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

Extraintestinal pathogenic Escherichia coli (ExPEC) frequently produce Group 2 or 3 capsules that enhance virulence, yet genetic regulation and specific roles of Group 3 capsules remain poorly understood. Here, we demonstrate that the K96 Group 3 capsule in ExPEC strain M12 confers resistance to human serum complement. Using genetic screens, we identified phase-variable mutations in kpsC and essential roles for transcriptional regulators OxyR and RfaH in activating capsule synthesis via a distant promoter. Mutants lacking OxyR or RfaH failed to produce capsule, exhibited complement sensitivity, and showed attenuated virulence in Galleria mellonella . These findings reveal novel mechanisms controlling Group 3 capsule expression and underscore the capsule’s contribution to immune evasion. IMPORTANCE 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 the 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.