Richard D. Hayward

Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella.

Salmonella causes severe gastroenteritis in humans, entering non‐phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation (‘bundling’) of actin filaments into cables. This is stimulated by contact‐induced delivery of an array of bacterial effector proteins, including the four Sips (Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N‐terminal domain, while additionally the C‐terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.

Cooperation between actin‐binding proteins of invasive Salmonella : SipA potentiates SipC nucleation and bundling of actin

Pathogen‐induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non‐phagocytic intestinal epithelial cells. Two Salmonella actin‐binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F‐actin bundling, we demonstrate that SipA stimulates substantially SipC‐mediated nucleation of actin polymerization. SipA additionally enhances SipC‐ mediated F‐actin bundling, and SipC–SipA collaboration generates stable networks of F‐actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC‐induced reorganization of the actin cytoskeleton in vivo was confirmed using semi‐ permeabilized cultured mammalian cells.

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen–host cross‐talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol‐binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell‐associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane‐integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol‐dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease worldwide. Pathogen adherence to host cells induces reorganization of the actin cytoskeleton into pedestal-like pseudopods beneath the extracellular bacteria. This requires two bacterial virulence factors that mimic a ligand-receptor interaction. EPEC delivers its own receptor, the translocated intimin receptor (Tir), into the target cell plasma membrane, which is phosphorylated on interaction with the bacterial surface protein intimin. Tir phosphorylated on Tyr 474 (ref. 4) binds the cellular adaptor Nck, triggering actin polymerization. Nevertheless, despite its critical role, the mechanism of Tir Tyr 474 phosphorylation remains unknown. Here, by artificially uncoupling Tir delivery and activity, we show that Tir phosphorylation and Nck-dependent pedestal formation require the Src-family kinase (SFK) c-Fyn. SFK inhibitors prevent Tyr 474 phosphorylation, and cells lacking c-fyn are resistant to pedestal formation. c-Fyn exclusively phosphorylates clustered Tir in vitro, and kinase knockdown suppresses Tir phosphorylation and pedestal formation in cultured cells. These results identify the transient interaction with host c-Fyn as a pivotal link between bacterial Tir and the cellular Nck–WASP–Arp2/3 cascade, illuminating a tractable experimental system in which to dissect tyrosine kinase signalling.

Salmonella SPI1 Effector SipA Persists after Entry and Cooperates with a SPI2 Effector to Regulate Phagosome Maturation and Intracellular Replication

Summary Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.

Control of actin turnover by a salmonella invasion protein

Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.

The Salmonella pathogenicity island 1 secretion system directs cellular cholesterol redistribution during mammalian cell entry and intracellular trafficking.

The bacterial pathogen Salmonella triggers its own uptake into non‐phagocytic mammalian cells. Entry is induced by the delivery of bacterial effector pro‐teins that subvert signalling and promote cytoskeletal rearrangement, although the molecular mechanisms that co‐ordinate initial pathogen‐host cell recognition remain poorly characterized. Here we show that cholesterol is essential for Salmonella uptake. Depletion and chelation of plasma membrane cholesterol specifically inhibited bacterial internalization but not adherence. Cholesterol accumulated at bacterial entry sites in cultured cells, and was retained by Salmonella‐containing vacuoles following pathogen internalization. Cellular cholesterol redistribution required bacterial effector protein delivery mediated by the Salmonella pathogenicity island (SPI) 1 type III secretion system, but was independent of the SPI2‐encoded system.

Exploiting pathogenic Escherichia coli to model transmembrane receptor signalling

Many microbial pathogens manipulate the actin cytoskeleton of eukaryotic target cells to promote their internalization, intracellular motility and dissemination. Enteropathogenic and enterohaemorrhagic Escherichia coli, which both cause severe diarrhoeal disease, can adhere to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into pedestal-like pseudopods beneath the extracellular bacteria. As pedestal assembly is triggered by E. coli virulence factors that mimic several host cell-signalling components, such as transmembrane receptors, their cognate ligands and cytoplasmic adaptor proteins, it can serve as a powerful model system to study eukaryotic transmembrane signalling. Here, we consider the impact of recent data on our understanding of both E. coli pathogenesis and cell biology, and the rich prospects for exploiting these bacterial factors as versatile tools to probe cellular signalling pathways.

Direct modulation of the host cell cytoskeleton by Salmonella actin-binding proteins

Invasive Salmonella trigger their own uptake into non-phagocytic eukaryotic cells by delivering virulence proteins that stimulate signaling pathways and remodel the actin cytoskeleton. It has recently emerged that Salmonella encodes two actin-binding proteins, SipC and SipA, which together efficiently nucleate actin polymerization and stabilize the resulting supramolecular filament architecture. Therefore, Salmonella might directly initiate actin polymerization independently of the cellular Arp2/3 complex early in the cell entry process. This is an unprecedented example of a direct intervention strategy to facilitate entry of a pathogen into a target cell. Here, we discuss the Salmonella actin-binding proteins and how they might function in combination with entry effectors that stimulate Rho GTPases. We propose that membrane-targeted bacterial effector proteins might trigger actin polymerization through diverse mechanisms during cell entry by bacterial pathogens.

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion.

An early event in Salmonella infection is the invasion of non‐phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact‐dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full‐length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N‐terminal protease‐resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli‐derived liposomes into the cytosol of living mammalian cells.