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.     

Identification of functional regions within invasion plasmid antigen C (IpaC) of Shigella flexneri

Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host-pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N-terminus and an immunogenic central region are not required for IpaC-dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N-terminus is essential, because IpaC mutants lacking the N-terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane-associating protein. Meanwhile, deletion of the C-terminus eliminates the mutant protein's ability to establish protein-protein contacts with full-length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact-mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.     

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.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri.

The enteric pathogens Salmonella typhimurium and Shigella flexneri differ in most virulence attributes including infectivity, pathology and host range. We have identified a new assemblage of genes responsible for invasion properties of Salmonella which is remarkably similar in order, arrangement and sequence to the gene cluster controlling the presentation of surface antigens (spa) on the virulence plasmid of Shigella. In Salmonella, this chromosomally encoded complex consists of over 12 genes, mutations in which abolish bacterial entry into epithelial cells. Although these genera use distinct invasion antigens, a non-invasive spa mutant of Salmonella could be rescued by the corresponding Shigella homolog. While spa promotes equivalent functions in Shigella and Salmonella, this constellation of genes has been acquired independently by each genus and displays motifs used by diverse antigen export systems including those required for flagellar assembly and protein secretion.     

The effect of the potential PhoQ histidine kinase inhibitors on Shigella flexneri virulence

PhoQ/PhoP is an important two-component system that regulates Shigella virulence. We explored whether the PhoQ/PhoP system is a promising target for new antibiotics against S. flexneri infection. By using a high-throughput screen and enzymatic activity coupled assay, four compounds were found as potential PhoQ inhibitors. These compounds not only inhibited the activity of SF-PhoQc autophosphorylation but also displayed high binding affinities to the SF-PhoQc protein in the Surface Plasmon Resonance response. A S. flexneri cell invasion assay showed that three of these potential PhoQ inhibitors inhibit the invasion of HeLa cells by S. flexneri 9380. In a Mouse Sereny test, mice inoculated with S. flexneri 9380 pre-treated with the potential PhoQ inhibitors 1, 2, 3 or 4 displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed severe keratoconjunctival inflammation. All four potential PhoQ inhibitors showed no significant cytotoxicity or hemolytic activity. These data suggest that the four potential PhoQ inhibitors inhibited the virulence of S. flexneri and that PhoQ/PhoP is a promising target for the development of drugs against S. flexneri infection.     

IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole.

By creating mutations within the Shigella flexneri ipaB gene, we have demonstrated that the invasion of epithelial cells is a three-step process encompassing adhesion on the cell surface, entry and lysis of the phagocytic vacuole allowing subsequent access to the cytoplasm. SC403, an insertion mutant which lacks expression of IpaB but still expresses downstream genes, has been particularly studied. It is non-invasive, does not elicit actin polymerization, but binds to HeLa cells indicating that an adhesion step occurs immediately prior to the entry process. The consequence of the inactivation of ipaB on the intracellular behaviour of S.flexneri was investigated using the macrophage cell line J774. SC403 was unable to lyse the phagocytic vacuole; moreover, this strain did not display the contact mediated haemolytic activity characteristics of Shigella. In addition to being a major component of the invasion complex, IpaB acts as a membrane-lysing toxin enabling escape to the cytoplasmic compartment.     

Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication

Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.     

Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells

Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720-726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell-cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions.     

The modulation of hela cells secretory patterns by invasive Shigella spp. and enteroinvasive E. coli bacterial cells and their soluble components

Considering the important role of cytokines in the initiation and evolution of the inflammatory process induced by Shigella and EIEC strains, the purpose of this study was the characterization of the secretory patterns of HeLa cells induced by Shigella ssp. and EIEC strains and to link the obtained results with the invasiveness level of bacterial strains on this cellular line. During this study there were analyzed two EIEC strains and 12 strains of the following Shigella species: 2 Sh. flexneri 2a, 2 Sh. flexneri 3a, 2 Sh. flexneri 4a, 2 Sh. boydii, 2 Sh. sonnei strains isolated in Romania during 2005 from children with dysentery and diarrhoea and confirmed for their invasive ability by Sereny test. The level of the main pro and anti-inflammatory cytokines, IL-1 beta, IL-6, IL-10, IL-13, IL-17 and TNF-alpha induced by whole bacterial cultures as well as by their soluble mediators was determined by an ELISA test. Our results showed that HeLa cells can be used not only for the qualitative and quantitative assessment of Shigella and EIEC strains invasion ability, but also as a simple work procedure for the investigation of an in vitro complex crosstalk communication mechanisms that involves physical interactions between bacterial cells and epithelial cells (adhesins and complementary receptors) and pro- and anti-inflammatory molecules regulation.The majority of the analyzed Shigella serogroups, with the exception of Shigella sonnei and EIEC strains, inhibited the inflammatory response by reducing the expression of majority of pro-inflammatory cytokines, i.e., IL-1 beta, IL-6, TNF-alpha and IL-17. The reduced cost of the in vitro procedure, the possibility of results interpretation and the strict regulations concerning the use of animals for experimental purposes are the main reasons that support the implementation of such an in vitro test in the research labs.     

