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 ( ), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a -null strain (L2Δ ) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2Δ lacked Kdo. L2Δ reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO 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 L2Δ -infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2Δ -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 L2Δ 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.