Functional characterisation of SOD isozymes in Klebsiella pneumoniae KR3167

Klebsiella pneumoniae is the causative agent of several nosocomial and community acquired infections. Some of its virulence determinants have been identified but it is not known how it copes with damaging effects of reactive oxygen species. Superoxide dismutase (SOD) is responsible for removal of toxic superoxide radicals, and K. pneumoniae genome contains three superoxide dismutase genes, sodA, sodB, and sodC coding for Mn-, Fe- and CuZn- co-factored SODs, respectively. This work was designed to investigate the role of each SOD isozyme in K. pneumoniae for oxidative stress response, mucoviscosity, biofilm formation, metabolism, and in vivo colonisation, and persistence. Lambda Red system and Flp-recombinase mediated excision were used to construct markerless single, double, and triple SOD, and they were tested in vitro and in vivo. All sod genes contribute to total SOD activity, but sodB codes for the major activity. While sodB is non-inducible, sodA and sodC are inducible. The presence of Mn-SOD is critical for K. pneumoniae growth in oxidative conditions whereas no detectable impact of Fe- and Cu/Zn-SOD could be recorded in vitro. Mutation of sodA, but not sodB, had an impact on the fermentation profile of the microbe. sodC contributed in metabolism when both sodA and sodB were absent. The presence of sodA, but not sodB and sodC, is required for normal cell morphology and size, exo-polysacharide production, aggregation and biofilm formation. It was also found that the absence of Mn-SOD leads to a significant increase in β-galactosidase activity, and cell elongation. In vivo results showed that while only Mn-SOD is needed for K. pneumoniae colonisation, for persistence in the lungs Cu/Zn SOD is required. These results demonstrate that SOD isozymes in K. pneumoniae have unique roles in oxidative stress resistance, metabolism, cell morphology, biofilm architecture, colonisation and virulence