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 byM. tuberculosis . However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation ofwhiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of thewhiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation ofwhiB3 differs among mycobacteria. A sequence analysis of thewhiB3 promoters (whiB3p ) fromM. tuberculosis and two closely related species, namely,M. marinum andM. smegmatis , showed that thewhiB3p regions fromM. tuberculosis andM. marinum contain a new type of PhoP box that is absent in theM. smegmatis whiB3p . Direct binding of PhoP towhiB3p fromM. tuberculosis andM. marinum but not that fromM. smegmatis was validated byin vitro protein-DNA binding assays. The direct activation ofwhiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis inM. marinum . Moreover, mutating the residues important for the phosphorylation pathway of PhoPR inM. marinum abolished the activation ofwhiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription ofwhiB3 . Since the PhoP box was only identified inwhiB3p 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 inMycobacterium 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 activateswhiB3 expression in response to low pH. An atypical PhoP box in thewhiB3 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.