Bacterial signals and cell responses during Shigella entry into epithelial cells

Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial–host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy-terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC-induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella -induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.     

Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src-overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c-src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain.     

Signaling mechanisms that regulate actin-based motility processes in the nervous system

Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood.     

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.     

Tyrosine kinase signaling and type III effectors orchestrating Shigella invasion

Upon epithelial cell contact, Shigella type III effectors activate complex signaling pathways that induce localized membrane ruffling, resulting in Shigella invasion. Bacterial induced membrane ruffles require a timely coordination of cytoskeletal processes, including actin polymerization, filament reorganization and depolymerization, orchestrated by Rho GTPases and tyrosine kinases. An emerging concept is that multiple Shigella effectors act in synergy to promote actin polymerization in membrane extensions at the site of bacterial entry. Recent advances point to the role of Abl/Arg and Src tyrosine kinases as key regulators of bacterial induced cytoskeletal dynamics.     

IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.     

Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells

The in vivo site of influenza virus infection is a polarized epithelium, and it is well established that the virus preferentially enters from the apical surface of polarized epithelial cells; however, many of the molecular events involved during the endocytosis of the virus into polarized epithelia remain unclear. Here we examined the role of actin microfilaments and the myosin VI motor protein during influenza entry into a panel of polarized and non-polarized cells. By treatment of cells with cytochalasin D and jasplakinolide, we show that influenza virus entry into all the polarized epithelial cells tested requires actin dynamics, with a specific role for the actin cytoskeleton in the process of virus internalization from the plasma membrane. In contrast, influenza could still could efficiently enter and infect all non-polarized cells tested after disruption or stabilization of the actin cytoskeleton. To examine the role of the actin motor protein, myosin VI, we expressed a dominant-negative construct in both polarized and non-polarized cells. Influenza virus infectivity in myosin VI tail mutant-transfected cells was significantly decreased in polarized epithelial cells, but not in non-polarized cells. As a whole, our data suggest indispensable roles of a dynamic actin cytoskeleton for influenza virus entry into polarized epithelial cells, a feature not shared with non-polarized cells.     

IpaA targets beta1 integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.     

Molecular bases of epithelial cell invasion by Shigella flexneri

The pathogenesis of shigellosis is characterized by the capacity of the causative microorganism, Shigella, to invade the epithelial cells that compose the mucosal surface of the colon in humans. The invasive process encompasses several steps which can be summarized as follows: entry of bacteria into epithelial cells involves signalling pathways that elicit a macropinocitic event. Upon contact with the cell surface, S. flexneri activates a Mxi/Spa secretory apparatus encoded by two operons comprising about 25 genes located on a large virulence plasmid of 220 kb. Through this specialized secretory apparatus, Ipa invasins are secreted, two of which (IpaB, 62 kDa and IpaC, 42 kDa) form a complex which is itself able to activate entry via its interaction with the host cell membrane. Interaction of this molecular complex with the cell surface elicits major rearrangements of the host cell cytoskeleton, essentially the polymerization of actin filaments that form bundles supporting the membrane projections which achieve bacterial entry. Active recruitment of the protooncogene pp 60^c-src has been demonstrated at the entry site with consequent phosphorylation of cortactin. Also, the small GTPase Rho is controlling the cascade of signals that allows elongation of actin filaments from initial nucleation foci underneath the cell membrane. The regulatory signals involved as well as the proteins recruited indicate that Shigella induces the formation of an adherence plaque at the cell surface in order to achieve entry. Once intracellular, the bacterium lyses its phagocytic vacuole, escapes into the cytoplasm and starts moving the inducing polar, directed polymerization of actin on its surface, due to the expression of IcsA, a 120 kDa outer membrane protein, which is localized at one pole of the microorganism, following cleavage by SopA, a plasmid-encoded surface protease. In the context of polarized epithelial cells, bacteria then reach the intermediate junction and engage their components, particularly the cadherins, to form a protrusion which is actively internalized by the adjacent cell. Bacteria then lyse the two membranes, reach the cytoplasmic compartment again, and resume actin-driven movement.     

