Methylation of PhoP by CheR Regulates Salmonella Virulence.

Yang Su,Rui Huang,Xiaoyun Liu,Jianhui Li,Jinjing Ni,Zhixin Tian,Wenjuan Wu,Sen Cheng,Qiuxiang Zhou,Christoph M Tang,Yu-Feng Yao,Zuoqiang Wang,Donghai Lin,Jie Lu,Yue Wang,Wenting Zhang

doi:10.1128/mbio.02099-21

Abstract

ABSTRACTThe two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence.

Highlights

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence
Our previous data showed that PhoP could be acetylated, and this Posttranslational modifications (PTMs) plays an important role in regulating Salmonella virulence [17, 18]
We explored the growth and viability of engineered wild-type, eE8A, eD9A, eE107A, eE108A, and eR112A strains subjected to low concentrations of Mg21 since these strains showed no growth defects in lysogeny broth (LB) broth and M9CA medium (M9 medium broth powder, 11.3 g/L; casamino acids, 1 g/L; glycerol, 2.8 ml/L) supplemented with high concentrations of Mg21(10 mM) (Fig. S2)

Summary

Introduction

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. To determine whether Mg21 concentration alters the methylation levels of E8 or R112 in vivo, His-tagged PhoP proteins were purified from Salmonella grown in M9CA medium supplemented with different concentrations of Mg21 and analyzed by Western blotting. Autoradiography showed that PhoP could be methylated by CheR and YfcB (Fig. 4A).

Results

Conclusion