Vol. 59, No. 8

INFECTION AND IMMUNITY, Aug. 1991, p. 2853-2855

0019-9567/91/082853-03$02.00/0 Copyright © 1991, American Society for Microbiology

Cloning and Sequence Analysis of the Major Outer Membrane Protein Gene of Chlamydia psittaci 6BC KARIN D. E. EVERETT,' ARTHUR A. ANDERSEN,2 MARIANNE PLAUNT,1 AND THOMAS P. HATCH'* Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163,1 and National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 500102 Received 25 February 1991/Accepted 21 May 1991

The gene encoding the major outer membrane protein (MOMP) of the psittacine Chlamydia psittaci strain 6BC was cloned and sequenced. N-terminal protein sequencing of the mature MOMP indicated that it is posttranslationally processed at a site identical to the site previously identified in the MOMP of Chlamydia trachomatis L2. The nucleotide sequence of the C. psittaci 6BC MOMP gene was found to be 67 to 68% identical to those of human C. trachomatis strains, 73% identical to that of Chiamydia pneumoniae IOL-207, 79% identical to that of the C. psittaci guinea pig inclusion conj unctivitis strain, GPIC, and 83% identical to that of the C. psittaci ovine abortion strain S26/3. In contrast, the 6BC sequence was found to be greater than 99% identical to the sequences reported for two strains of C. psittaci, A22/M and Cal-10 meningopneumonitis, believed to be of nonpsittacine avian origin. Monoclonal antibody analysis confirmed the nonpsittacine avian origin of A22/M but identified the Cal-10 strain from which the MOMP gene was previously sequenced as a psittacine strain. These results confirm that psittacine and nonpsittacine avian strains of C. psittaci are closely related and distinct from the mammalian guinea pig inclusion conjunctivitis and ovine abortion strains of C. psittaci.

The bacterial genus Chlamydia is made up of three species, C. trachomatis, C. psittaci, and C. pneumoniae (13, 19). C. pneumoniae and C. trachomatis, with the exception of the mouse pneumonitis biovariant, are human parasites. C. psittaci has been isolated from a wide variety of nonhuman mammalian and avian species and is believed to encompass a diverse group of loosely related organisms which produce numerous disease symptoms including pneumonitis, enteritis, conjunctivitis, abortion, arthritis, and encephalomyelitis. Humans are accidental hosts and are most commonly infected from avian sources. The paucity of biochemical, nutritional, and morphological markers has made classification of Chlamydia spp. difficult. Traditionally, organisms have been classified as Chlamydia spp. if they possess a typical chlamydial intracellular life cycle and react with genus-specific antiserum directed against chlamydial lipopolysaccharide (19). Identification of species has been based on intraCellular inclusion morphology, the accumulation of glycogen within the inclusion, differential susceptibility to antibiotics, and the natural host of origin (19). The development of analytical methods such as monoclonal antibody (MAb), restriction endonuclease, and DNA reassociation analyses has permitted a more precise examination of the relationships among the chlamydiae. Many of these techniques are based on the diversity among chlamydial strains of the major outer membrane protein (MOMP), which is immunodominant (6, 25) and includes four antigenically variable domains (3, 8, 24, 28). Consequently, MAbs raised against the MOMP have been used to serotype C. trachomatis (4, 5, 22, 27). Also, antibodies directed against the MOMP of C. trachomatis have been shown to be protective in animal studies (29), and a MOMP subunit vaccine has been demonstrated to protect ewes from disease caused by ovine abortion strains of C. psittaci (26). The genes encoding the MOMPs (ompi genes) of four C. psittaci strains (15, 23, *

Corresponding author.

