The type 3 secretion effector IpgD promotes S. flexneri dissemination.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.

Shigella flexneri subverts host polarized exocytosis to enhance cell-to-cell spread.

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers.

Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread.

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes. Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO's pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4(-/-) mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.

The class II phosphatidylinositol 3-phosphate kinase PIK3C2A promotes Shigella flexneri dissemination through formation of vacuole-like protrusions.

Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions.

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-ß-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Secretome of the biocontrol agent metarhizium anisopliae induced by the cuticle of the cotton pest Dysdercus peruvianus reveals new insights into infection.

Metarhizium anisopliae is an entomopathogenic fungus that has evolved specialized strategies to infect insect hosts. Here we analyzed secreted proteins related to Dysdercus peruvianus infection. Using shotgun proteomics, abundance changes in 71 proteins were identified after exposure to host cuticle. Among these proteins were classical fungal effectors secreted by pathogens to degrade physical barriers and alter host physiology. These include lipolytic enzymes, Pr1A, B, C, I, and J proteases, ROS-related proteins, oxidorreductases, and signaling proteins. Protein interaction networks were generated postulating interesting candidates for further studies, including Pr1C, based on possible functional interactions. On the basis of these results, we propose that M. anisopliae is degrading host components and actively secreting proteins to manage the physiology of the host. Interestingly, the secretion of these factors occurs in the absence of a host response. The findings presented here are an important step in understanding the host-pathogen interaction and developing more efficient biocontrol of D. peruvianus by M. anisopliae.

Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-ß-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

Yersinia type III secretion: send in the effectors.

Pathogenic Yersinia spp (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) have evolved an exquisite method for delivering powerful effectors into cells of the host immune system where they inhibit signaling cascades and block the cells' response to infection. Understanding the molecular mechanisms of this system has provided insight into the processes of phagocytosis and inflammation.

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.

Pseudomonas aeruginosa-induced bleb-niche formation in epithelial cells is independent of actinomyosin contraction and enhanced by loss of cystic fibrosis transmembrane-conductance regulator osmoregulatory function.

UNLABELLED: The opportunistic pathogen Pseudomonas aeruginosa can infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic pathogen problematic in hospitalized patients and those with cystic fibrosis (CF). Previously, we showed that P. aeruginosa can enter epithelial cells and replicate within them and traffics to the membrane blebs that it induces. This "bleb-niche" formation requires ExoS, previously shown to cause apoptosis. Here, we show that the driving force for bleb-niche formation is osmotic pressure, differentiating P. aeruginosa-induced blebs from apoptotic blebs. Either CFTR inhibition or CFTR mutation (as seen in people with CF) causes P. aeruginosa to make more bleb niches and provides an osmotic driving force for blebbing. CFTR inhibition also enhances bacterial occupation of blebs and intracellular replication. Since CFTR is targeted for removal from the plasma membrane when P. aeruginosa invades a healthy cell, these findings could relate to pathogenesis in both CF and healthy patient populations.

Listeria exploits damage and death to spread bad news.

Pathogenic Listeria monocytogenes replicates within the host cytosol; little is known about how it transits from cell to cell, spreading infection. A recent study implicates infection-induced membrane damage as a trigger for efferocytosis, the recognition and uptake of dead cells, thereby tricking neighboring cells into taking up the invader.

Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria.

The bacterial pathogen Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42. These three human proteins form a complex that generates tension at apical cell junctions. Listeria relieves this tension and facilitates protrusion formation by secreting a protein called InlC. InlC interacts with a Src Homology 3 (SH3) domain in Tuba, thereby displacing N-WASP from this domain. Interaction of InlC with Tuba is needed for efficient Listeria spread in cultured human cells and infected animals. Recent structural data has elucidated the mechanistic details of InlC/Tuba interaction, revealing that InlC and N-WASP compete for partly overlapping binding surfaces in the Tuba SH3 domain. InlC binds this domain with higher affinity than N-WASP, explaining how InlC is able to disrupt Tuba/N-WASP complexes.

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell.

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.

crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus.

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.