The serine/threonine protein kinases of Burkholderia pseudomallei

Burkholderia pseudomallei is a Gram-negative bacterium that is endemic in tropical and sub-tropical regions. B. pseudomallei is an opportunistic pathogen and is the causative agent of the human disease, melioidosis, which accounts for 20% of all septicaemic deaths in Thailand. The phosphorylation via serine/threonine protein kinases (STKs) coordinates a vast array of signal pathways in both eukaryotic and prokaryotic organisms. In bacteria these pathways include: biofilm formation, cell wall biosynthesis, metabolism, sporulation, stress response and virulence. In this study, I identified six genes in the B. pseudomallei K96243 genome, bpsl0220, bpsl0571, bpsl0597, bpsl1828, bpss1584 and bpss2102 encoding putative STKs. The importance of these enzymes in B. pseudomallei was not determined to date, and thus the aim of this study is to Understand the roles of these proteins in B. pseudomallei virulence and regulations of Cellular processes. Four of the genes, bpsl0220, bpsl0597, bpsl1828, and, bpss2102, were successfully cloned and over-expressed the putative STKs in Escherichia coli and the recombinant proteins were purified. Each of the recombinant proteins exhibited autophosphorylation as well as phosphorylation of a general kinase substrate in vitro, thus demonstrating that each of the four proteins has protein kinase activity. Furthermore, I constructed specific mutants in each of the four STK-encoding genes in B. pseudomallei. These mutants were assessed in a variety of in vivo and in vitro, assays. The inactivation of the genes in B. pseudomallei, identified a switch in colony morphotype may be associated with a change in lipopolysaccharide structure and the motility of the bacteria, which warrants further study. One of the mutants, Δbpsl1828 displayed a conditional temperature sensitivity in distilled water, and thus the protein encoded by this gene could potentially contribute to the ability of B. pseudomallei to persist in the tropical and sub-tropical environments. However, future work is required to confirm a role of BPSL1828 in the regulation of environmental stress. A second mutant, Δbpsl0220, was avirulent in the Galleria mellonella model of disease, and introducing the full-length gene into the mutant complemented the phenotype. This result suggests that BPSL0220 may play a role in virulence. However, future work should assess the Δbpsl0220 deletion mutant in the mouse model of melioidosis. If these results confirm the role of BPSL0220 in pathogenicity, subsequent work could investigate if BPSL0220 maybe used as a target for therapeutic treatment of B. pseudomallei.