TargeTron inactivation of Chlamydia trachomatis gseA results in a lipopolysaccharide KDO-deficient strain that is cytotoxic for cells

All members of the family Chlamydiaceae lipopolysaccharide (LPS) possess a shared carbohydrate trisaccharide antigen 3-deoxy-D-manno-oct-2-ulosonic (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 (L2deltagseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a KDO-specific monoclonal antibody demonstrated that L2deltagseA lacked KDO. L2deltagseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating 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 microcopy of L2deltagseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol treated L2deltagseA infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide crosslinking, suggesting elementary body outer membrane structure was affected by the lack of KDO. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2deltagseA was significantly more cytotoxic to host cells than wild type. The cytotoxic phenotype may result from altered outer membrane biogenesis structure and/or function, or conversely by 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 post-infection host cell survival.