Arginine GlcNAcylation and Activity Regulation of PhoP by a Type III Secretion System Effector in Salmonella.

Juan Xue,Kun Meng,Jiaqingzi Hu,Xing Pan,Yuxuan Huang,Ting Peng,Jun Lv,Shan Li,Hua Zhang

doi:10.3389/fmicb.2021.825743

Abstract

Salmonella type III secretion system (T3SS) effector SseK3 is a glycosyltransferase delivered directly into the host cells to modify host protein substrates, thus manipulating host cellular signal transduction. Here, we identify and characterize the Arg-GlcNAcylation activity of SseK3 inside bacterial cells. Combining Arg-GlcNAc protein immunoprecipitation and mass spectrometry, we found that 60 bacterial proteins were GlcNAcylated during Salmonella infection, especially the two-component signal transduction system regulatory protein PhoP. Moreover, the Arg-GlcNAcylation of PhoP by SseK3 was detected in vivo and in vitro, and four arginine residues, Arg65, Arg66, Arg118, and Arg215 were identified as the GlcNAcylation sites. Site-directed mutagenesis showed that the PhoP R215A change significantly reduced the DNA-binding ability and arginine to alanine change at all four sites (PhoP 4RA) completely eliminated the DNA-binding ability, suggesting that Arg215 is essential for the DNA-binding activity of PhoP and GlcNAcylation of PhoP affects this activity. Additionally, GlcNAcylation of PhoP negatively regulated the activity of PhoP and decreased the expression of its downstream genes. Overall, our work provides an example of the intra-bacterial activities of the T3SS effectors and increases our understanding of endogenous Arg-GlcNAcylation.

Highlights

Salmonella, a Gram-negative bacterium, can cause a broad range of food-borne diseases, including gastroenteritis, enteric fever, and bacteremia in a large range of mammalian hosts (Bell et al, 2005)
The results showed that activation of sseK1/2/3 increased the levels of Arg-GlcNAcylation and caused more endogenous Arg-GlcNAcylation of Salmonella (Figure 1C)
These results suggested that LPM media induced Salmonella SPI-2 genes expression, especially sseK1/2/3, increasing the levels of bacterial endogenous Arg-GlcNAcylation

Summary

Introduction

Salmonella, a Gram-negative bacterium, can cause a broad range of food-borne diseases, including gastroenteritis, enteric fever, and bacteremia in a large range of mammalian hosts (Bell et al, 2005). Typhimurium) encodes two specific type III secretion systems (T3SSs) within Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that display functions during infection (Hansen-Wester and Hensel, 2001). Intra-Bacterial Arg-GlcNAcylation by SseK3 cells, the bacterial cells remain within a modified phagosome called the Salmonella-containing vacuole (SCV), in which they will survive and replicate with nutrient limitations (Dandekar et al, 2014). Typhimurium has evolved a large amount of regulatory circuits that facilitate them to adapt to the nutrient-limited environment. One important regulatory system is the PhoQ/PhoP two-component signal transduction system, which is essential for Salmonella virulence (Groisman, 2001)

Methods

Results

Conclusion