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

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)P 2 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 Δ sopF 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.

Presence of SopE and mode of infection result in increasedSalmonella‐containing vacuole damage and cytosolic release during host cell infection bySalmonella enterica

“…Recent work identified SopF as a novel SPI1‐T3SS effector protein with roles in controlling the SCV integrity (Lau et al, ) and inhibition of the host cell xenophagic machinery by modifying subunit of the vATPase (Xu et al, ). These findings underline the critical importance of controlling SCV integrity and host cell response to intracellular Salmonella .…”

“…Salmonella maintains the integrity of the SCV by actions of the SPI2-T3SS and its effector protein SifA, and sifAdeficient strains are highly attenuated in systemic virulence (Beuzon et al, 2000). This would argue against an overall importance of cyto- Recent work identified SopF as a novel SPI1-T3SS effector protein with roles in controlling the SCV integrity (Lau et al, 2019) and inhibition of the host cell xenophagic machinery by modifying subunit of the vATPase (Xu et al, 2019). These findings underline the critical importance of controlling SCV integrity and host cell response to intracellular Salmonella.…”

“…A reporter system for quantitative analyses of intracellular subpopulation was established using a promoter responsive to host cell cytosolic glucose‐6‐phosphate (G6P) to drive fluorescent protein expression. The G6P reporter was introduced by Schmutz et al (Schmutz et al, ) and subsequently applied in several studies (Chong, Starr, Finn, & Steele‐Mortimer, ; Cooper, Chong, Starr, Finn, & Steele‐Mortimer, ; Lau et al, ; Spinnenhirn et al, ). Here, we demonstrate that SCV rupture and cytosolic release of Salmonella in the early intracellular phase are functions of the experimental settings in cell culture models and linked to the presence of SPI1‐T3SS effector protein SopE.…”

Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression

“…Previous work has shown that a subfraction of S. Tm may escape the SCV over time and become cytosolic. 81,85 When we analyzed the cytosolic subpopulation with the help of S. Tm localizer , carrying a reporter for cytosolic escape (pCK100, glucose-6-phosphate (Glc6P)-driven expression of mCherry), [86][87][88][89] we found that at 7 hpi, around one quarter of all the cytosolic bacteria was FliC + (Fig S7B). Strikingly, we frequently detected bacteria with protruding flagella (Fig S7A, S7C).…”

The use of hybrid data-dependent and -independent acquisition spectral libraries empower dual-proteome profiling

“…Since most intracellular bacteria replicate inside the host, bacteria enumeration varies during the time course of an infection. More specifically, in in vitro infection models using Salmonella infected epithelial HeLa cells, bacterial enumeration estimates <10 bacteria per HeLa cell at early times and up to 100 bacteria at late times in infection [22].…”