Differential virulence and immune recognition of <i>Klebsiella pneumoniae</i> O-antigen subtypes O2α and O2β

<i>Klebsiella pneumoniae</i> infections are sharply on the rise among at-risk populations. <i>K. pneumoniae</i> has nine serogroups of O-antigens. Recently, additional O-antigen subtypes within these serogroups have been identified; the contributions of these subtypes to pathogenic fitness and their immunogenicity, functional antibody responses, and cross-reactivity are unknown. We investigated how the addition of the single-branched galactose in O-antigen subtype O2b compared to O2a alters its virulence and host immune responses. We deleted the <i>gmlABC</i> region of an O2b strain of <i>K. pneumoniae</i>, converting it to an otherwise isogenic O2a strain. Complementation of this mutant allowed us to identify the specific genes responsible for the addition of the single branched galactose of O2b. Experiments using the O2a mutant and its parent O2b strain confirmed similar phenotypic expression of virulence factors beyond the O-antigen. Well-established murine models of pneumonia were used to determine the pulmonary fitness of the strains and assess the host innate immune responses. Complement-mediated killing assays suggested differences in susceptibility to innate immune defenses, with the O2a mutant being more susceptible to serum killing. Lastly, using polysaccharide-protein bioconjugate vaccines against these specific O-antigen subtypes, we determined that only partial cross-reactivity and protection are elicited. These studies advance our understanding of the immune response to <i>K. pneumoniae</i> O-antigens by defining a fitness advantage of O2b compared to O2a and informing vaccine design to combat this drug-resistant pathogen.