28) have been cloned and sequenced: those of GPIC (28), the agent of guinea pig inclusion conjuctivitis (20), a strain reported to be Cal-10 meningopneumonitis (28), originally isolated from a ferret inoculated with respiratory secretions from a human presenting with influenzalike symptoms (11), S26/3, an ovine abortion isolate (15), and A22/M (23), originally thought to be related by serial passage to an ovine abortion isolate, A22 (23). The nature of the original isolation of Cal-10 renders it difficult to ascertain the natural host of this strain; however, restriction endonuclease profiles, DNA reassociation properties, and antigenic analysis suggest that Cal-10 is an avian strain of C. psittaci (9, 15-18). The sequence of ompi of A22/M differs significantly from that of S26/3, and, on the basis of restriction endonuclease analysis, it has been suggested that A22/M is of avian rather than ovine origin (15). We have cloned and sequenced ompi of C. psittaci 6BC (12) to better understand the relationship of this psittacine isolate and C. psittaci strains isolated from other hosts. C. psittaci 6BC was prepared from mouse L cells (14), and DNA was isolated as previously described (10). pFEN50 (3), a clone encoding the MOMP of C. trachomatis L2, was used to probe EcoRI-digested C. psittaci 6BC genomic DNA. A single band of about 7 kbp reacted with the probe and was cloned into the EcoRI site of pBluescript KS(+) (Stratagene, La Jolla, Calif.), yielding in pCPM2. The sequence of both strands of pCPM2 DNA encoding the MOMP of C. psittaci 6BC was determined by the dideoxy chain termination method, using oligonucleotide primers. The sequence of ompi of C. psittaci 6BC was found to be 67 to 68% identical to those of human isolates of C. trachomatis (28), 73% identical to that of C. pneumoniae IOL-207 (7), 79% identical to that of C. psittaci GPIC (28), and 83% identical to that of C. psittaci S26/3 (15). In contrast, the encoding region of ompi of 6BC (1,206 nucleotides) was found to differ from the sequenced Cal-10 strain gene (28) by only two nucleotide base pairs (two amino acids) and from that of A22/M (23) by 2853




6BC Cal-10 A22/M


.....E............................................ ....................

...................s ...------------.






+190 ATLGAEFQYAQSNPKIDU1NV .....................



FIG. 1. Amino acid differences between ompl gene product of C. psittaci 6BC and those of C. psittaci A22/M (23) and Cal-10 (29). Variable domains II and IV are boxed. Numbering is from the first amino acid in the mature proteins.

nine nucleotide base pairs (eight amino acids). These differences, largely confined to variable domains II and IV, are shown in Fig. 1. Although it is difficult to distinguish psittacine from nonpsittacine avian strains by restriction endonuclease and DNA reassociation analyses, the near identity between the MOMP gene sequence of 6BC and the MOMP gene sequences of A22/M (99.3% identical) and Cal-10 (99.8% identical) is striking. In an attempt to further establish the relationships among the C. psittaci strains used in the ompi sequencing studies, we reacted these strains in the indirect microimmunofluorescence test against a battery of 10 serovar-specific MAbs previously described (2). These MAbs represented four avian (psittacine, pigeon, duck, and turkey) and six mammalian (abortion [type I], polyarthritis [type II], feline pneumonitis, Wolfson cattle, muskrat, and GPIC) serovars. The strains were reacted with an additional MAb, MP/A9, produced to the ATCC meningopneumonitis VR-122 strain of C. psittaci. The results shown in Table 1 indicate that the psittacine MAb (VS1/E8) reacted with our 6BC strain, the ATCC 6BC strain, and the Cal-10 strain used in the ompi sequence study (28) but not with the ATCC VR-122 strain or the sequenced A22/M strain (23). In conTABLE 1. Reaction of C. psittaci strains with servar-specific MAbs in indirect immunofluorescence test Reaction of MAba:

C. psittaci strain

ATCC 6BC (VR-125) Sequenced 6BC Sequenced Cal-10 (29) ATCC meningo.b (VR-122) Sequenced A22/M (23) Ovine abortion (B577) (2)



