PhoPR Positively Regulates whiB3 Expression in Response to Low pH in Pathogenic Mycobacteria

During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.