Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells.

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen.

Insights into cell division using Listeria monocytogenes infections of PtK2 renal epithelial cells.

The assembly of actin into a cleavage furrow is accompanied by disassembly of the interphase actin cytoskeleton. A variation of this actin filament disassembly/assembly cycle is seen during cell division in PtK2 cells infected with the intracellular pathogen, Listeria monocytogenes, where F-actin associates with the bacteria either as a halo surrounding nonmoving bacteria, or as an array of filaments that encases the sides of moving baceteria and extends behind them like a tail. The moving Listeria are found both in the cytoplasm and in the distal ends of undulating filopodia. When infected cells enter mitosis, the distribution of moving and stationary bacteria changes. In the transition from prophase to metaphase, there is a decrease in the number of bacteria with tails of actin in the cytoplasm. The nonmoving bacteria surrounded with F-actin are excluded from the mitotic spindle and moving bacteria are seldom seen in the cytoplasm during mitosis, although small thin filopodia cluster at the edges of the cells. After completion of cytokinesis, strong tail reformation first becomes obvious in the filopodia with Listeria moving back into the cytoplasm as the daughter cells spread. In summary, the disassembly and reassembly of actin tails extending from Listeria in dividing cells is a variation of the changes in actin organization produced by stress fiber and myofibril disassembly/assembly cycles during cell division. We suggest that the same unknown factors that regulate the disassembly/assembly of stress fibers and myofibrils during mitosis and post cytokinesis also affect the movement of Listeria inside mitotic cells.

Filopodium retraction is controlled by adhesion to its tip.

Filopodia are thin cell extensions sensing the environment. They play an essential role during cell migration, cell-cell or cell-matrix adhesion, by initiating contacts and conveying signals to the cell cortex. Pathogenic microorganisms can hijack filopodia to invade cells by inducing their retraction towards the cell body. Because their dynamics depend on a discrete number of actin filaments, filopodia provide a model of choice to study elementary events linked to adhesion and downstream signalling. However, the determinants controlling filopodial sensing are not well characterized. In this study, we used beads functionalized with different ligands that triggered filopodial retraction when in contact with filopodia of epithelial cells. With optical tweezers, we were able to measure forces stalling the retraction of a single filopodium. We found that the filopodial stall force depends on the coating of the bead. Stall forces reached 8 pN for beads coated with the ß1 integrin ligand Yersinia Invasin, whereas retraction was stopped with a higher force of 15 pN when beads were functionalized with carboxyl groups. In all cases, stall forces increased in relation to the density of ligands contacting filopodial tips and were independent of the optical trap stiffness. Unexpectedly, a discrete and small number of Shigella type three secretion systems induced stall forces of 10 pN. These results suggest that the number of receptor-ligand interactions at the filopodial tip determines the maximal retraction force exerted by filopodia but a discrete number of clustered receptors is sufficient to induce high retraction stall forces.

Shigella gets captured to gain entry.

The type III secretion system-dependent epithelial invasion and dissemination of Shigella is stimulated by ATP released through hemichannels. Romero et al. (2011) show that prior to epithelial contact, Shigella is captured by nanometer-thin micropodial extensions at a distance from the cell surface, in a process involving ATP and connexin-mediated signaling.

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.

A novel pseudopodial component of the dendritic cell anti-fungal response: the fungipod.

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the "fungipod", formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7+/-5.6 microm long and 1.8+/-0.67 microm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225+/-55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 microm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion.

TLR4-mediated podosome loss discriminates gram-negative from gram-positive bacteria in their capacity to induce dendritic cell migration and maturation.

Chronic infections are caused by microorganisms that display effective immune evasion mechanisms. Dendritic cell (DC)-dependent T cell-mediated adaptive immunity is one of the mechanisms that have evolved to prevent the occurrence of chronic bacterial infections. In turn, bacterial pathogens have developed strategies to evade immune recognition. In this study, we show that gram-negative and gram-positive bacteria differ in their ability to activate DCs and that gram-negative bacteria are far more effective inducers of DC maturation. Moreover, we observed that only gram-negative bacteria can induce loss of adhesive podosome structures in DCs, a response necessary for the induction of effective DC migration. We demonstrate that the ability of gram-negative bacteria to trigger podosome turnover and induce DC migration reflects their capacity to selectively activate TLR4. Examining mice defective in TLR4 signaling, we show that this DC maturation and migration are mainly Toll/IL-1 receptor domain-containing adaptor-inducing IFNbeta-dependent. Furthermore, we show that these processes depend on the production of PGs by these DCs, suggesting a direct link between TLR4-mediated signaling and arachidonic metabolism. These findings demonstrate that gram-positive and gram-negative bacteria profoundly differ in their capacity to activate DCs. We propose that this inability of gram-positive bacteria to induce DC maturation and migration is part of the armamentarium necessary for avoiding the induction of an effective cellular immune response and may explain the frequent involvement of these pathogens in chronic infections.

