Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells

Abstract

Intestinal epithelial cells provide an important colonization niche for Salmonella enterica serovar Typhimurium during gastrointestinal infections. In infected epithelial cells, a subpopulation of S. Typhimurium bacteria damage their internalization vacuole, leading to escape from the Salmonella-containing vacuole (SCV) and extensive proliferation in the cytosol. Little is known about the bacterial determinants of nascent SCV lysis and subsequent survival and replication of Salmonella in the cytosol. To pinpoint S. Typhimurium virulence factors responsible for these steps in the intracellular infectious cycle, we screened a S. Typhimurium multigene deletion library in Caco-2 C2Bbe1 and HeLa epithelial cells for mutants that had an altered proportion of cytosolic bacteria compared to the wild type. We used a gentamicin protection assay in combination with a chloroquine resistance assay to quantify total and cytosolic bacteria, respectively, for each strain. Mutants of three S. Typhimurium genes, STM1461 (ydgT), STM2829 (recA), and STM3952 (corA), had reduced cytosolic proliferation compared to wild-type bacteria, and one gene, STM2120 (asmA), displayed increased cytosolic replication. None of the mutants were affected for lysis of the nascent SCV or vacuolar replication in epithelial cells, indicating that these genes are specifically required for survival and proliferation of S. Typhimurium in the epithelial cell cytosol. These are the first genes identified to contribute to this step of the S. Typhimurium infectious cycle.

Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population

“…( Fig. 3A ), respectively, and also for the proportion of infected cells containing cytosolic bacteria (≥100 bacteria) ( 20 , 43 , 44 ) at 8 h p.i. (6.6% ± 2.4% and 6.3% ± 2.0%) (data are means ± standard deviations [SD]) ( Fig.…”

IntracellularSalmonellametabolism

“…Most Salmonella remain initially in phagosomes where they slowly proliferate, but some Salmonella escape to the host cell cytosol where they can vigorously proliferate and overgrow the phagosomal Salmonella subpopulation, (Knodler, ; Knodler et al, ). Although their differential growth rates must correspond to striking differences in biomass generation and the entire metabolism network (Knodler, ; Knodler et al, ; Wrande et al, ), and recent evidence reveals distinct gene expression patterns and genetic determinants for the two subpopulations (Knodler, ; Knodler et al, ; Wrande et al, ), most HeLa infection studies merely determined average properties. Such data initially reflect mostly phagosomal Salmonella , but later predominantly the overgrowing cytosolic Salmonella subset.…”

Salmonella Populations inside Host Cells

“…The elegant studies of Steele-Mortimer, Knodler et al favor the idea of cytosolic replication as a trait that may have integrated during Salmonella evolution to trigger the extrusion of the heavily-infected epithelial cell and to load the lumen with large bacterial numbers (Knodler et al, 2010 ; Sellin et al, 2014 ). The existence of Salmonella factors that regulate positively or negatively the growth rate in the cytosol of epithelial cells such as YdgT, CorA, RecA, or AsmA (Wrande et al, 2016 ), favors the notion of a specific program involved in the differentiation of the cytosolic population. Recent data also supports the idea of cytosolic bacteria targeting the pro-survival kinase Akt to prolong the lifespan of the infected cell (Finn et al, 2017 ).…”