A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven <i>Salmonella</i> Typhimurium Host Cell Invasion

<div><p><i>Salmonella</i> Typhimurium (<i>S</i>. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and <i>S</i>. Tm^SipA, a <i>sopBsopE2sopE</i> mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that <i>S</i>. Tm^SipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the <i>S</i>. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of <i>S</i>. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between <i>S</i>. Tm and SPIRE proteins or to an indirect effect.</p></div

Infection step assays using iMEF cell lines.

<p>(A) Binding assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the data from the wild type iMEF control cell line. <i>Spire1</i>^gt/gt show reduced <i>Salmonella</i> binding. (B) Effector injection assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line. <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- cells show attenuation and increase in effector injection, respectively. (C) Modified gentamicin protection assay using <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line. <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- cell lines show decrease in invasion after 4h. Invasion is not dependent on <i>Salmonella</i> effectors or invasion type. (D) Intracellular replication in <i>Spire1</i>^gt/gt and <i>Spire2</i>^-/- iMEFs normalized to the wild type iMEF control cell line measure by plating assay. <i>Spire2</i>^-/- cell line shows decrease of intracellular bacterial replication. (E) Absolute number of CFUs corresponding to D. A-C show data from 3 independent experiments with 2 replicates each in HeLa Kyoto cells. D and E show data from 2 independent experiments with 3 replicates each. Asteriscs indicate significant differences. *: p<0.05.</p

Bacterial strains.

<p>Bacterial strains.</p