Characterisation of the Burkholderia pseudomallei two-component signal transduction system BprRS

Abstract

Burkholderia pseudomallei is a Gram negative facultative intracellular bacterium that is the causative agent of the disease Melioidosis. Melioidosis has a high prevalence in many tropical countries and a mortality rate of 14% in Australia and 40% in Thailand. B. pseudomallei is highly resistant to antibiotics and has a number of virulence factors which help it escape being effectively targeted and killed by host defences. In many bacterial pathogens, virulence factors are often controlled by two-component signal transduction systems (TCSTS). These systems involve a membrane-bound sensor histidine kinase (SHK) that detects stimuli, and a cytoplasmic response regulator (RR) that can alter expression of a particular set of genes allowing for control over several different biological processes such as motility and chemotaxis. B. pseudomallei has many putative TCSTSs, one of which is the BprRS system. Inactivation of either the SHK bprS or the RR bprR attenuated B. pseudomallei virulence but inactivation of the whole bprRS system did not. These data suggest that removal of each single component results in aberrant crosstalk interactions with other B. pseudomallei TCSTSs that lead to dysregulation and attenuated virulence. Therefore, the BprR and BprS proteins may be suitable targets for therapeutic intervention. In silico analysis of the BprR and BprS proteins demonstrated that both were highly conserved in most B. pseudomallei strains and other Burkholderiaceae species members. Each protein displayed strong conservation of functionally important domains and motifs identified in characterised SHKs and RRs from other bacterial species. Attempts were made to express and purify the BprR and BprS proteins via heterologous expression in E. coli. Ni-NTA affinity purification of the BprS SHK protein was unsuccessful with only insoluble protein produced; future work should focus on optimising soluble expression with different fusion tags and expression conditions. Purification of the BprR RR protein using a combination of glutathione-based affinity, gel filtration, thrombin cleavage and Ni-NTA affinity purification was largely successful. It was shown that the purified BprR was primarily in a mostly folded monomeric form and functions to bind a 208-bp DNA fragment located directly upstream of the BPSS0688 gene. Further studies need to be conducted in order to further characterise the structural and functional properties of both the BprS and BprR protein in order to identify if these two proteins are appropriate targets for novel therapeutic techniques using small molecule inhibitors.