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half- maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella . Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella -induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB–CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.     

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin-based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin-based motility. IcsA is inserted into the outer membrane as a 120-kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95-kDa amino-terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin-based motility, with a mean speed 27% faster than the wild-type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin-based motility of wild-type serotype 2a S. flexneri .     

Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells

Shigella flexneri, the causative agent of bacillar dystentery, invades the colonic mucosa where it elicits an intense inflammatory reaction responsible for destruction of the epithelium. During cell invasion, contact with host cells activates the type-III secretion of the Shigella IpaB and IpaC proteins. IpaB and IpaC are inserted into host cell plasma membranes and trigger initial signals that result in actin polymerization, while allowing cytosolic access of other bacterial effectors that further reorganize the cytoskeleton. After internalization, Shigella moves intracellularly and forms protrusions that infect neighbouring cells, promoting bacterial dissemination across the epithelium. Here, we show that during cell invasion, Shigella induces transient peaks in intracellular calcium concentration that are dependent on a functional type-III secretory apparatus. In addition, Shigella invasion induces the opening of Connexin 26 (Cx26) hemichannels in an actin- and phospholipase-C-dependent manner, allowing release of ATP into the medium. The released ATP, in turn, increases bacterial invasion and spreading, as well as calcium signalling induced by Shigella. These results provide evidence that pathogen-induced opening of connexin channels promotes signalling events that favour bacterial invasion and dissemination.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IpaA invasin.

Shigella flexneri is the causative agent of bacillary dysentery in humans. Shigella invasion of epithelial cells is characterized by cytoskeletal rearrangements and formation of cellular projections engulfing the bacterium in a macropinocytic process. We show here that vinculin, a protein involved in linking actin filaments to the plasma membrane, is a direct target of Shigella during cell invasion. IpaA, a Shigella protein secreted upon cell contact, rapidly associates with vinculin during bacterial invasion. Although defective for cell entry, an ipaA mutant is still able to induce foci of actin polymerization, but differs from wild-type Shigella in its ability to recruit vinculin and alpha-actinin. Presumably, IpaA-vinculin interaction initiates the formation of focal adhesion-like structures required for efficient invasion.     

Roles for T and NK cells in the innate immune response to Shigella flexneri

Shigella flexneri, an enteroinvasive Gram-negative bacterium, is responsible for the worldwide endemic form of bacillary dysentery. The host response to primary infection is characterized by the induction of an acute inflammation, which is accompanied by polymorphonuclear cell (PMN) infiltration, resulting in massive destruction of the colonic mucosa. However, PMN play a major role in the recovery from primary infection, by restricting the bacterial infection at the intestinal mucosa. In this study, we assessed the roles for T and NK cells in the control of primary S. flexneri infection, using an alymphoid mouse strain (Rag null gamma(c) null) devoid of B, T, and NK cells. Using the mouse pulmonary model of Shigella infection, we showed that alymphoid Rag null gamma(c) null mice were highly susceptible to S. flexneri infection in comparison with wild-type (wt) mice. Whereas PMN recruitment upon infection was similar, macrophage recruitment and production of proinflammatory cytokines were significantly decreased in Rag null gamma(c) null mice compared with wt mice. Upon selective engraftment of Rag null gamma(c) null mice with polyclonal alphabeta T cells, but not with alphabeta T cells from IFN-gamma null , S. flexneri infection could be subsequently controlled. Rag null mice devoid of B and T cells but harboring NK cells could control infection. Local IFN-gamma production by T and NK cells recruited to the lung was demonstrated in S. flexneri-infected wt mice. These data demonstrate that both alphabeta T cells and NK cells contribute to the early control of S. flexneri infection through amplification of an inflammatory response. This cellular lymphocyte redundancy assures IFN-gamma production, which is central to innate immunity against Shigella infection.