TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

Microscopic Analysis of the Chlamydia abortus Inclusion and Its Interaction with Those Formed by Other Chlamydial Species.

The Chlamydiae are obligate intracellular pathogens that develop and multiply within a poorly characterized parasitophorous vacuole (the inclusion) during growth. Chlamydia abortus is a major pathogen of sheep and other ruminants, and its inclusion development is poorly characterized. We used immunofluorescence microscopy, quantitative culture, and qPCR to examine C. abortus inclusion development and to examine the interaction of C. abortus inclusions with those formed by other species. Antibodies used in these studies include sera from ewes from production facilities that were naturally infected with C. abortus. Multiple inclusions are often found in C. abortus-infected cells, even in populations infected at very low multiplicity of infection. Labeling of fixed cells with sera from infected sheep revealed fibrous structures that extend away from the inclusion into the cytoplasm of the host cell. C. abortus inclusions fused with C. caviae and C. psittaci inclusions in coinfected cells. Inclusions formed by C. abortus and C. caviae did not fuse with inclusions formed by C. trachomatis, C. pneumoniae, or C. pecorum. The ability of inclusions to fuse was correlated with the overall genomic relatedness between species, and with sequence similarity in the inclusion membrane protein IncA. Quantitative PCR data demonstrated that C. abortus grows at a decreased rate during coinfections with C. caviae, while C. caviae growth was unaffected. The collected data add depth to our understanding of inclusion development in this significant zoonotic veterinary pathogen.