BackgroundThe chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars.ResultsA plasmid transfection approach was used to examine the function of the product of CT223 and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that CT223, and, to a lesser extent, adjacent inc genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells.ConclusionThese studies suggest that certain Inc proteins block cytokinesis in C. trachomatis-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.

The chlamydiae are obligate intracellular bacteria that occupy a nonacidified vacuole (the inclusion) during their entire developmental cycle. These bacteria produce a set of proteins (Inc proteins) that localize to the surface of the inclusion within infected cells. Chlamydia trachomatis IncA is also commonly found in long fibers that extend away from the inclusion. We used standard and confocal immunofluorescence microscopy to demonstrate that these fibers extend to newly developed inclusions, termed secondary inclusions, within infected cells. Secondary inclusions observed at early time points postinfection were devoid of chlamydial reticulate bodies. Later in the developmental cycle, secondary inclusions containing variable numbers of reticulate bodies were common. Reticulate bodies were also observed within the IncA-laden fibers connecting primary and secondary inclusions. Quantitative differences in secondary inclusion formation were found among clinical isolates, and these differences were associated with serovar. Isolates of serovar G consistently produced secondary inclusions at the highest frequency (P < 0.0001). Similar quantitative studies demonstrated that secondary inclusion formation was associated with segregation of inclusions to daughter cells following cytokinesis. We conclude that the production of secondary inclusions via IncA-laden fibers allows chlamydiae to generate an expanded intracellular niche in which they can grow and may provide a means for continuous infection within progeny cells following cell division.The chlamydiae are obligate intracellular bacteria that develop within a nonacidified vacuole, the inclusion, which provides them with a unique intracellular environment. The inclusion membrane is not passively permeable to low molecular weight compounds (13), yet intravacuolar ion concentrations are very similar to those found in the cytoplasm (11). The events leading to inclusion biogenesis are unclear, but the process is likely controlled by proteins produced by chlamydiae and delivered to the inclusion membrane and/or the host cell cytosol (29). All chlamydiae produce a set of proteins, termed Inc proteins, which are localized to the inclusion membrane (20) and are likely important in inclusion development. The IncA proteins of Chlamydia trachomatis, Chlamydophila caviae, and Chlamydophila pneumoniae have also been localized to discrete filamentous structures (IncA-laden fibers) that extend away from the inclusion membrane into the cytosol of host cells (3). Under conditions of stress, these fibers can accumulate antigens normally localized to the outer membrane of intracellular developmental forms (5). We undertook the studies reported here to elucidate the development and function of these unique IncA-laden fibers. Utilizing both standard and confocal immunofluorescence microscopy, we present evidence demonstrating that these fibers participate in the generation of secondary inclusions within cells. Additionally, we report that isolates of different C. trachomatis serovars p...

A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis-and Chlamydophila caviaeinfected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-a-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect a-adducin that was localized to the margins of the inclusion. The demonstration of a-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host-pathogen interactions in this system.