Common Themes in Cytoskeletal Remodeling by Intracellular Bacterial Effectors.

Bacterial pathogens interact with various types of tissues to promote infection. Because it controls the formation of membrane extensions, adhesive processes, or the junction integrity, the actin cytoskeleton is a key target of pathogens during infection. We will highlight common and specific functions of the actin cytoskeleton during bacterial infections, by first reviewing the mechanisms of intracellular motility of invasive Shigella, Listeria, and Rickettsia. Through the models of EPEC/EHEC, Shigella, Salmonella, and Chlamydia spp., we will illustrate various strategies of diversion of actin cytoskeletal processes used by these bacteria to colonize or breach epithelial/endothelial barriers.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip.

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.

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.

A Retro-orbital Sinus Injection Mouse Model to Study Early Events and Reorganization of the Astrocytic Network during Pneumococcal Meningitis.

Pneumococcal (PN) meningitis is a life-threatening disease with high mortality rates that leads to permanent neurological sequelae. Studies of the process of bacterial crossing of the blood brain barrier (BBB) are hampered by the lack of relevant in vitro and in vivo models of meningitis that recapitulate the human disease. PN meningitis involves bacterial access to the bloodstream preceding translocation across the BBB. A large number of PN meningitis models have been developed in mice, with intravenous administration via the lateral tail vein representing the main way to study BBB crossing by PN. While in humans, meningitis is not always associated with bacteremia, PN meningitis after intravenous injection in mice usually develops following sustained and very high bacteremic titers. High grade bacteremia, however, is known to favor inflammation and BBB permeabilization, thereby increasing PN translocation across the BBB and associated damages. Therefore, specific processes associated with early events of PN translocation may be blurred by overall changes in the inflammatory environment and potentially systemic dysfunction in the case of severe sepsis. Here, we report a mouse meningitis model induced by PN injection in the retro-orbital (RO) sinus. We show that, in this model, mice appear to control bacteremic levels during the first 13 h post-infection, while PN crossing of the BBB can be clearly detected by fluorescence confocal microscopy analysis of brain slices as early as 6 h post-infection. Because of the low frequency of events, however, PN translocation across brain parenchymal vessels at early time points requires a rigorous and systematic examination of the brain volume.

WAFL, a new protein involved in regulation of early endocytic transport at the intersection of actin and microtubule dynamics.

We have previously identified a new gene with sequence homology to the WASP-family of actin regulators denoted WAFL (WASP and FKBP-like). Here we report a possible biological function for WAFL, by demonstrating an association to early endosomes via its central coiled-coil domain. Further we show by functional and structural studies that WAFL is associated with both microtubules and the actin filament system, the two means of transport of early endosomes. In addition, WAFL interacts with WASP-interacting protein (WIP) and actin, thus linking WAFL to actin dynamics. The use of RNAi depletion of WAFL shows that WAFL-deficient cells display delayed transport of endosomal cargo. Our findings are compatible with a model whereby WAFL is involved in the transport of early endosomes at the level of transition between microfilament-based and microtubule-based movement.

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.

IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri.

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.

Interplay Between Intracellular Ca(2+) Oscillations and Ca(2+)-stimulated Mitochondrial Metabolism.

Oscillations of cytosolic Ca(2+) concentration are a widespread mode of signalling. Oscillatory spikes rely on repetitive exchanges of Ca(2+) between the endoplasmic reticulum (ER) and the cytosol, due to the regulation of inositol 1,4,5-trisphosphate receptors. Mitochondria also sequester and release Ca(2+), thus affecting Ca(2+) signalling. Mitochondrial Ca(2+) activates key enzymes involved in ATP synthesis. We propose a new integrative model for Ca(2+) signalling and mitochondrial metabolism in electrically non-excitable cells. The model accounts for (1) the phase relationship of the Ca(2+) changes in the cytosol, the ER and mitochondria, (2) the dynamics of mitochondrial metabolites in response to cytosolic Ca(2+) changes, and (3) the impacts of cytosol/mitochondria Ca(2+) exchanges and of mitochondrial metabolism on Ca(2+) oscillations. Simulations predict that as expected, oscillations are slowed down by decreasing the rate of Ca(2+) efflux from mitochondria, but also by decreasing the rate of Ca(2+) influx through the mitochondrial Ca(2+) uniporter (MCU). These predictions were experimentally validated by inhibiting MCU expression. Despite the highly non-linear character of Ca(2+) dynamics and mitochondrial metabolism, bioenergetics were found to be robust with respect to changes in frequency and amplitude of Ca(2+) oscillations.

CD44 Promotes intoxication by the clostridial iota-family toxins.

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

ATP-mediated Erk1/2 activation stimulates bacterial capture by filopodia, which precedes Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery in humans, invades epithelial cells, using a type III secretory system (T3SS) to inject bacterial effectors into host cells and remodel the actin cytoskeleton. ATP released through connexin hemichanels on the epithelial membrane stimulates Shigella invasion and dissemination in epithelial cells. Here, we show that prior to contact with the cell body, Shigella is captured by nanometer-thin micropodial extensions (NMEs) at a distance from the cell surface, in a process involving the T3SS tip complex proteins and stimulated by ATP- and connexin-mediated signaling. Upon bacterial contact, NMEs retract, bringing bacteria in contact with the cell body, where invasion occurs. ATP stimulates Erk1/2 activation, which controls actin retrograde flow in NMEs and their retraction. These findings reveal previously unappreciated facets of interaction of an invasive bacterium with host cells and a prominent role for Erk1/2 in the control of filopodial dynamics.

When Shigella tells the cell to hang on.

OspE, a Shigella type III effector binds to integrin-like kinase and enhances cell adhesion to better disseminate and colonize the intestinal epithelium. Because of the existence of OspE orthologues in other enteropathogens such as enteropathogenic Escherichia coli or Salmonella sp., maintenance of cell adhesion appears as a widespread strategy for bacteria that interact with the intestinal epithelium.

Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy.

Intracellular pathogens like Shigella flexneri enter host cells by phagocytosis. Once inside, the pathogen breaks the vacuolar membrane for cytosolic access. The fate and function of the vacuolar membrane remnants are not clear. Examining Shigella-infected nonmyeloid cells, we observed that proteins associated with vacuolar membrane remnants are polyubiquinated, recruit the autophagy marker LC3 and adaptor p62, and are targeted to autophagic degradation. Further, inflammasome components and caspase-1 were localized to these membranes and correlated with dampened inflammatory response and necrotic cell death. In Atg4B mutant cells in which autophagosome maturation is blocked, polyubiquitinated proteins and P62 accumulated on membrane remnants, and as in autophagy-deficient Atg5(-/-) cells, the early inflammatory and cytokine response was exacerbated. Our results suggest that host membranes, after rupture by an invading cytoplasm-targeted bacterium, contribute to the cellular responses to infection by acting as a signaling node, with autophagy playing a central role in regulating these responses.