The Interrogation of a Proposed Multikinase-Network in Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with very poor clinical outcomes, including an up to 40% fatality rate and a significant relapse rate. The pathogen has a wide endemic range across South-east Asia and Northern Australia. At present the melioidosis is poorly managed, with no rapid and effective diagnostic tools available, no vaccine either available or in clinical trials, and current antibiotic therapy often proving inadequate.

One possible target for developing a novel therapeutic agent for B. pseudomallei is multikinase networks (MKNs). These networks consist of multiple two component systems, a ubiquitous signalling system in prokaryotes, which interact with one another. MKNs have been found to control many features related to virulence and major microbial lifestyle choices, and it is therefore likely they would make effective antibiotic targets. By examining the amino acid residues which govern molecular recognition within two component systems, a putative multikinase network has been predicted in B. pseudomallei.

In this dissertation three branches of experimentation used to investigate this network are presented: phosphorylation assays to determine the flow of phosphoryl groups between the members of the network, two hybrid assays to detect other interactions within the network and confirm any interactions detected by the phosphorylation assays in vivo, and creation and phenotypic analysis of knockout mutants in the model organism Burkholderia thailandensis. The phosphorylation assays confirmed the existence of the MKN, with multiple interactions between the proposed members being observed. The two hybrid assays provided a limited further conformation, whilst the knock out mutants demonstrated the importance of the network for the formation of biofilms and for swarming motility. This study thus validated the method used to predict the multikinase network, demonstrated the existence of one such network in B. pseudomallei, and revealed two phenotypes for which elements of the signalling network are vital.