SifA permits survival and replication of Salmonella typhimurium in murine macrophages

Abstract

SifA was originally identified as a virulence factor required for formation of Salmonella‐induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella‐containing vacuole in infected epithelial cells. Here, we demonstrate that deletion mutants of ssaR, a component of the SPI‐2 type III secretion system, do not form Sifs in HeLa epithelial cells. This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs. In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP‐1‐positive compartments. Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures. ΔsifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants. Our findings suggest that SifA is an effector of the SPI‐2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals. A family of SifA‐related proteins and their importance to Salmonella pathogenesis is also discussed.

The Salmonella virulence protein SifA is a G protein antagonist

“…To do this, we transfected HeLa cells with gfp, gfp-rab9, and gfp-dnrab9, infected these cells with Salmonella typhimurium expressing a fluorescent monomeric mCherry reporter, and analyzed infected, transfected cells for proliferation of LAMP1-positive bacteria within the SCV and for Sif production. As indicated earlier, upon Salmonella infection LAMP1 is recruited to SCV and Sif membranes (7,(22)(23)(24). Our data indicate that GFP-Rab9 expression had no obvious impact on SCV integrity, based on LAMP1-staining of intracellular bacteria (Fig.…”

“…As indicated earlier, LAMP1 is a transmembrane glycoprotein that localizes primarily to lysosomes and to late endosomes and is detected in punctuate spots dispersed throughout the cell including its periphery. Further, SifA recruits LAMP1 which becomes clustered into perinuclear regions of the cells (22) (Fig. 3E).…”

“…They further used bioinformatics to predict a diverse and larger family of 24 proteins detected in pathogenic bacteria such as enteropathogenic E. coli, Shigella, Citrobactor and Salmonella. This larger family included SifA (4), an effector known to be essential for intracellular replication of Salmonella in macrophages (8,22,25), but there was no knowledge on whether and how it mimicked a G protein. We propose that SifA antagonizes Rab9 by binding to the SKIP PH domain.…”

The Salmonella effector protein PipB2 is a linker for kinesin-1

“…Deletion of sifA leads to several diverse phenotypes, including redistribution of SCVs from a juxtanuclear position to the cell periphery (12), loss of vacuole integrity (8), a replication defect in macrophages (8,16), and a marked attenuation of virulence in mice (17). The first two phenotypes can be partially rescued by inhibiting host kinesin activity (12,18), suggesting that the accumulation of this microtubule motor onto the sifA Ϫ SCV was responsible for its loss of positioning and membrane integrity.…”

Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages

“…Additionally, SifA is implicated in maintaining the integrity of the SCV, and it is well documented that infection with the sifA mutant leads to loss of SCV integrity (25). In contrast to S. flexneri and L. monocytogenes, S. Typhimurium is unable to survive in the cytosol of macrophages, which results in severe attenuation of the sifA mutant (22).…”