LAUSR

Significance Genetic analysis of Rickettsia has been difficult. We developed a transposon and selection scheme to facilitate the isolation of Rickettsia conorii mutants with insertional lesions. Here, we demonstrate that the R. conorii polysaccharide synthesis operon (pso) encompasses genetic determinants for biosynthesis of the O antigen, which also affect the composition of outer-membrane proteins, invasion of host cells, and pathogenesis. The O antigen provides essential barrier functions and plays a major role in host–pathogen interactions. Our findings suggest that infected hosts develop protective immunity against R. conorii via the production of antibodies targeting the O antigen. Conservation of pso among rickettsial species suggests that it may play a universal role in O-antigen synthesis, disease pathogenesis, and the development of immunity. Rickettsial diseases have long been diagnosed with serum antibodies cross-reactive against Proteus vulgaris (Weil–Felix reaction). Although Weil–Felix antibodies are associated with the development of immunity, their rickettsial target and contribution to disease pathogenesis are not established. Here, we developed a transposon for insertional mutagenesis of Rickettsia conorii, isolating variants defective for replication in cultured cells and in spotted fever pathogenesis. Mutations in the polysaccharide synthesis operon (pso) abolish lipopolysaccharide O-antigen synthesis and Weil–Felix serology and alter outer-membrane protein assembly. Unlike wild-type R. conorii, pso mutants cannot elicit bactericidal antibodies that bind O antigen. The pso operon is conserved among rickettsial pathogens, suggesting that bactericidal antibodies targeting O antigen may generate universal immunity that could be exploited to develop vaccines against rickettsial diseases.