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

Abstract

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.

Keywords: Mycobacterium; transcriptional regulation; two-component regulatory systems.

FIG 1

Acidic stress activates the expression of whiB3 in M. marinum but not in M. smegmatis. Transcriptional levels of the whiB3 gene in M. marinum (Mma) (A) and M. smegmatis (Msm) (B) at neutral or low pH (pH 6.8 and pH 4.5, respectively). RNA levels were first normalized to either the 16S rRNA (in M. marinum) or the sigA levels (in M. smegmatis), and the relative RNA levels at pH 6.8 in these strains were normalized to 1. Mean values with standard deviations from two independent tests in duplicates are shown. *, P < 0.05.

FIG 2

PhoP binds to a promoter upstream of the whiB3 gene. (A) Sequence conservation of the DNA fragments adjacent to the whiB3 gene in M. tuberculosis, M. marinum, and M. smegmatis. (B) Putative PhoP box in the noncoding region of the whiB3 gene. (C) Interaction between M. tuberculosis PhoP and whiB3p from M. smegmatis, M. tuberculosis, and M. marinum tested by a gel retardation assay. Acetyl phosphate (Acp) was added at a final concentration of 100 mM in all tests. PhoP at concentrations of 0.05, 0.25, and 0.5 μM was used. (D) Effect of mutation of the putative PhoP box in whiB3p on PhoP binding. R1 represents a fragment in the noncoding region 1 upstream of whiB3p. Mutated sequences of the PhoP box are shown in gray in the upper panels.

FIG 3

PhoPR is required for the activation of whiB3 under acidic conditions in M. marinum. (A) Schematic diagram of mutant construction in mycobacteria. (B) PCR confirmation of the deletion of the whiB3 and phoP genes from M. marinum. (C and D) Expression levels of M. marinum whiB3 in M. marinum ΔwhiB3 ΔphoP strains complemented with whiB3 transcribed from the native promoter (C) or from the promoter with a mutation of the PhoP box (Mma pm) (D). Strains with or without complementation of M. marinum PhoPR were tested. PhoPR represents strains transformed with the plasmid pMV306-PhoPR and CK represents strains transformed with the plasmid pMV306. Mean values with standard deviations from two independent tests in duplicates are shown. *, P < 0.05; n.s., not significant.

FIG 4

Phosphorylation of PhoPR is important for the regulation of whiB3 expression. (A) Schematic diagram of the proposed model for the roles of the PhoPR phosphorylation pathway in the regulation of whiB3. (B) Expression levels of M. marinum whiB3 in M. marinum ΔwhiB3 ΔphoP strains complemented with wild-type M. marinum PhoPR or the phosphorylation-defective PhoP_D64NR. Complementation with whiB3 transcribed from the native promoter was also performed. (C) PCR verification of the M. smegmatis ΔwhiB3 ΔphoPR strain. (D) Expression levels of M. marinum whiB3 in M. smegmatis ΔwhiB3 ΔphoPR strains complemented with M. marinum whiB3 transcribed from the native promoter. Complementation with wild-type M. marinum PhoPR or the phosphorylation-defective PhoP_D64NR and PhoPR_H259Q were tested. Mean values with standard deviations from two independent tests in duplicates are shown. *, P < 0.05; n.s., not significant.

FIG 5

Sequence analysis of noncoding regions upstream of the whiB3 genes in mycobacteria. Conserved sequences of the PhoP box and the −35 and −10 elements of the promoter are indicated with a gray background. Nucleotides not conserved in the PhoP box are shown in a hollow font.