Genetic control of Group 3 (K96) capsule synthesis and complement resistance in an extraintestinal pathogenic...

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 .

Group 2 capsules are established extraintestinal pathogenic Escherichia coli (ExPEC) virulence factors and are the type most often associated with human isolates. Group 3 capsules have previously been considered a sub-type of Group 2, although nothing was known about factors controlling their expression. Capsule serotype K96-encoding ExPEC strains are increasingly isolated from human infections and inhabit numerous other hosts. It is critical to understand the factors that enable their pathogenic versatility and survival in specific environments. Our study shows that K96 capsule of strain M12 is required for human complement resistance. Additionally, we have identified genetic factors that control Group 3 capsule synthesis, including a potential phase-variable mechanism as well as transcriptional control by OxyR. Co-regulation of Group 3 capsule synthesis genes with genes necessary for oxidative stress resistance may increase the virulence of some versatile ExPEC strains.