Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent Mycobacterium tuberculosis

Abstract

The DevR response regulator of is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the operon using H37Ra and H37Rv overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant gene of LIX48 with the WT gene. PhoP dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoP to the promoter and further revealed that DevR and PhoP proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoP but not with the mutant PhoP protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.

Metabolic switching ofMycobacterium tuberculosisduring hypoxia is controlled by the virulence regulator PhoP

“…While the eluent showed clear presence of DosR (lane 3), we were unable to detect DosR from the cell lysate of Δ phoP carrying the empty vector (p19Kpro) (lane 2, see Table S3). In agreement with recently reported PhoP-DosR interaction by Vashist et al (23), these results suggest specific in vivo interaction between PhoP and DosR.…”

“…Considering the above results, recently-reported PhoP-DosR interaction (23) emerged as a possible explanation to account for the regulation of hypoxia-inducible genes. To examine this, whole cell lysate of ΔphoP expressing His-tagged PhoP, as described previously (35), was incubated with Ni-NTA, and proteins were eluted from Ni-NTA column using imidazole (Fig.…”

“…To examine PhoP dependent dosR expression, we probed PhoP binding site within the dosR promoter (dosRup spanning −1438 to −774 relative to the start of the ORF). Based on the knowledge of consensus PhoP binding site (31, 32), and more recent results on PhoP binding (23) we could locate a region spanning −1117 to −1100 within dosRup as the likely PhoP box. Next, mutant dosRup (dosRupmut) was generated by introducing mutations at the PhoP box as shown in Fig.3A, and transcriptional fusions of the WT (dosRup) and the mutant promoter (dsoRupmut) were cloned at the ScaI site of pSM128 (33), a promoter-less integrative lacZ reporter vector with a streptomycin resistance gene.…”

“…While different experimental conditions and varying quantitative approaches appear to account for this discrepancy, clearly our results are consistent with Asensio et al showing a significant effect of PhoP on the expression of dosR . More recently, Vashist et al have shown that PhoP functions as a repressor of dosR (23). Although both our study and this report have investigated the regulation of dosR by PhoP, use of laboratory attenuated Mtb H37Ra, a different genetic background comprising genomic copy of a mutant PhoP (S219L) by Vasisht et al versus a clean mutant used in our study-most likely accounts for this discrepancy.…”

“…In keeping with this, DosR-SigA interaction remains essential in the dormancy survival program of Mtb (17). Although previous studies suggest that Mtb dosR is under the regulation of PhoP (18–20) with results supporting PhoP recruitment to dosR upstream regulatory region (21–23), role of PhoP during mycobacterial hypoxia remains unknown.…”