Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins

Abstract

Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD‐sphingomyelin. Both fusion of exocytic vesicles and long‐term avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis‐infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial‐specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co‐transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD–G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis demonstrated that IncD–G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

“…IncG is expressed already 2 h after infection (19). This correlates well with our finding that infected cells treated 8 h in the infectious cycle result in SIB able to recruit 14-3-3␤ to the membrane, indicative of a normal IncG translocation.…”

“…(19). It localizes to the inclusion membrane and there interacts with the mammalian protein 14-3-3␤ (12).…”

Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

“…These data are consistent with detection of CopB2 within intra-inclusion chlamydiae via indirect immunofluorescence (15). In contrast, both CopB and IncG are efficiently secreted and are not detected accumulating within bacteria at this stage of development (15,47). While these data do not indicate precise localization for CopB or CopB2, they indicate that both proteins are capable of associating with membranes.…”

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

“…We have shown recently that IncA transcript is not detected until sometime between 6 h and 12 h after infection (Scidmore-Carlson et al, 1999). A more detailed temporal analysis revealed that IncA transcript is detectable by 10 h after infection (Fig.…”

“…Production of rabbit polyclonal monospeci®c antibodies to IncA, F and G has been described previously (Scidmore-Carlson et al, 1999). Subcon¯uent monolayers of HeLa 229 cells on gridded CELLocate glass coverslips (Eppendorf) were infected with C. trachomatis L2 EBs at MOIs of either <2±3 or 0.1, as stated in the text.…”