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United States Department of Agriculture

Agricultural Research Service

Title: Diversity Within Inc Genes of Clinical Chlamydia Trachomatis Variant Isolates That Occupy Non-Fusogenic Inclusions

Authors
item Rockey, Daniel - OREGON STATE UNIVERSITY
item Viratyosin, Wasna - OREGON STATE UNIVERSITY
item BANNANTINE, JOHN
item Suchland, Robert - UNIVERSITY OF WASHINGTON
item Stamm, Walter - UNIVERSITY OF WASHINGTON

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 25, 2002
Publication Date: N/A

Interpretive Summary: Chlamydia trachomatis is an obligate intracellular bacterium that develops and multiplies within a nonacidified vacuole termed an inclusion. A unique characteristic of C. trachomatis is the fusion of homotypic chlamydia- containing inclusions within infected cells. Recently, in a large-scale serotyping study of clinical isolates, a collection of C. trachomatis with unusual multiple nonfusogenic inclusions (nonfusogenic isolates) were identified. Immunofluorescence staining of selected nonfusogenic isolates revealed that IncA, a protein localized to the inclusion membrane, was undetectable in these mutants (Suchland et al., Infect. Immun. 68:360-367). In this report we expand on the characterization of the nonfusogenic isolates through immunofluorescence, immunoblotting, and PCR amplification/ DNA sequence analysis. Nucleotide sequence analysis of incA from 26 nonfusogenic isolates identified a variety of lesions within incA, leading to either truncated or full length but altered predicted IncA sequence. While patterns were evident in these analyses, no single common mutation was associated with the defect in IncA production. Surprisingly, these studies also identified 3 nonfusogenic isolates that re IncA-positive by immunofluroescence and immunoblot. Finally, parallel studies with a different Inc protein, CT223p, demonstrated that selected isolates did not accumulate this protein within infected cells. No correlation was observed between fusogenicity and presence or absence of CT223p. Collectively these data demonstrated that multiple genotypes of incA are associated with the fusogenicity defect. While over 90% of isolates are IncA-negative, in some .

Technical Abstract: Immunofluorescence staining of selected nonfusogenic chlamydia isolates revealed that IncA, a protein localized to the inclusion membrane, was undetectable in these mutants (Suchland et al., Infect. Immun. 68:360-367). In this report we expand on the characterization of the nonfusogenic isolates through immunofluorescence, immunoblotting, and PCR amplification/ /DNA sequence analysis. Nucleotide sequence analysis of incA from 26 nonfusogenic isolates (representing serovars B, D, D-, E, F, G, H, Ia, J and K) identified a variety of lesions within incA, leading to either truncated or full length but altered predicted IncA sequence. While patterns were evident in these analyses, no single common mutation was associated with the defect in IncA production. Surprisingly, these studies also identified 3 nonfusogenic isolates that are IncA-positive by immunofluorescence and immunoblot. Each of these isolates had a unique incA sequence but each encoded intact IncA. The morphology of mature inclusions produced by these IncA-positive nonfusogenic isolates was altered relative to both the wild type and IncA-negative nonfusogenic stains. Finally, parallel studies with a different Inc protein, CT223p, demonstrated that selected isolates did not accumulate this protein within infected cells. No correlation was observed between fusogenicity and presence or absence of CT223p. Collectively these data demonstrated that multiple genotypes of IncA are associated with the fusogenicity defect. While over 90% of isolates are IncA-negative, in some cases IncA- positive isolates are also nonfusogenic.

Last Modified: 9/10/2014
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