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

Abstract

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.

The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei

“…Our observation of replication-deficient B. pseudomallei 1026b lacking either the NarX histidine kinase or the NarL DNA-binding regulator suggests that nitrate metabolism is important for intracellular survival. This observation parallels a similar study showing decreased, but not significant reductions in intracellular replication of a Δ narL mutant strain of an environmental B. pseudomallei strain E8, which was isolated from soil 35 . The intracellular growth deficiencies of Δ narX and Δ narL B. pseudomallei 1026b observed in our study can be explained by lack of active NarG and subsequent nitrate reductase activity, which is important for Mycobacterium tuberculosis intracellular growth 59 , although this definitive connection remains to be characterized in B. pseudomallei.…”

“…S3 and Supplementary Table S3 ). Recently, another group has examined the transcriptional regulation of Δ narL in B. pseudomallei E8, an environmental isolate, after 30 min of anaerobic growth supplemented with 50 mM NaNO 3 suggesting that NarL is important for activating electron transport and nitrite reduction to nitric oxide under hypoxic conditions 35 . Here, we present the global expression patterns of B. pseudomallei 1026b during aerobic biofilm formation grown with either 10 mM NaNO 3 or 10 mM NaNO 2 to gain further insights into the NarX-NarL TCS as biofilms form.…”

Two-component systems regulate bacterial virulence in response to the host gastrointestinal environment and metabolic cues

“…The link between NarL and biofilm formation is apparent in multiple pathogens and suggests a clear bacterial signalling and activity cascade: the activation of NarL by nitrate is a positive indication to the cell of sufficient exogenous nitrogen sources for pathogenic growth, and therefore the protection biofilm formation provides is not necessary for survival in these nitrogen sufficient environments. Similar biofilm control by the NarLX/NarPQ TCSs can be seen in human pathogen Burkholderia pseudomallei [ 71 , 89 ]. As nitrate and nitrite control and metabolism is central to many cellular processes, it is not surprising that many symbiotic and pathogenic bacteria have one or both of the previously described nitrogen-sensing TCSs or a homolog of these TCSs [ 22 , 25 , 90 ], though more detailed mechanistic research is necessary to link these nitrogen related TCSs directly to the modulation of virulence in more pathogens.…”

DksA, ppGpp and RegAB regulate nitrate respiration inParacoccus denitrificans

“…A recent study has shown that RegAB acts both positively and negatively under hypoxic conditions in Burkholderia pseudomallei , an α-proteobacterium quite closely related to P. denitrificans (57). Specifically, RegAB are required for upregulation of Nar under hypoxic conditions in this organism, though this may involve both direct regulation by RegA and indirect regulation, via FNR (57). Indirect effects of RegAB via NarR and/or FnrP are also possible in P. denitrificans ; we are able to discern a potential RegA binding site in the narR - narK intergenic region that controls expression of the narGHJI genes encoding Nar (50).…”

“…A recent study has shown that RegAB acts both positively and negatively under hypoxic conditions in Burkholderia pseudomallei , an α-proteobacterium quite closely related to P. denitrificans (57). Specifically, RegAB are required for upregulation of Nar under hypoxic conditions in this organism, though this may involve both direct regulation by RegA and indirect regulation, via FNR (57).…”