Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2.

Andrew Tze Fui Liew,Moirangthem Kiran Singh,Parisa Zangoui,Linda J Kenney,Ranjit Gulvady,Yong Hwee Foo,Yunfeng Gao

doi:10.7554/elife.45311

Abstract

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

Highlights

Salmonella enterica serovar Typhimurium is a pathogen that causes gastroenteritis in humans and a typhoid-like disease in the mouse
In order to characterize the effect of acid pH on SsrB and SsrA function, we determined the number of SsrB and SsrA molecules using PALM imaging of Salmonella grown under Salmonella pathogenicity islands (SPIs)-2-inducing
PALM localization studies combined with Spt-PALM to track protein dynamics under different environmental conditions will enhance our understanding of the signaling repertoire in bacteria

Summary

Introduction

Salmonella enterica serovar Typhimurium is a pathogen that causes gastroenteritis in humans and a typhoid-like disease in the mouse. The most well characterized genomic islands are SPI-1 and SPI-2, which encode two distinct type-three secretion systems (T3SS), as well as genes encoding secreted effectors that are important for pathogenesis (Hensel, 2000; Lee et al, 1992). The SPI-1 T3SS aids in the initial attachment and invasion of the intestinal epithelium (Zhou and Galan, 2001), while SPI-2 genes play an essential role in survival of Salmonella within the macrophage vacuole and its subsequent maturation into a Salmonella-containing vacuole (SCV) (Feng et al, 2003; Garmendia et al, 2003; Lee et al, 2000). Previous reports showed that in vitro, SPI-2 is only detected in a minority of the population (~13%) (Chakraborty et al, 2015). There are one or at most two injectisomes/cell, most often they are located at the cell pole (Chakraborty et al, 2015; Chakravortty et al, 2005)

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