Functional Characterisation Of Asparaginase And Glutaminase In Klebsiella Pneumoniae KR3167

Asparaginase and glutaminase are sets of enzymes that assist Klebsiella pneumoniae in acquiring necessary nitrogen sources when ammonia (NH3), the preferred nitrogen source, is low or absent. K. pneumoniae contains four putative asparaginase and glutaminase genes (yneH, ansA, ybiK, and KPN_01165) but their functions are unknown. Therefore, the aim of my Ph.D. project was to investigate the contribution of these enzymes to K. pneumoniae KR3167 biology and virulence by creating unmarked mutant strains using Lambda Red system and Flp-recombinase-mediated excision mutagenesis. A phenotypic assessment of the mutants was undertaken to determine the impact of asparaginase and glutaminase enzymes in survival and virulence of K. pneumoniae by using in vitro and in vivo assays. In growth medium not limited in nitrogen content, there was no significant difference in growth between the wild type and the mutants. In all assays, the yneH mutant showed no difference in phenotype compared to the wild type. However, all the other stains showed variation in asparaginase/glutaminase activity levels. In particular, the KPN_01165 mutation produced the greatest effects. The mutant strain suffered reduced growth and had low asparaginase and glutaminase activities. Moreover, this strain had less capsule synthesis and was attenuated in growth in serum-containing medium. My results showed that Lectin Pathway of complement (LP) is activated on the surface of K. pneumoniae KR3167 following the binding of the carbohydrate recognition molecule Collectin-11 (CL-11) which enhances C3 deposition. The role of LP was confirmed by using different mouse sera that were deficient of mannan-binding lectin serine protease (MASP-2), Ficolin A, and CL-11. Survival and killing assays using G.mellonella also revealed significant attenuation in virulence of ΔansA, ΔybiK , ΔKPN_01165, and ΔybiK/ΔKPN_01165 strains compared to wild-type KR3167. Collectively, these results show that asparaginase and glutaminase enzymes are important in survival and virulence of K. pneumoniae.