SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella-containing vacuole.

Nicole Lau,Amanda L Haeberle,Hayley J Newton,Leigh A Knodler,Jean Celli,Brittany J O’Keeffe,Eleanor A Latomanski,Denise M Monack

doi:10.1371/journal.ppat.1007959

Abstract

The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole.

Highlights

Many pathogenic bacteria of public health significance undergo an intracellular cycle as part of their virulence strategy
Typhimurium relies on two type III secretion systems (T3SS)
We show that SopF associates with host cell membranes by binding to phosphoinositides, which are specialized lipids present in eukaryotic cellular membranes, and that SopF is required for maintaining the integrity of the nascent Salmonella-containing vacuole (SCV) membrane

Summary

Introduction

Many pathogenic bacteria of public health significance undergo an intracellular cycle as part of their virulence strategy. Typhimurium), a common cause of foodborne gastroenteritis, is a facultative intracellular pathogen that can colonize epithelial cells, dendritic cells, macrophages and fibroblasts. Typhimurium translocates ~40 effector proteins [3,4,5,6] using two T3SSs, T3SS1 and T3SS2, which are encoded on Salmonella Pathogenicity Island (SPI)-1 and SPI-2, respectively Based upon their timing of expression, T3SS1 effectors are primarily associated with early events in Salmonella-host cell interactions such as promoting bacterial entry into non-phagocytic cells and establishment of the nascent SCV [7], whereas T3SS2 effectors contribute to later events including maturation of the SCV and bacterial replication and survival within the host cell, phagocytic cells [8,9]

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Discussion

Conclusion