Joëlle Mounier

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.

Modulation of Shigella virulence in response to available oxygen in vivo

Bacteria coordinate expression of virulence determinants in response to localized microenvironments in their hosts. Here we show that Shigella flexneri, which causes dysentery, encounters varying oxygen concentrations in the gastrointestinal tract, which govern activity of its type three secretion system (T3SS). The T3SS is essential for cell invasion and virulence. In anaerobic environments (for example, the gastrointestinal tract lumen), Shigella is primed for invasion and expresses extended T3SS needles while reducing Ipa (invasion plasmid antigen) effector secretion. This is mediated by FNR (fumarate and nitrate reduction), a regulator of anaerobic metabolism that represses transcription of spa32 and spa33, virulence genes that regulate secretion through the T3SS. We demonstrate there is a zone of relative oxygenation adjacent to the gastrointestinal tract mucosa, caused by diffusion from the capillary network at the tips of villi. This would reverse the anaerobic block of Ipa secretion, allowing T3SS activation at its precise site of action, enhancing invasion and virulence.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4‐phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5)P). We show that PtdIns(5)P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri‐induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short‐chain penetrating PtdIns(5)P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5)P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD‐expressing cells. Accordingly, IpgD and PtdIns(5)P production specifically activates a class IA PI 3‐kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3‐kinase/Akt activation via PtdIns(5)P production that plays an important role in host cell responses such as survival.

icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized the icsB gene which is located on the virulence plasmid pWR100. After inactivation of icsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco‐2 islets, and was unable to provoke a keratoconjunctivitis in guinea‐pigs. Examination of infected HeLa cells showed that the icsB mutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spread.

Galectin-3, a marker for vacuole lysis by invasive pathogens

Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin‐3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta‐galactoside‐binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non‐classical secretion bind extracellular beta‐galactosides. Here we analysed the localization of galectin‐3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin‐3 accumulated in structures in vicinity to internalized bacteria. By using immuno‐electron microscopy analysis we identified galectin‐3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin‐3 to host N‐acetyllactosamine‐containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system‐dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram‐positive Listeria and Salmonella sifA mutant. We suggest that galectin‐3‐containing structures may serve as a potential novel tool to spot vacuole lysis.

Cadherin expression is required for the spread of Shigella flexneri between epithelial cells

Shigella flexneri, a gram-negative pathogen, invades the human colonic epithelium. After entering epithelial cells, bacteria escape into the cytoplasm, move intracellularly, and pass from cell to cell. The bacterium diverts actin and associated actin-binding proteins to generate a cytoskeleton-based motor that pushes forward the bacterium. As the moving bacterium reaches the inner face of the host-cell cytoplasmic membrane, a protrusion forms that allows passage of this bacterium into a neighboring cell. We show here that components of the intermediate junction are used by the bacterium to allow this passage. Using S180, a mouse fibroblastic sarcoma cell line that does not produce cell adhesion molecules (CAM), and S180L and S180cadN, the same cell line transfected with L-CAM and N-cadherin cDNA, respectively, we demonstrate that expression of a cadherin is required for cell-to-cell spread to occur.

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.

Secretion of type III effectors into host cells in real time.

Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time–lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif–tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.

Shigella flexneri Infection Generates the Lipid PI5P to Alter Endocytosis and Prevent Termination of EGFR Signaling

Shigella keeps infected cells alive by preventing the lysosomal trafficking and degradation of an activated growth factor receptor. Launching a Lipid Weapon Dysentery caused by the pathogenic bacterium Shigella flexneri causes considerable mortality, especially among infants in developing nations. S. flexneri injects several proteins into infected host cells, including an enzyme called IpgD that generates PI5P, a lipid that enhances host cell survival through the PI3K-Akt signaling pathway. Ramel et al. found that IpgD-generated PI5P activated the epidermal growth factor receptor (EGFR) and that this lipid prevented the receptor from being trafficked to lysosomes, subcellular compartments in which activated EGFR is broken down. The authors note that other pathogenic bacteria also produce enzymes that generate PI5P, thus raising the possibility that this survival strategy may be conserved and therefore a potential therapeutic target. The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase–Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.

The IpaC carboxyterminal effector domain mediates Src-dependent actin polymerization during Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by locally reorganizing the actin cytoskeleton. Shigella invasion requires actin polymerization dependent on the Src tyrosine kinase and a functional bacterial type III secretion (T3S) apparatus. Using dynamic as well as immunofluorescence microscopy, we show that the T3S translocon component IpaC allows the recruitment of the Src kinase required for actin polymerization at bacterial entry sites during the initial stages of Shigella entry. Src recruitment occurred at bacterial-cell contact sites independent of actin polymerization at the onset of the invasive process and was still observed in Shigella strains mutated for translocated T3S effectors of invasion. A Shigella strain with a polar mutation that expressed low levels of the translocator components IpaB and IpaC was fully proficient for Src recruitment and bacterial invasion. In contrast, a Shigella strain mutated in the IpaC carboxyterminal effector domain that was proficient for T3S effector translocation did not induce Src recruitment. Consistent with a direct role for IpaC in Src activation, cell incubation with the IpaC last 72 carboxyterminal residues fused to the Iota toxin Ia (IaC) component that translocates into the cell cytosol upon binding to the Ib component led to Src-dependent ruffle formation. Strikingly, IaC also induced actin structures resembling bacterial entry foci that were enriched in activated Src and were inhibited by the Src inhibitor PP2. These results indicate that the IpaC effector domain determines Src-dependent actin polymerization and ruffle formation during bacterial invasion.