Mechanism of Shigella entry into epithelial cells

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by reorganizing the cell cytoskeleton during bacterial entry. This entry process requires the Shigella Ipa proteins that are secreted by a type III secretion apparatus and that act in concert to fine tune cell responses. Actin polymerization at the site of entry is dependent on the IpaB and IpaC proteins, whereas IpaA further modulates cytoskeletal rearrangements by binding to vinculin.     

Rho-dependent membrane folding causes Shigella entry into epithelial cells.

The small GTPase rho is functionally involved in the formation of cytoskeletal structures like stress fibers or focal adhesion plaques. Shigella entry into HeLa cells induces a blossom-like membrane structure at the bacterial entry site. We show here that this membrane-folding process is rho-dependent. The three rho isoforms were recruited into bacterial entry sites with differential localization relative to the membrane structure. A rho-specific inhibitor abolished Shigella-induced membrane folding and impaired bacterial entry accordingly. S1-myosin labeling indicated that rho was involved in Shigella-induced actin polymerization but not actin nucleation in the bacterial invasion site. This provides a major link in the signalization cascade allowing entry of a bacterial pathogen into a eukaryotic cell.     

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.     

Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.

Entry into host cells is an essential feature in the pathogenicity of Salmonella spp. The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells. We report here the identification of several proteins whose secretion into the culture supernatant of S. typhimurium is dependent on the function of the inv-encoded translocation apparatus. Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp. invasins IpaB and IpaC, respectively. An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC. Nonpolar mutations in sicA, sipB, and sipC rendered S. typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.     

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.     

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.     

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1‐encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin–Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F‐actin in tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin‐stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild‐type and sipA mutant strains of S. typhimurium. Time‐lapse phase‐contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild‐type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild‐type bacteria or failed to invade at all. Furthermore, although wild‐type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild‐type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.     

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.     

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.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

Molecular genetic bases of Salmonella entry into host cells

Salmonella spp. can enter into non-phagocytic cells, a property that is essential for their pathogenicity. Recently, considerable progress has been made in the understanding of the molecular genetic bases of this process. It is now evident that Salmonella entry functions are largely encoded on a 35–40 kb region of the Salmonella chromosome located at centisome 63. The majority of the loci in this region encode components of a type III or contact-dependent secretion system homologous to those described in a variety of animal and plant-pathogenic bacteria as well as a number of proteins that require this system for their export to the extracellular environment. A somewhat unexpected finding has been the remarkable homology between the Salmonella and Shigella proteins that mediate the entry of these organisms into cultured epithelial cells.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.     

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.     

The versatility of Shigella effectors

When Shigella infect the intestinal epithelium, they deliver several effectors through the type III secretion system (T3SS) into the surrounding space and directly into the host-cell cytoplasm, where they can mimic and usurp host cellular functions or subvert host-cell signalling pathways and the immune response. Although bacterial strategies and mechanisms of infection vary greatly, recent studies of Shigella effectors have revealed that Shigella possess a highly evolved strategy for infection.     

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.     

SseA is required for translocation of Salmonella pathogenicity island-2 effectors into host cells

The Salmonella pathogenicity island-2 (SPI2) is a virulence locus on the bacterial chromosome required for intracellular proliferation and systemic infection in mice. Cell culture models and a murine model of systemic infection were used to address the role of an uncharacterized SPI2 open reading frame, designated as sseA, in Salmonella virulence. A Salmonella strain with an unmarked internal deletion of sseA displayed a phenotype that was similar to an SPI2-encoded type III secretion system apparatus mutant. Moreover, SseA was required for survival and replication within epithelial cells and macrophages. Murine infection studies confirmed that the DeltasseA strain was severely attenuated for virulence. Using immunofluorescence microscopy, the virulence defect in the DeltasseA strain was attributed to an inability to translocate SPI2 effector proteins into host cells. These data demonstrate that SseA is essential for SPI2-mediated translocation of effector proteins.