+++ +++ +++ -





+++ +++ -



trast, a MAb to the ATCC VR-122 strain recognized A22/M but not our 6BC strain, the ATCC 6BC strain, or the Cal-10 strain used in the sequence study. The abortion MAb (B577/ F3) reacted with B577 but not with the A22/M strain. No reactions were observed with any of the remaining eight serovar-specific MAbs (data not shown). These serotyping experiments support the conclusion that the Cal-10 strain used in the ompl sequence study (28), the ATCC 6BC strain, and our 6BC strain are of psittacine origin and that the ATCC VR-122 strain is of avian but nonpsittacine origin. The results also support the previous suggestion that A22/M also is a nonpsittacine avian strain of C. psittaci. The N terminus of the mature MOMP of C. psittaci was determined by N-terminal peptide sequencing. It was found to be Leu-Pro-Val-Gly-Asn-Pro-Ala-Glu-Pro-Ser-Leu-LeuIle, indicating that the MOMP of C. psittaci 6BC is posttranslationally processed at a site identical to the processing site in the MOMP of C. trachomatis L2 (21). Our studies demonstrate for the first time at the level of a specific gene sequence that a psittacine strain of C. psittaci is remarkably closely related to suspected nonpsittacine avian strains. They also confirm that avian and mammalian strains can be distinguished on the basis of variation in MOMP gene sequences. Nucleotide sequence accession number. The nucleotide sequence encoding the MOMP of C. psittaci 6BC is available from GenBank (accession no. X56980). We thank Jerry Seyer for his help in N-terminal peptide sequence analysis and the Molecular Resource Center at the University of Tennessee, Memphis, for supplying oligonucleotides. We also thank M. E. Ward for providing us with A22/M antigen and Harlan Caldwell for providing us with the previously sequenced Cal-10 meningopneumonitis strain. This work was supported by Public Health Service research grant

AI-19570 from the National Institutes of Health.

a +++, bright fluorescence observed with ascitic fluids at a dilution of 1:1,600 or higher; -, no visible reaction at a 1:10 dilution. VS1/E8 was prepared against psittacine strain VS1, MP/A9 was prepared against ATCC meningopneumonitis strain VR-122, and B577/F3 was prepared against ovine abortion strain B577 (1, 2). bmeningo., meningopneumonitis.

REFERENCES 1. Andersen, A. A. 1991. Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies. J. Clin. Microbiol. 29:244-249. 2. Andersen, A. A. 1991. The serotyping of Chlamydia psittaci isolates using serovar-specific monoclonal antibodies with the


VOL. 59, 1991

microimmunofluorescence test. J. Clin. Microbiol. 29:707-711. 3. Baehr, W. B., Y.-X. Zhang, T. Joseph, H. Su, F. E. Nano, K. D. E. Everett, and H. D. Caldwell. 1988. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc. Natl. Acad. Sci. USA 85:40004004. 4. Barnes, R. C., S.-P. Wang, C.-C. Kuo, and W. E. Stamm. 1985. Rapid immunotyping of Chlamydia trachomatis with monoclonal antibodies in a solid-phase enzyme immunoassay. J. Clin. Microbiol. 22:609-613. 5. Batteiger, B. E., W. J. Newhall V, P. Terho, C. E. Wilde, and R. B. Jones. 1986. Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies. Infect. Immun. 53:530-533. 6. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 31:11611176. 7. Carter, M. W., S. A. H. Al-Mahdawi, I. G. Giles, J. D. Treharne, M. E. Ward, and I. N. Clarke. 1991. Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207. J. Gen. Microbiol. 137:465-475. 8. Conlan, J. W., I. N. Clarke, and M. E. Ward. 1988. Epitope mapping with solid-phase peptides: identification of type-, subspecies-, and genus-reactive antibody binding domains of the major outer membrane protein of Chlamydia trachomatis. Mol. Microbiol. 2:673-679. 9. Cox, R. L., C.-C. Kuo, J. T. Grayston, and L. A. Campbell. 1988. Deoxyribonucleic acid relatedness of Chlamydia sp. strain TWAR to Chlamydia trachomatis and Chlamydia psittaci. Int. J. Syst. Bacteriol. 38:265-268. 10. Crenshaw, R. W., M. J. Fahr, D. G. Wichian, and T. P. Hatch. 1990. Developmental cycle-specific host-free RNA synthesis in Chlamydia spp. Infect. Immun. 58:3194-3201. 11. Francis, T., and T. P. Magill. 1938. An unidentified virus producing acute meningitis and pneumonia. J. Exp. Med. 68: 147-160. 12. Gordon, F. B., and A. L. Quan. 1965. Occurrence of glycogen in inclusions of the psittacosis-lymphogranuloma venereum-trachoma agent. J. Infect. Dis. 115:186-196. 13. Grayston, J. T., C.-C. Kuo, L. A. Campbell, and S.-P. Wang. 1989. Chlamydia pneumoniae sp. nov. for Chlamydia sp. strain TWAR. Int. J. Syst. Bacteriol. 39:88-90. 14. Hatch, T. P., D. W. Vance, and E. Al-Hossainy. 1981. Identification of a major envelope protein in Chlamydia spp. J. Bacteriol. 146:426-429. 15. Herring, A. J., T. W. Tan, S. Baxter, N. F. Inglis, and S. Dunbar. 1989. Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci. FEMS Microbiol. Lett. 65:153-158. 16. McClenaghan, M., A. J. Herring, and I. D. Aitken. 1984. Comparison of Chlamydia psittaci isolates by DNA restriction


