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

Abstract

The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular Salmonella.

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells

“…To better understand this post-entry role of SipA in epithelial cells, we first compared proliferation of WT Salmonella and an isogenic ΔsipA mutant in HeLa cells using two complimentary approaches (gentamicin protection assays vs real time imaging). In agreement with previous studies (Brawn et al, 2007;Klein et al, 2017), the gentamicin protection assay showed that, at 6-8 h pi, the ΔsipA mutant has a proliferation defect (Fig. 1A).…”

“…Other activities that have been attributed to SipA include Caspase‐3 cleavage and activation (Cain et al , ; McIntosh et al , ), induction of CXC chemokine expression through phosphorylation of IL‐8‐transcription regulatory proteins, JUN and p38MAK (Figueiredo et al , ) and promoting fusion with early endosomes (Singh et al , ). Of relevance to this study, several groups have shown that SipA is also involved in intracellular replication, with the consensus being that it promotes vacuolar replication (Brawn et al , ; Klein et al , ; Singh et al , ; Zhang et al , ). Here, we found that SipA has no effect on replication per se .…”

“…Cytosolic Salmonella initiates replication earlier and replicates faster than those in the SCV and expresses invasive virulence factors (T3SS1 and flagella) rather than those associated with intravacuolar survival (T3SS2). Thus far, only one T3SS1 effector, SopB, has been shown to have a post‐invasion function in this population (Finn et al , ), although SipA has also been implicated in intracellular replication (Brawn et al , ; Klein et al , ). Here, we show that SipA facilitates survival of the emergent cytosolic Salmonella and/or initiation of replication in the cytosol, with little influence on vacuolar replication, and therefore is an important player in cytosolic colonization and intracellular growth.…”

“…SipA contributes to invasion by catalyzing actin polymerization and bundling of actin filaments, but this multi‐functional protein has also been shown to have several post‐invasion roles including phagosome maturation and intracellular replication (Brawn et al , ). In vitro studies have shown that SipA can impact net intracellular proliferation of Salmonella (Brawn et al , ; Klein et al , ) and we have shown that cytosolic bacteria secrete SipA, as well as SopB, via a non‐canonical T3SS1‐dependent mechanism (Finn et al , ). Since these effectors are actively delivered by cytosolic bacteria, they presumably also have functions relevant to this population.…”

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

“…time points if the SPI1‐T3SS was functional. A later study used a system for controlled SPI1‐T3SS activity in intracellular Salmonella and observed a role of effector proteins SopB and SipA in cytosolic proliferation (Klein et al, ). Our data show that the SPI1‐T3SS is involved in damage of the early SCV, and we propose a key role of SopE in the early phase of internalisation and intracellular presence.…”