Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA/AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.     

Rearrangements of actin cytoskeleton during infection with Escherichia coli O157 in macrophages

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as a fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as a fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.     

Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] into phosphatidylinositol 5-monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P(2) into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site.     

Effect of lipoteichoic acid on the uptake of Streptococcus pyogenes by HEp-2 cells

Lipoteichoic acid (LTA) is thought to play a role in the interactions between Streptococcus pyogenes and host cells. We have examined the effect of exogenous LTA on the adherence and entry of S. pyogenes JRS4 strain into HEp-2 epithelial cells. LTA markedly inhibited bacterial entry in a concentration-dependent manner, up to 250 microg ml(-1). In contrast, LTA had only a slight inhibitory effect on adherence. LTA also inhibited the entry but not adherence of Salmonella typhimurium strain into HEp-2 cells. Binding experiments showed a dose-dependent binding of LTA to cells up to 10 microg ml(-1). Confocal laser microscopy imaging and analysis revealed that LTA was internalized by the epithelial cells and colocalized with F-actin. These results might imply that, following binding, exogenous LTA enters HEp-2 cells and exerts a cytotoxic effect that interferes with bacterial internalization. A possible target for LTA activity might be the actin cytoskeleton, which is known to be essential for bacterial uptake.     

Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell

Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells. Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization. The translocated Salmonella protein SopE is an activator for Cdc42. Because SopE is absent from most S. typhimurium strains it remains unclear whether all S. typhimurium strains rely on activation of Cdc42 to invade host cells. We have identified SopE2, a translocated effector protein common to all S. typhimurium strains. SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE. Analysis of S. typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion. Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization. In conclusion, as a result of SopE2 all S. typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry.     

Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization

Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation.     

Invasive phenotype and apoptosis induction of Plesiomonas shigelloides P-1 strain to Caco-2 cells

AIMS: The mechanism of the host cell invasion of Plesiomonas shigelloides and its capability to induce apoptosis were investigated. METHODS AND RESULTS: We performed a time course experiment on the bacterial adherence and invasion of the P. shigelloides P-1 strain into Caco-2 cells using an invasion assay and flow cytometry. The adherence of P. shigelloides to the Caco-2 cells was almost completed within 10 min after the infection. Thereafter, P. shigelloides starts internalization within the Caco-2 cells, which was completed within 60 min after the infection. Based on the invasion assay using nocodazole, cytochalasin D, and genistein, it became clear that the mechanism of the internalization depended on the signal transduction followed by the rearrangement of the cytoskeletal protein. Based on the DNA laddering and TUNEL methods, the cytotoxicity of the Caco-2 cells by the invasion of P. shigelloides occurred through the induction of apoptosis. CONCLUSIONS: This work demonstrated that the mechanism of invasion of P. shigelloides into Caco-2 cells and the invasion of P. shigelloides induces apoptotic cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the virulence factor, which may be important for understanding of the pathogenesis of P. shigelloides.     

Cellular actin is affected by interaction with Candida albicans

Attachment of Candida albicans, an important opportunistic pathogen, to host tissues is an initial step in the development of the infection. The events occurring in the fungal and in the host cells after interaction are poorly understood. In this study we concentrated on the events occurring in the mammalian cells after the interaction with Candida, with emphasis on the cytoskeleton actin. Human cell line cells (HEp2) were exposed to C. albicans or C. albicans-secreted material (culture filtrate) (actin-rearranging Candida-secreted factor, arcsf). The HEp2 cells were examined for cellular changes using confocal laser microscopy (CLSM), transmission and scanning electron microscopy (TEM and SEM). The CLSM studies, using fluorescein isothiocyanate-labeled C. albicans and rhodamine phalloidin actin staining, revealed yeasts adhering to the HEp2 cells or internalized into the cells, with actin surrounding the fungi. Furthermore, actin rearrangement from filamentous network to actin aggregates was noticed. Interaction between the HEp2 cells and C. albicans could be demonstrated also by SEM and TEM after a 2-4-h exposure of the cells to the fungus. Yeasts and hyphae were found attaching to the surface and within the cells. CLSM studies revealed that exposure of HEp2 cells to arcsf was also followed by cellular actin rearrangement, reduced membrane ruffling and decreased cellular motility. The effect was dose- and time-dependent. All these data indicate that the interaction of Candida with HEp2 cells involves signaling events and affects the cellular actin.     

Host cell responses of Salmonella typhimurium infected human dendritic cells

Live attenuated Salmonella are attractive vaccine candidates for mucosal application because they induce both mucosal immune responses and systematic immune responses. After breaking the epithelium barrier, Salmonella typhimurium is found within dendritic cells (DC) in the Peyer's patches. Although there are abundant data on the interaction of S. typhimurium with murine epithelial cells, macrophages and DC, little is known about its interaction with human DC. Live attenuated S. typhimurium have recently been shown to efficiently infect human DC in vitro and induce production of cytokines. In this study, we have analysed the morphological consequences of infection of human DC by the attenuated S. typhimurium mutant strains designated PhoPc, AroA and SipB and the wild-type strains of the American Type Culture Collection (Manassas, VA, USA), ATCC 14028 and ATCC C53, by electron microscopy at 30 min, 3 h and 24 h after exposure. Our results show that genetic background of the strains profoundly influence DC morphology following infection. The changes included (i) membrane ruffling; (ii) formation of tight or spacious phagosomes; (iii) apoptosis; and (iv) spherical, pedunculated membrane-bound microvesicles that project from the plasma membrane. Despite the fact that membrane ruffling was much more pronounced with the two virulent strains, all mutants were taken up by the DC. The microvesicles were induced by all the attenuated strains, including SipB, which did not induce apoptosis in the host cell. These results suggest that Salmonella is internalized by human DC, inducing morphological changes in the DC that could explain immunogenicity of the attenuated strains.     

Promotion of Salmonella typhimurium adherence and membrane ruffling in MDCK epithelia by staurosporine

Abstract Infection of Madin-Darby canine kidney epithelial cell monolayers with Salmonella typhimurium SL1344 for 60 min results in widespread bacterial invasion which is associated with remodelling of the apical cell membrane to form 'membrane ruffles'. Treatment of Madin-Darby canine kidney cell monolayers with the protein kinase inhibitor staurosporine resulted in a 12-fold increase in the number of adhered bacteria without significantly affecting bacterial invasion. Staurosporine treatment also significantly increased both the number and size of membrane ruffles. As S. typhimurium adhere preferentially to these areas of membrane lacking microvilli, the increased extent of membrane ruffling may explain the increased bacterial adherence. These data provide evidence that the propagation of membrane ruffles during S. typhimurium infection is modulated by changes in the phosphorylation state of host proteins.     

Augmentation of microsporidia adherence and host cell infection by divalent cations

The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.     

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.     

The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.     

Characterization of Human Astrovirus Cell Entry

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.     

Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency

Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.     

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

Background

Increased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.

Methods and Findings

Here, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.

Conclusions

These results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.     

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.     

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.     

Relationship between organization of the actin cytoskeleton and the cell cycle in normal and adenovirus-infected rat cells.

Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.