Yates J.R., Lampe M.F. Expression, processing, and localization of PmpD of Chlamydia trachomatis Serovar L2 during the chlamydial developmental cycle. PLoS One. (2007) Jun 27;2(6):e568. doi: 10.1371/journal.pone.0000568 17593967

57. Swanson K.A., Taylor L.D., Frank S.D., Sturdevant G.L., Fischer E.R., Carlson J.H. et al. Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure. (2009) Infect Immun. 77: 508–16. doi: 10.1128/IAI.01173-08 19001072

58. Wheelhouse N.M., Sait M., Aitchison K., Livingstone M., Wright F., McLean K. et al. Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle. PLoS One. (2012) 7 (11): e49190. doi: 10.1371/journal.pone.0049190 23145118

59. Peters J., Wilson J.P., Myers G., Timms P., Bavoil P.M. Type III secretion à la Chlamydia. Trends in Microbiology (2007) 15: 241–251. doi: 10.1016/j.tim.2007.04.005 17482820

60. Hueck C.J. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev. (1998) 62: 379–433. 9618447

61. Brinkworth A.J., Malcolm D.S., Pedrosa A.T., Roguska K., Shahbazian S., Graham J.E., et al. Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP. Mol Microbiol. (2011) 82: 131–144. doi: 10.1111/j.1365-2958.2011.07802.x 21883523

62. Pais S.V., Milho C., Almeida F., Mota L.J. Identification of novel type III secretion chaperone-substrate complexes of Chlamydia trachomatis. PLoS One. (2013) 8:e56292. doi: 10.1371/journal.pone.0056292 23431368

63. Hatch T.P. Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae? J. Bacteriol. (1996) 178: 1–5. doi: 10.1128/jb.178.1.1-5.1996 8550401

64. Everett K.D., Hatch T.P. Architecture of the cell envelope of Chlamydia psittaci 6BC. J. Bacteriol. (1995) 177: 877–82. doi: 10.1128/jb.177.4.877-882.1995 7532170

65. Tanzer R.J., Longbottom D., Hatch T.P. Identification of polymorphic outer menbrane proteins of Chlamydia psittaci 6BC. Infect. Immun. (2001) 69: 2428–2434. doi: 10.1128/IAI.69.4.2428-2434.2001 11254603

66. Fadel S., Eley A. Chlamydia trachomatis OmcB protein is a surface-exposed glycosaminoglycan-dependent adhesin, J. Med. Microbiol. (2007) 56:15–22. doi: 10.1099/jmm.0.46801-0 17172511

67. Moelleken K., Hegemann J.H. The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding, Mol. Microbiol. (2008) 67: 403–419. doi: 10.1111/j.1365-2958.2007.06050.x 18086188

68. Gervassi A.L., Grabstein K.H., Probst P., Hess B., Alderson M.R., Fling S.P. Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis. J. Immunol. (2004) 173: 6905–6913. doi: 10.4049/jimmunol.173.11.6905 15557186

69. Gentle I.E., Burri L., Lithgow T. Molecular architecture and function of the Omp85 family of proteins. Mol. Microbiol. (2005) 58:1216–1225. doi: 10.1111/j.1365-2958.2005.04906.x 16313611

70. Stephens R.S., Lammel C.J. Chlamydia outer membrane protein discovery using genomics. Current Opinion in Microbiology (2001) 4: 16–20. 11173028

71. Carey A.J., Timms P., Rawlinson G., Brumm J., Nilsson K., Harris J.M. et al. A multi-subunit chlamydial vaccine induces antibody and cell-mediated immunity in immunized koalas (Phascolarctos cinereus): comparison of three different adjuvants. Am. J. Reprod. Immunol. (2010) 63:161–172. doi: 10.1111/j.1600-0897.2009.00776.x 20064144

72. Myers G.S.A., Grinvalds R., Booth S., Hutton S.I., Binks M., Kemp D.J. et al. Expression of two novel proteins in Chlamydia trachomatis during natural infection, Microb. Pathog. (2000) 29: 63–72. doi: 10.1006/mpat.2000.0359 10906261

73. Perez-Melgosa M., Kuo C.C., Campbell L.A. Isolation and characterization of a gene encoding a Chlamydia pneumoniae 76- kilodalton protein containing a species-specific epitope. Infect. Immun. (1994) 62: 880–886. 7509320

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden