| dc.description.abstract | Two-component systems (TCSs) are a primary means of responding to environmental cues across the bacterial domain, and usually act independently of one another, forming discrete, isolated units. Multikinase networks (MKNs) are systems of multiple TCSs which can interact to integrate numerous signals into a decided cellular output, conferring an advantage to the cell. This work focuses on a novel MKN from the opportunistic pathogen, Burkholderia cenocepacia. Through purification of each putative TCS, their ability to undergo non-cognate phosphorylation with one another was assessed in vitro through the phosphotransfer assay. This revealed that three sensor kinases, BCAM0442, BCAM0715 and BCAS0585 are able to phosphorylate one another’s response regulators. This non-cognate phosphotransfer was also possible, albeit to a lesser extent, in the presence of the cognate response regulator protein. Deletion mutants of each TCS and investigation of TCS promoter induction revealed that BCAM0442/3 is implicated in copper resistance, and BCAM0714/5 is implicated in cadmium and zinc resistance. Exposure of B. cenocepacia to copper or zinc enhances resistance to imipenem, a phenomenon in which this MKN is implicated. A MKN deletion mutant is heavily attenuated in virulence in Galleria mellonella, linking the metal response with virulence in B. cenocepacia. Additionally, exploration of the copper response by RNA-seq analysis revealed substantial upregulation of the genes surrounding BCAM0442/3. A CopABCDE-like system was strongly upregulated in a BCAM0442/3-dependent manner, suggesting that BCAM0442/3 directly regulates this system. Previous work identified that BCAM0714/5 regulates a downstream gene region, BCAM0716-21, deletion of which confers susceptibility to zinc in B. cenocepacia. Complementation with the BCAM0716/17 gene region restored resistance to zinc, implicating these genes in the zinc response of B. cenocepacia. Given the environmental impact of heavy metal pollution and the use of metals as both an antibacterial strategy by the immune system and in medical devices, understanding the mechanisms of bacterial metal resistance is vital. This work has identified a novel metal-sensing MKN in B. cenocepacia, linking the cadmium, zinc and copper responses with virulence and carbapenem resistance. | en_GB |
