RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence

Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host. Author summary Oxygen often becomes a highly limiting factor within tissues, in particular during inflammation and abscesses formation, leading to hypoxic or even strictly anaerobic conditions. Consequently, B. pseudomallei, a highly neglected and difficult to treat human pathogen has to adjust its metabolism accordingly in order to survive in oxygen-restricted environments. Here, we identify key regulators of anaerobic B. pseudomallei metabolism and demonstrate the fundamental role of the RegAB redox sensing two-component signal transduction system as the master regulator of anaerobic physiology and its extensive effect on virulence. Further investigations of two RegAB-dependend redox regulators, NarL and FNR, allowed us to provide a first comprehensive insight into the complex regulatory network of anaerobic gene expression in B. pseudomallei. Integrating these results, we propose a regulatory model of gene expression under oxygen-limiting conditions in B. pseudomallei. The high level of conservation of the RegAB two-component system in the proteobacteria indicates that our findings are relevant for a broad range of bacteria including may important human and animal pathogens.