Functional role for the class IX myosin myr5 in epithelial cell infection by Shigella flexneri

Efficient control of Shigella -induced, rho-dependent cytoskeletal rearrangements seems to be required to shape the delicate cellular structures associated with bacterial invasion of epithelial cells. We therefore studied a class IX myosin and rho antagonist, the GTPase-activating protein (GAP) myr5, for a potential role in the bacterial entry process. We show that myr5 is recruited into bacterial entry spots. The recruitment pattern resembled that of rhoC or ezrin, but not rhoA, rac or CDC42, while in vitro GAP activity of myr5 was similar for rhoA, B or C. Analysis of myr5 mutants suggested that GTPase- or ATP-binding activites are not required for Shigella -induced recruitment of this atypical myosin to the bacterial entry site. Functional studies revealed a potential dual role of the myosin functions and the GAP module of myr5 for bacterial internalization.     

Ras-related GTPases and the cytoskeleton.

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.     

Escherichia coli producing CNF1 toxin hijacks Tollip to trigger Rac1-dependent cell invasion

Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.     

The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction

The pattern-recognition molecule Nod1 is a critical sensor for bacterial derived diaminopimelic acid-containing peptidoglycan fragments which induces innate immune responses in epithelial cells. Here we report the subcellular localization of this protein in human epithelial cells. Nod1 is localized in the cytosol and at the plasma membrane in human cells. This membrane association is dependent on the integrity of the protein, on its signalling capacity and on an intact actin cytoskeleton. Signalling-inactive mutants of Nod1 or disruption of the actin cytoskeleton interferes with this localization pattern and impacts on downstream NF-κB activation. Moreover, the invasive bacterium Shigella flexneri was used as a model for physiological activation of Nod1. Imaging revealed that Nod1 is recruited to the site of bacterial entry, where it colocalizes with NEMO. Our data provide evidence that membrane association is linked to Nod1 function and, in view of recent findings on Nod2, that this may be a common feature of NLR family members.     

Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells

Shigella flexneri is an enteroinvasive bacterium which causes bacillary dysentery in humans. A major feature of its pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization process requiring polymerization of actin. Here we describe the cytoskeletal rearrangements during S. flexneri invasion of HeLa cells. After an initial contact of the bacterium with the cell surface, distinct nucleation zones of heavy chain actin polymerization appear in close proximity to the contact site underneath the parasite with long filaments being polymerized. These structures then push cellular protrusions that rise beside the entering bacterium, being sustained by tightly bundled long actin filaments organized in parallel orientation with their positive ends pointing to the cytoplasmic membrane. Finally, the cellular projections coalesce above the bacterial body, leading to its internalization. In addition, we found the actin-bundling protein plastin to be concentrated in these protrusions. Since plastin is known to bundle actin filaments in parallel orientation, colocalization of parallel actin filaments and plastin in the cellular protrusions strongly suggested a functional role of this protein in the architecture of parasite-induced cellular projections. Using transfection experiments, we show the differential recruitment of the two plastin isoforms (T- and L-) into Shigella entry zones. By transient expression of a truncated T-plastin which is deprived of one of its actin-binding sites, we also demonstrate the functional role of T-plastin in Shigella entry into HeLa cells.     

rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo.

rho family GTPases link extracellular signals to changes in the organization of cytoskeletal actin. Serum stimulation of quiescent Swiss 3T3 fibroblasts leads to rho-dependent actin stress fibre formation and focal adhesions, whilst several growth factors initiate signalling pathways leading to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro. We have directly compared the activities of the isolated GAP domains of these three proteins in regulating different rho family GTPases, both by in vitro assays and by microinjection, to address their possible physiologic functions. We show that bcr accelerates the GTPase activity of rac, but not rho in vitro, and inhibits rac-mediated membrane ruffling, but not rho-mediated stress fibre formation, after microinjection into Swiss 3T3 fibroblasts. In vitro, rhoGAP has a striking preference for G25K as a substrate, whilst p190GAP has marked preferential activity for rho. Furthermore, p190 preferentially inhibits rho-mediated stress fibre formation in vivo. Our data suggest that p190, rhoGAP and bcr play distinct roles in signalling pathways mediated through different rho family GTPases.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca2+-activated K+ (BK(Ca)) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK(Ca) channels opener NS1619 reversed thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; BK(Ca) channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca2+ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK(Ca) channels may play a regulatory role in it.     

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

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.     

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 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.