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Diversity within inc genes of clinical Chlamydia trachomatis variant isolates that occupy non-fusogenic inclusions^a

Department of Microbiology, Oregon State University, Corvallis OR 97331-3804, USA¹
NADC/ARS/USDA Ames, IA 50010, USA²
Division of Allergy and Infectious Diseases, School of Medicine, University of Washington, Seattle, WA 98195, USA³

Author for correspondence: Daniel D. Rockey. Tel: +1 541 737 1848. Fax: +1 541 737 0496. e-mail: rockeyd{at}orst.edu

The obligately intracellular chlamydiae are bacterial pathogens that occupy intracellular vacuoles, termed inclusions, as they develop and multiply. Typical Chlamydia trachomatis isolates occupy inclusions that fuse with other C. trachomatis inclusions within cells infected with multiple elementary bodies (wild-type phenotype). The authors of this study have recently described C. trachomatis isolates that form multiply-lobed, non-fusogenic inclusions within single cells infected with multiple elementary bodies (variant phenotype). Inclusions formed by these isolates uniformly lacked the protein IncA on the inclusion membrane (IM). In the present work, the study of the C. trachomatis inclusion phenotype has been expanded to include 27 variant and 13 wild-type isolates. Twenty-four of the 27 variant isolates were IncA-negative, as detected by fluorescence microscopy and immunoblotting, but three variants localized IncA to the IM. The IncA-positive variants formed inclusions that fused, at a reduced rate, with those occupied by wild-type isolates and with inclusions formed by other IncA-positive variants. Nucleotide-sequence analysis of the incA sequences from the variant isolates identified a variety of distinct sequence polymorphisms relative to incA from wild-type strains. The authors also demonstrate that a second Inc protein, CT223p, is not found in the IM in selected C. trachomatis isolates. No change in the structure or the fusogenicity of the inclusions was associated with the presence or absence of CT223p.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Sequence analysis of the chlamydial isolates examined in this study. For each isolate, the strain designation is indicated in the left column and the accession number for the incA sequence is indicated in the right column. The complete incA sequence was determined for the isolates shown in bold, whereas the first 450–500 nt were analysed for all other isolates. The sequences of incA from five additional wild-type isolates (E9332, J2364, J3464, J9311 and J9325) were identical to that of the serovar D sequence in the Chlamydia genome-project database (Stephens et al., 1998 ), and these sequences are not represented in the figure. One recent IncA-positive variant, J(s)9045, was not subjected to sequence analysis. The results of the fluorescence-microscopy analysis are listed under IncA IF, with a + indicating labelling of the IM in cells infected with the isolate. The length of the IncA sequence in the ORF predicted from the nucleotide-sequence analysis is indicated by the open bar for each isolate. , Silent base changes in incA; *, a base change that leads to an amino-acid substitution; , single base deletions or insertions that lead to premature truncation of the protein; , single base changes that lead directly to a stop codon. Solid bars indicate larger deletions and are drawn to scale.

Abbreviations: DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; HSP60, 60kDa heat-shock protein; IM, inclusion membrane; MOMP, major outer-membrane protein; (s), isolates that formed variant inclusions

^a The GenBank accession numbers for the sequences reported in this paper can be found in Fig. 1.

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