Genetic control of Group 3 (K96) capsule synthesis and complement resistance in an extraintestinal pathogenic Escherichia coli ST69 strain

Extraintestinal pathogenic Escherichia coli (ExPEC) often produce capsules belonging to Groups 2 or 3, which contribute to invasive disease in humans and other animals. Group 3 capsule loci contain conserved kps genes flanking serotype-specific glycosyltransferase and nucleotide-sugar biosynthesis genes, but little is known about genetic factors that control synthesis, or the specific roles that K96 capsules play in virulence. Previously, we identified a Group 3 serotype K96 capsule in a mastitis-associated strain M12 that is critical for its survival in some host tissues. In this study, we show that the K96 capsule of strain M12 confers resistance to human serum complement. We conducted genetic screens to determine how K96 capsule expression is controlled, which led to two principal findings. First, Group 3 capsule synthesis is unstable. Sequencing of spontaneously appearing mutants revealed potential phase-variable control of capsule expression. Some mutants harbored reversible frameshift mutations in a homopolymeric site within kpsC , which were also identified in other K96-encoding ExPEC isolates. Second, a transposon mutagenesis screen revealed that capsule synthesis requires proteins encoded outside of the kps locus, including the RfaH antiterminator and OxyR transcriptional regulator, which control activation of an unusually distant promoter. An Δ oxyR mutant of strain M12 failed to produce capsule, was extremely sensitive to complement-mediated killing, and avirulent in Galleria mellonella .