Exploitation of the ubiquitin system by invading bacteria

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.     

Perturbation of host cell cytoskeleton by cranberry proanthocyanidins and their effect on enteric infections

Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions.     

The unrelated surface proteins ActA of Listeria monocytogenes and lcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli respectively

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the lcsA (VirG) surface protein in S. flexneri . ActA and lcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli . In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing lcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in lcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.     

Involvement of SipA in modulating actin dynamics during Salmonella invasion into cultured epithelial cells

Salmonella entry into epithelial host cells results from the host actin cytoskeleton reorganization that is induced by a group of bacterial proteins delivered to the host cells by the Salmonella type III secretion system. SopE, SopE2 and SopB activate CDC42 and Rac1 to intercept the signal transduction pathways involved in actin cytoskeleton rearrangements. SipA and SipC directly bind actin to modulate the actin dynamics facilitating bacterial entry. Biochemical studies have indicated that SipA decreases the critical concentration for actin polymerization and may be involved in promoting the initial actin polymerization in Salmonella-induced actin reorganization. In this report, we conducted experiments to analyze the in vivo function(s) of SipA during Salmonella invasion. SipA was found to be preferentially associated with peripheral cortical actin filaments but not stress fibres using permeabilized epithelial cells. When polarized Caco-2 cells were infected with Salmonella, actin cytoskeleton rearrangements induced by the wild-type strain had many filopodia structures that were intimately associated with the bacteria. In contrast, ruffles induced by the sipA null mutant were smoother and distant from the bacteria. We also found that the F-actin content in cells infected with the sipA mutant decreased nearly 80% as compared to uninfected cells or those infected with the wild-type Salmonella strain. Furthermore, expression of either the full-length or the SipA(459-684) actin-binding fragment induced prominent punctuate actin assembly in the cortical region of COS-1 cells. These results indicate that SipA is involved in modulating actin dynamics in cultured epithelial cells during Salmonella invasion.     

Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively study actin rearrangements triggered by Salmonella Typhimurium (S. Tm). Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape avoiding strong cell‐to‐cell variations, a major limitation in classical cell culture conditions. S. Tm induced F‐actin‐rich ruffles and invaded micropatterned cells similar to unconstrained cells. Yet, standardized conditions allowed fast and unbiased comparison of cellular changes triggered by the SipA and SopE bacterial effector proteins. Intensity measurements in defined regions revealed that the content of pre‐existing F‐actin remained unchanged during infection, suggesting that newly polymerized F‐actin in bacteria‐triggered ruffles originates from the G‐actin pool. Analysing bacterial target sites, we found that bacteria did not show any preferences for the local actin cytoskeleton specificities. Rather, invasion was constrained to a specific ‘cell height’, due to flagella‐mediated near‐surface swimming. We found that invasion sites were similar to bacterial binding sites, indicating that S. Tm can induce a permissive invasion site wherever it binds. As micropatterned cells can be infected by many different pathogens they represent a valuable new tool for quantitative analysis of host–pathogen interactions.     

Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures

The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate.     

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.     

The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.

Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.     

沙门菌侵袭研究进展

鼠伤寒沙门菌表达两个不同的Ⅲ型分泌系统(typeⅢsecretion/translocation systems, TTSS),分别由致病岛1和2(pathogenicityi slands 1 and 2, SPI-1 and SPI-2)编码。细菌依赖TTSS将效应蛋白转运至宿主细胞,通过“触发”机制诱导细菌进入宿主细胞。这些效应蛋白可诱导细胞骨架重排,导致“巨吞饮”,促使细菌入侵。本综述依据多种沙门菌效应蛋白的功能,建立沙门菌侵袭模型。TTSS活化并转运效应蛋白进入宿主细胞发挥功能(Ⅰ)。小G蛋白交换因子SopE和肌醇磷酸酯酶SopB通过激活CDC42和Rac1,诱导内陷相关的蛋白聚集(Ⅱ)。SipA和SipC通过降低肌动蛋白临界浓度、刺激网素成束、稳定纤维状肌动蛋白(fibrousactin, F-actin)以及使肌动蛋白核化等功能,促使细菌入侵(Ⅲ)。SopB可使膜内陷区PIP2的浓度降低以及VAMP8聚集,促使细胞膜分裂(Ⅳ)。这些效应蛋白的联合作用,使膜皱褶在局部向外显著延伸,使沙门菌被细胞内形成的特殊膜结构包裹。沙门菌的另一种效应蛋白SptP,通过刺激小G蛋白内源性GTPase的活性,抑制小G蛋白的活化,使细胞膜恢复至原有状态(Ⅴ)。     

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.     

Analysis of the mechanisms of Salmonella-induced actin assembly during invasion of host cells and intracellular replication

Salmonella enterica serovar Typhimurium (S. typhimurium) induces actin assembly both during invasion of host cells and during the course of intracellular bacterial replication. In this study, we investigated the involvement in these processes of host cell signalling pathways that are frequently utilized by bacterial pathogens to manipulate the eukaryotic actin cytoskeleton. We confirmed that Cdc42, Rac, and Arp3 are involved in S. typhimurium invasion of HeLa cells, and found that N-WASP and Scar/WAVE also play a role in this process. However, we found no evidence for the involvement of these proteins in actin assembly during intracellular replication. Cortactin was recruited by Salmonella during both invasion and intracellular replication. However, RNA interference directed against cortactin did not inhibit either invasion or intracellular actin assembly, although it resulted in increased cell spreading and a greater number of lamellipodia. We also found no role for either the GTPase dynamin or the formin family member mDia1 in actin assembly by intracellular bacteria. Collectively, these data provide evidence that signalling pathways leading to Arp2/3-dependent actin nucleation play an important role in S. typhimurium invasion, but are not involved in intracellular Salmonella-induced actin assembly, and suggest that actin assembly by intracellular S. typhimurium may proceed by a novel mechanism.     

