Differential virulence and immune recognition of Klebsiella pneumoniae O-antigen subtypes O2α and O2β

Abstract

Klebsiella pneumoniae infections are sharply on the rise among at-risk populations. K. pneumoniae 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 gmlABC region of an O2b strain of K. pneumoniae, 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 K. pneumoniae O-antigens by defining a fitness advantage of O2b compared to O2a and informing vaccine design to combat this drug-resistant pathogen.

Keywords: Klebsiella pneumoniae; O-antigen polysaccharide; bioconjugation; pulmonary infections; vaccine.

Fig 1

Construction of isogenic strains of the O2-antigen subtypes O2β and O2α. (A) Pictogram representation of the O2α and O2β subtypes. (B) KR74 parent strain was mutated by knocking out the gmlABC locus to transform the strain from O2β producing to O2α (ΔgmlABC). O-antigen production was verified via western blot of full cell lysates of the bacteria using antibodies against each subtype (4). Red band is RNA polymerase loading control.

Fig 2

gmlABC complementation in Klebsiella. (A) Each individual gene—gmlA, gmlB, and gmlC—along with gmlBC and gmlABC, was cloned into pACT3 expression plasmid and electroporated into the ΔgmlABC strain. Complementation constructs were verified by PCR using check primers to amplify the expected fragments as displayed on the agarose gel. (B) Whole-cell lysates of the ΔgmlABC strain expressing each construct were probed via immunoblot with α-O2β antibody to test O-antigen expression and determine which constructs convert the strain to O2β expression.

Fig 3

Strain survival in normal human serum. Strains were incubated in 75% normal human serum for 1 h, serially diluted to enumerated colonies, and counted. Percent survival was determined as output CFU compared to input CFU. Replicates from two independent experiments are shown. Statistics were performed using unpaired t-test. **P < 0.005, *P < 0.05; ns, not significant.

Fig 4

Antibody production after immunization with O2α and O2β conjugate vaccines. (A) CD-1 female mice were immunized with either O2α-EPA or O2β-EPA at time points depicted in the immunization schematic. Blood samples were taken at indicated days. (B) Serum IgG at day 42 was measured via ELISA on plates coated with glycoengineered E. coli producing each O2 subtype. The x-axis indicates the immunization group. ELISA depicts serum concentrations at a 1:100 dilution.

Fig 5

Organ titers after infection with KR74 and DgmlABC. Female CD-1 mice were immunized with either EPA, O2α-EPA, or O2β-EPA and at day 42 were infected with either (A) KR74 or (B) DgmlABC isolates. At 24 h post-infection, lungs were harvested, homogenized, and CFU enumerated. At 6 h post-infection, blood was drawn from (C) KR74-infected mice or (D) DgmlABC-infected mice, and CFU were enumerated. Statistics were performed using Mann-Whitney. *P < 0.05; ns, not significant. L.O.D. = limit of detection of 20 CFU.