endonuclease analysis. Infect. Immun. 45:384-389. 17. McClenaghan, M., J. R. Honeycombe, B. J. Bevan, and A. J. Herring. 1988. Distribution of plasmid sequences in avian and mammalian strains of Chlamydia psittaci. J. Gen. Microbiol.

134:559-565. 18. Manire, G. P., and K. F. Meyer. 1950. The toxins of the psittacosis-lymphogranuloma group of agents. III. Differentiation of strains in the toxin neutralization test. J. Infect. Dis. 86:241-250. 19. Moulder, J. W., T. P. Hatch, C.-C. Kuo, J. Schachter, and J. Storz. 1984. Genus I. Chlamydia Jones, Rake and Steams 1945, 55AL, p. 729-739. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 20. Murray, E. S. 1964. Guinea pig inclusion conjunctivitis virus. I. Isolation and identification as a member of the psittacosislymphogranuloma-trachoma group. J. Infect. Dis. 114:1-12. 21. Nano, F. E., P. A. Barstad, L. W. Mayer, J. E. Coligan, and H. D. Caldwell. 1985. Partial amino acid sequence and molecular cloning of the encoding gene for the major outer membrane protein of Chlamydia trachomatis. Infect. Immun. 48:372-377. 22. Newhall, W. J., V, P. Terho, C. E. Wilde III, B. E. Batteiger, and R. B. Jones. 1986. Serovar determination of Chlamydia trachomatis isolates using type-specific monoclonal antibodies. Infect. Immun. 23:333-338. 23. Pickett, M. A., J. S. Everson, and I. N. Clarke. 1988. Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene. FEMS Microbiol. Lett. 55:229-234. 24. Stephens, R. S., R. Sanchez-Pescador, E. A. Wagar, C. Inouye, and M. S. Urdea. 1987. Diversity of Chlamydia trachomatis major outer membrane protein genes. J. Bacteriol. 169:38793885. 25. Stephens, R. S., M. R. Tam, C.-C. Kuo, and R. C. Nowinski. 1982. Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization. J. Immunol.

128:1083-1089. 26. Tan, T.-W., A. J. Herring, I. E. Anderson, and G. E. Jones. 1990. Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein. Infect. Immun. 58:3101-3108. 27. Wang, S.-P., C.-C. Kuo, R. C. Barnes, R. S. Stephens, and J. T. Grayston. 1985. Immunotyping of Chlamydia trachomatis with monoclonal antibodies. J. Infect. Dis. 152:791-800. 28. Zhang, Y.-X., S. G. Morrison, H. D. Caldwell, and W. B. Baehr. 1989. Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains. Infect. Immun. 57:1621-1625. 29. Zhang, Y.-X., S. Stewart, T. Joseph, H. R. Taylor, and H. D. Caldwell. 1987. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J. Immunol. 138:575-581.

Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC.

The gene encoding the major outer membrane protein (MOMP) of the psittacine Chlamydia psittaci strain 6BC was cloned and sequenced. N-terminal protein...
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