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.     

Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.     

Mechanisms of Salmonella entry into host cells

Salmonella enterica is an enteric bacterial pathogen that causes a variety of food and water-borne diseases ranging from gastroenteritis to typhoid fever. Ingested bacteria colonize the intestinal epithelium by triggering their own phagocytosis, using a sophisticated array of effector proteins that are injected into the host cell cytoplasm through a type III secretion apparatus. The synergistic action of these secreted effectors leads to a dramatic reorganization of the host actin cytoskeleton, resulting in vigorous membrane protrusion and the engulfment of attached bacteria. Analysis of these effector proteins and identification of their cellular targets has provided insight into the molecular mechanisms by which bacteria can subvert the host signalling and cytoskeletal machinery for their own purposes. This review is intended to summarize our current understanding of the tools used by Salmonella to enter host cells, with a focus on effectors that modulate the actin cytoskeleton.     

Regulation of Salmonella-induced membrane ruffling by SipA differs in strains lacking other effectors

The Salmonella pathogenicity island 1 (SPI-1) type three secretion system (TTSS) is essential for Salmonella invasion of host cells through its triggering of actin-dependent membrane ruffles. The SPI-1 effectors SipA, SopE, SopE2 and SopB all have actin regulating activities and contribute to invasion. The precise role of actin regulation by SipA in Salmonella invasion remains controversial since divergent data have been presented regarding the relationship between SipA and membrane ruffling. We hypothesized that the contribution of SipA to membrane ruffling and invasion might vary between Salmonella strains. We compared the effects of SipA deletion on Salmonella enterica serovar Typhimurium ( S.  Typhimurium) strains that possess or lack SopE. Loss of SipA reduced invasion in the early stages of infection by SopE⁺ and SopE^- strains but the number of membrane ruffles elicited was unaffected. Salmonella strains lacking both SipA and SopE induced ruffles with very different morphology from those induced by wild-type strains or ones lacking single effectors, including the presence of highly dynamic finger-like protrusions and numerous filopodia. A similar phenotype was found for sipA ^- sopE ^-, sipA ^- sopE2 ^- and sipA ^- sopB ^- mutants. Thus, SipA plays a more prominent role in induction of invasion-competent membrane ruffles by Salmonella lacking a full complement of SPI-1 effectors.     

The cytoskeletal scaffold Shank3 is recruited to pathogen-induced actin rearrangements

The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions.     

Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton

The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.     

Autophagy targeting of Listeria monocytogenes and the bacterial countermeasure

Autophagy acts as an intrinsic defense system against intracellular bacterial survival. Recently, multiple cellular pathways that target intracellular bacterial pathogens to autophagy have been described. These include the Atg5/LC3 pathway, which targets Shigella, the ubiquitin (Ub)-NDP52-LC3 pathway, which targets Group A Streptococcus (GAS) and Salmonella typhimurium, the Ub-p62-LC3 pathway, which targets Mycobacterium tuberculosis, Listeria monocytogenes and S. typhimurium, and the diacylglycerol-dependent pathway, which targets S. typhimurium. In addition, the bacterial invasion process is targeted by the NOD1 or NOD2-Atg16LLC3 pathway. Bacterial pathogens with an intracytosolic lifestyle, i.e., those capable of inducing actin polymerization and cell-to-cell spreading, also employ diverse tactics to evade autophagic recognition. Thus, Shigella, L. monocytogenes and Burkholderia pseudomallei deploy highly evolved systems to evade autophagic recognition and growth restriction. Here, we briefly review current knowledge of host recognition of L. monocytogenes by the innate immune system, and highlight how autophagic recognition by the host is overcome by bacterial countermeasures.     

Src, cortactin and Arp2/3 complex are required for E-cadherin-mediated internalization of Listeria into cells

Listeria monocytogenes is a food-borne pathogen able to invade non-phagocytic cells. InlA, a L. monocytogenes surface protein, interacts with human E-cadherin to promote bacterial entry. L. monocytogenes internalization is a dynamic process involving co-ordinated actin cytoskeleton rearrangements and host cell membrane remodelling at the site of bacterial attachment. Interaction between E-cadherin and catenins is required to promote Listeria entry, and for the establishment of adherens junctions in epithelial cells. Although several molecular factors promoting E-cadherin-mediated Listeria internalization have been identified, the proteins regulating the transient actin polymerization required at the bacterial entry site are unknown. Here we show that the Arp2/3 complex acts as an actin nucleator during the InlA/E-cadherin-dependent internalization. Using a variety of approaches including siRNA, expression of dominant negative derivatives and pharmacological inhibitors, we demonstrate the crucial role of cortactin in the activation of the Arp2/3 complex during InlA-mediated entry. We also show the requirement of the small GTPase Rac1 and that of Src-tyrosine kinase activity to promote Listeria internalization. Together, these data suggest a model in which Src tyrosine kinase and Rac1 promote recruitment of cortactin and activation of Arp2/3 at Listeria entry site, mimicking events that occur during adherens junction formation.     

A role for cofilin and LIM kinase in Listeria-induced phagocytosis

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.