Summary
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and ...replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella‐containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi‐function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion‐associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra‐cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.
The bacterial pathogen Salmonella Typhimurium invades intestinal epithelial cells. Following invasion, the bacteria can either replicate within a membrane bound vacuole or escape into the cytosol. We show here that SipA, a secreted bacterial protein, facilitates the early survival and/or initiation of replication of cytosolic Salmonella.
Salmonella enterica serovar Typhimurium is a common facultative intracellular pathogen that causes food-borne gastroenteritis in millions of people worldwide. Intracellular survival and replication ...are important virulence determinants and the bacteria can be found in a variety of phagocytic and non-phagocytic cells in vivo. Invasion of host cells and intracellular survival are dependent on two type III secretion systems, T3SS1 and T3SS2, each of which translocates a distinct set of effector proteins. However, other virulence factors including ion transporters, superoxide dismutase, flagella and fimbriae are also involved in accessing and utilizing the intracellular niche.
THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, ...particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages.
Summary
The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non‐phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which ...delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin‐driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium‐ and phospholipid‐binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS‐mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho‐family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.
Yersinia pestis survives and replicates within PMNs, which are taken up by macrophages in vitro.
Yersinia pestis, the bacterial agent of plague, is transmitted by fleas. The bite of an infected flea ...deposits Y. pestis into the dermis and triggers recruitment of innate immune cells, including phagocytic PMNs. Y. pestis can subvert this PMN response and survive at the flea‐bite site, disseminate, and persist in the host. Although its genome encodes a number of antiphagocytic virulence factors, phagocytosis of Y. pestis by PMNs has been observed. This study tests the hypotheses that Y. pestis, grown at the ambient temperature of the flea vector (21°C), where the major antiphagocytic virulence factors are not produced, can survive and replicate within human PMNs and can use PMNs as a route to infect macrophages subsequently. We show that Y. pestis is localized within PMN phagosomes, predominately as individual bacteria, and that intracellular bacteria can survive and replicate. Within 12 h of infection, ∼70% of infected PMNs had PS on their surface and were plausibly competent for efferocytosis. With the use of live cell confocal imaging, we show that autologous HMDMs recognize and internalize infected PMNs and that Y. pestis survives and replicates within these HMDMs following efferocytosis. Addition of HMDMs to infected PMNs resulted in decreased secretion of inflammatory cytokines (compared with HMDMs incubated directly with pCD1− Y. pestis) and increased secretion of the anti‐inflammatory cytokine IL‐1ra. Thus, Y. pestis can survive and replicate within PMNs, and infected PMNs may be a route for noninflammatory infection of macrophages.
Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. ...However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens.
Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild ...type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.
Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first ...line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface.
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•Enteric pathogens activate an epithelial cell-intrinsic noncanonical inflammasome•Caspase-4 mediates infected epithelial cell extrusion via induction of pyroptosis•Caspase-4 mediates IL-18 cleavage in human epithelial cells•Caspase-11 activation controls bacterial burdens in the intestine of mice
Intestinal epithelial cells are the first line of defense against pathogens. Knodler et al. report that a noncanonical inflammasome in these cells recognizes intracellular and extracellular bacterial pathogens. Inflammasome activation limits Salmonella colonization via infected epithelial cell extrusion and proinflammatory cytokine release, representing a potent mucosal antimicrobial defense mechanism.
A variety of bacterial intracellular pathogens target the host cell ubiquitin system during invasion, a process that involves transient but fundamental changes in the actin cytoskeleton and plasma ...membrane. These changes are induced by bacterial proteins, which can be surface associated, secreted or injected directly into the host cell. Here, the invasion strategies of two extensively studied intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, are used to illustrate some of the diverse ways by which bacterial pathogens intersect the host cell ubiquitin pathway.
Efficient invasion of non-phagocytic cells, such as intestinal epithelial cells, by Salmonella Typhimurium is dependent on the Salmonella Pathogenicity Island 1 (SPI-1)-encoded Type Three Secretion ...System. The environmental cues involved in SPI-1 induction are not well understood. In vitro, various conditions are used to induce SPI-1 and the invasive phenotype. Although lysogeny broth (LB) is widely used, multiple formulations exist, and variation can arise due to intrinsic differences in complex components. Minimal media are also susceptible to variation. Still, the impact of these inconsistencies on Salmonella virulence gene expression has not been well studied. The goal of this project is to identify growth conditions in LB and minimal medium that affect SPI-1 induction in vitro using both whole population and single cell analysis. Here we show, using a fluorescent reporter of the SPI-1 gene prgH, that growth of Salmonella in LB yields variable induction. Deliberate modification of media components can influence the invasive profile. Finally, we demonstrate that changes in SPI-1 inducing conditions can affect the ability of Salmonella to replicate intracellularly. These data indicate that the specific media growth conditions impact how the bacteria interact with host cells.
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