Novel transcriptional regulators of virulence in the genus Burkholderia

Bacteria have evolved systems through which they can sense their environment, enabling them to turn genes on and off in response to environmental conditions and thus ensure their continued survival and success. One such class of sensors is the family of two-component systems (TCSs). Two-component systems comprise a sensor protein on the bacterial surface that detects the presence of a particular signal and relays that signal to a protein (termed the ?response regulator?) inside the bacteria. The response regulator then turns a variety of genes on or off, enabling the bacteria to respond to the original signal. These TCSs can switch on diverse genes that are necessary for the bacteria to cause disease. In some cases, bacteria which lack a particular TCS cannot cause disease at all, and can be used as a vaccine. This study will investigate the TCSs within a group of bacteria called Burkholderia. These bacteria are resistant to almost every available antibiotic and are capable of causing serious disease of humans, animals and plants. Two particular species within the Burkholderia group can be used as warfare agents. In stark contrast, other Burkholderia are harmless bacteria residing in soil where they can boost crop production, resulting in calls for their environmental application. How Burkholderia organisms control this remarkable diversity is unknown. From available genome sequences, it is apparent that Burkholderia organisms possess an unusually high number of TCSs. Preliminary studies show that the TCSs within Burkholderia behave very differently from equivalent systems within other bacterial species. The proposed research will investigate these novel Burkholderia TCSs, and identify those which are particularly important for causing disease. Having identified these key TCSs, studies will be performed that will reveal which genes are being turned on or off by these systems. Different Burkholderia species vary considerably in their ability to cause disease. Genome sequences of Burkholderia that can and cannot cause disease will be compared to see if these differing abilities might be explained by the presence or absence of particular TCSs and their associated genes. Finally, TCS inhibitors will be tested against Burkholderia to determine whether such inhibitors may be used as novel antibiotics. In conclusion, this research will identify Burkholderia systems that are essential for causing disease, thus identifying novel ways to treat and prevent Burkholderia infection.

Organisms of the Burkholderia genus exhibit diverse biological properties and are capable of occupying a wide variety of ecological niches. The genus, which exhibits innate resistance to the majority of antimicrobial agents, encompasses major pathogens of plants, humans and animals, including the biowarfare agents B. mallei and B. pseudomallei (causative agents of glanders and melioidosis respectively). Other Burkholderia species are capable of causing infection of both humans and plants, with certain species also having potential as biocontrol and bioremediation agents. The transcriptional pathways that regulate this remarkable versatility are uncharacterized. Bacterial two-component systems (TCSs) are important regulators of virulence factors in plant and animal pathogens. Their pivotal role in the regulation of virulence is exemplified by the fact that TCS-deficient Salmonella strains have been exploited as live vaccines. Additionally, TCSs have been recognised as relevant antimicrobial targets. Burkholderia genomes contain a remarkably high number of TCSs, highlighting the ability of these organisms to respond to diverse environmental stimuli. Preliminary studies show that the Burkholderia TCSs have different activities and different target genes from homologous TCSs in other bacterial species. Developing these preliminary findings, the proposed research aims to define the activity of novel TCSs within the Burkholderia genus and establish their role in virulence. Using B. cenocepacia as a model organism, TCS-deficient strains will be constructed and assessed in plant and animal models of infection, identifying the TCSs associated with virulence. Selected TCSs will be investigated by ChIP-on?chip analysis to identify the genes directly regulated by that system. Comparison of the promoter regions of those target genes will allow identification of consensus sequences likely to facilitate binding of the TCS response regulator. A combination of in silico analysis and dot-blot hybridizations will be applied to virulent and avirulent Burkholderia species to establish whether their differing virulence and biological properties correlate with the presence or absence of TCSs, relevant consensus sequences and/or their target genes. Finally, competitive inhibitors of bacterial histidine kinases will be investigated to determine their potential role in the treatment of Burkholderia infection. In conclusion, this programme of research will make a significant contribution to our understanding of the mechanisms which underlie the diversity of the Burkholderia. The research will highlight novel antimicrobial targets and guide the rational design of TCS-deficient strains that may be exploited as live vaccines, thus identifying novel strategies for the treatment and prevention of multi-drug resistant Burkholderia infection.