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. 2008 Oct;15(10):1606-15.
doi: 10.1128/CVI.00134-08. Epub 2008 Sep 3.

Chlamydophila felis CF0218 is a novel TMH family protein with potential as a diagnostic antigen for diagnosis of C. felis infection

Kenji Ohya  1 , Yu Takahara, Etsuko Kuroda, Saori Koyasu, Shigeyuki Hagiwara, Maki Sakamoto, Mitsuaki Hisaka, Kazuko Morizane, Shinryou Ishiguro, Tsuyoshi Yamaguchi, Hideto Fukushi
Affiliations

Chlamydophila felis CF0218 is a novel TMH family protein with potential as a diagnostic antigen for diagnosis of C. felis infection

Kenji Ohya et al. Clin Vaccine Immunol. 2008 Oct.

Abstract

Chlamydophila felis is a causative agent of acute and chronic conjunctivitis and pneumonia in cats (feline chlamydiosis). Also, C. felis is a suspected zoonotic agent of such diseases as non-Chlamydia trachomatis conjunctivitis in humans, although this is controversial. At present, there is no serodiagnostic system that specifically detects C. felis infection conveniently. Current systems use antigens such as lipopolysaccharide that cross-react with all chlamydia species. In addition, it is difficult to distinguish between cats that are vaccinated with the commercial vaccine against C. felis and cats that are infected with C. felis. Here, we describe a new candidate diagnostic antigen for diagnosis of C. felis infection, CF0218, that was obtained by screening a genomic expression library of C. felis Fe/C-56 with C. felis-immunized serum. CF0218 was a putative transmembrane head (TMH) family protein with bilobed hydrophobic motifs at its N terminus, and orthologues of CF0218 were not found in the Chlamydophila pneumoniae or Chlamydia trachomatis genomes. The recombinant CF0218 was not recognized by antiserum against C. trachomatis, suggesting that CF0218 is C. felis specific. CF0218 transcription during the course of C. felis infection was confirmed by reverse transcription-PCR. By indirect immunofluorescence analysis, CF0218 was colocalized with the C. felis-formed inclusion bodies in the infected cells. The antibody response against CF0218 was elevated following C. felis infection but not by vaccination in experimentally vaccinated and infected cats. These results suggest that CF0218, a novel TMH family protein of C. felis, possesses potential as a C. felis infection-specific diagnostic antigen.

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Figures

FIG. 1.
FIG. 1.
Gene arrangement of the TMH loci in the C. felis (Fe/C-56), C. abortus (S26/3), and C. caviae (GPIC) genomes and the analogous regions in the C. pneumoniae (J138) and C. trachomatis (D/UW-3/Cx) genomes. By screening of the C. felis genome library with anti-C. felis serum, the positive clone obtained was placed at the TMH locus (between the lpxB and pmp genes; an arrow at both ends) in the C. felis genome. The TMH loci are present in the C. felis, C. abortus, and C. caviae genomes but not present in the C. pneumoniae and C. trachomatis genomes. Representative locus tags (and gene names) are shown on each ORF (arrows). Black arrows show putative TMH family proteins.
FIG. 2.
FIG. 2.
Comparative analysis of the predicted amino acid sequences of CF0218, CAB764, CAB766, and CCA797. (A) Multiple alignment of CF0218, CAB764, CAB766, and CCA797. Boxes indicate identical residues. (B) Comparison of hydropathy profiles of CF0218, CAB764, CAB766, and CCA797. Profiles were determined using the algorithm developed by Kyte and Doolittle (18) with a window size of 11 amino acids. The relative hydrophobicity of each protein is shown on the vertical axis. Negative numbers indicate relative hydrophilicities. The bilobed hydrophobic region present in each protein is circled.
FIG. 3.
FIG. 3.
Production of recombinant CF0218 and its immunogenicity. (A) Purified GST alone, GST-CF0218, and GST-cleaved CF0218 were separated by SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B to E) Equal amounts of recombinant CF0218 shown in panel A and purified C. felis EB were separated by SDS-PAGE and analyzed by immunoblotting using rabbit antiserum raised against CF0218 (B), C. felis-hyperimmunized antiserum (C), C. trachomatis-hyperimmunized antiserum (D), and C. psittaci-hyperimmunized antiserum (E). Molecular mass standards are indicated in kilodaltons on the left sides of panels.
FIG. 4.
FIG. 4.
RT-PCR analysis of cf0218 expression in infected cells. Specific messages for ompA and cf0218 were detected from total RNA of HeLa cells infected with C. felis at the times indicated (including noninfected cells as negative control). DNase I-treated total RNA was applied to reactions in the absence (−) or presence (+) of reverse transcriptase (RT). Message for ompA was amplified as a control for chlamydial infection. C. felis genomic DNA was amplified as a PCR control.
FIG. 5.
FIG. 5.
Localization of CF0218 in C. felis-infected cells. HeLa cells infected with C. felis for 72 h were fixed and stained with DAPI for host cell nuclei and apparent chlamydial inclusion bodies (blue; white arrowheads show large chlamydial inclusion bodies), anti-chlamydial LPS (red), and anti-CF0218 (green). The rightmost columns show merged triple fluorescence images. Bars, 10 μm.
FIG. 6.
FIG. 6.
Antibody response against EB and CF0218 in experimentally vaccinated and C. felis-infected cats. Female specific-pathogen-free cats were inoculated with the vaccine containing formalin-inactivated C. felis EB (closed squares, solid line) or with saline (closed circles, dashed line) via the intramuscular route twice (at 0 and 3 weeks; open triangles). Subsequently, 5 weeks after the first vaccination (black triangles), all cats were inoculated with 104 50% embryo infectious doses of C. felis via the mucosal (conjunctival, oral, and nasal) route. To assess antibody responses against C. felis EB (A) and CF0218 (B) from sera at each time point, ELISAs were performed using detergent-inactivated C. felis EB and the recombinant CF0218 as antigens. Each sample was measured in duplicate. Data represent the mean OD value ± standard deviation. Significant differences (P < 0.01) between control serum (at 0 weeks) and at each time point are indicated as asterisks (single for the vaccinated cats and double for the nonvaccinated cats).
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References

    1. Azuma, Y., H. Hirakawa, A. Yamashita, Y. Cai, M. A. Rahman, H. Suzuki, S. Mitaku, H. Toh, S. Goto, T. Murakami, K. Sugi, H. Hayashi, H. Fukushi, M. Hattori, S. Kuhara, and M. Shirai. 2006. Genome sequence of the cat pathogen, Chlamydophila felis. DNA Res. 13:15-23. - PubMed
    1. Baker, J. A. 1942. A virus obtained from a pneumonia of cats and its possible relation to the cause of atypical pneumonia in man. Science 96:475-476. - PubMed
    1. Bannantine, J. P., R. S. Griffiths, W. Viratyosin, W. J. Brown, and D. D. Rockey. 2000. A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane. Cell. Microbiol. 2:35-47. - PubMed
    1. Bannantine, J. P., D. D. Rockey, and T. Hackstadt. 1998. Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane. Mol. Microbiol. 28:1017-1026. - PubMed
    1. Brade, L., O. Holst, P. Kosma, Y. X. Zhang, H. Paulsen, R. Krausse, and H. Brade. 1990. Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide. Infect. Immun. 58:205-213. - PMC - PubMed

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