Prevotella intermedia ATCC 25611 targets host cell lamellipodia in epithelial cell adhesion and invasion.

INTRODUCTION: The Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens, are phylogenetically closely related and potentially connected with oral and gastrointestinal tract disease pathogenesis. The aim of the present study was to examine whether these species differ in their capabilities of adhesion to and invasion of epithelial cells. METHODS: Adhesion and invasion were assayed by standard antibiotic/culture assays and fluorescent microscopy techniques. The effect of Prevotella strains on epithelial cell viability was measured using a commercial cell proliferation assay. RESULTS: The strains P. intermedia ATCC 25611 and P. nigrescens ATCC 33263 adhered to epithelial cells, the adhesion numbers of P. intermedia being twice as high as those of P. nigrescens. These strains invaded epithelial cells but invasion was weak. The adhesion of P. intermedia was specifically targeted to epithelial cell lamellipodia. The number of adhered P. intermedia cells increased or decreased when the formation of lamellipodia was stimulated or inhibited, respectively. None of the tested strains showed toxic effects on epithelial cells; a clinical P. intermedia strain even increased the number of viable cells by about 20%. CONCLUSION: The results suggest that among the P. intermedia group bacteria, P. intermedia and P. nigrescens type strains can adhere to and invade epithelial cells, the capability of P. intermedia ATCC 25611(T) being highest in this context. This strain proved to have a special affinity in binding to epithelial cell lamellipodia.

EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42.

Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 (Shigella), SifA, SifB (Salmonella) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism.

Micromechanics of filopodia mediated capture of pathogens by macrophages.

The biological function of filopodia has been extensively studied while only little work has been done on their mechanical properties. In the present study, we apply magnetic microbeads to explore the capturing and initial step of phagocytosis of pathogens by macrophages through filopodia. Microbeads were covered by the bacterial coat protein invasin which is known to trigger the invasion of the intestine by the bacteria Yersinia enterocolitica. These mimetics of bacteria were placed in the vicinity of J774 mouse macrophages exhibiting long filopodia. The specific adhesion of beads to the tip of a filopodium induced the retraction of the protrusion resulting in the dragging of the bead towards the cell body. The dynamics of the retraction process was analyzed by following the in-plane motion of the bead. We estimated the minimal force developed by filopodia and compared the results with previous magnetic tweezer studies of mechanical force induced growth of protrusions (Vonna et al. 2003). We show that very thin filopodia can generate astonishingly large retraction forces over large distances (>10 microm) and can act as an efficient mechanical tool to detach pathogens adhering on surfaces.

Morphology of intestinal colonization of Yersinia enterocolitica serovar O3 in mice.

The present study was made to know the morphology of the initial invasion and lesions involved in the intestinal colonization of Yersinia enterocolitica serovar O3 in the epithelium of Peyer's patches of mice. Microfold (M) cells formed a specific structure like a pseudopodium and the bacteria were observed on the surface of the pseudopodium-like structure 4 hr after oral administration of serovar O3. The colonies of serovar O3 were observed in the epithelium and the lamina propria of the Peyer's patches dome region, and the bacteria grown in the Peyer's patches were in direct contact with the lumen without covered with the host tissue 24 hr after the administration.

Uptake of killed Yersinia enterocolitica by pseudopodia of M cells in the Peyers' patches of the murine small intestines.

To understand the mechanisms of uptake of killed bacteria of Yersinia enterocolitica serovar O3 into the epithelium of Peyer's patches, the killed bacteria were perorally inoculated into mice and observation was carried out by scanning and transmission electron microscopy. Microfold (M) cells formed a specific pseudopodium-like structure and the bacteria were observed on the surface and the interior of the pseudopodium-like structure 8 hr after oral administration of killed bacteria of serovar O3.

Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome.

Vascular colonisation by Bartonella henselae may cause vaso-proliferative tumour growth with clumps of bacteria found in close association with proliferating endothelial cells. By using B. henselae-infected human umbilical vein endothelial cells as an in vitro model for endothelial colonisation, we report here on a novel mechanism of cellular invasion by bacteria. First, the leading lamella of endothelial cells establishes cellular contact to sedimented bacteria and mediates bacterial aggregation by rearward transport on the cell surface. Subsequently, the formed bacterial aggregate is engulfed and internalised by a unique host cellular structure, the invasome. Completion of this sequence of events requires 24 hours. Cortical F-actin, intercellular adhesion molecule-1 and phosphotyrosine are highly enriched in the membrane protrusions entrapping the bacterial aggregate. Actin stress fibres, which are anchored to the numerous focal adhesion plaques associated with the invasome structure, are typically found to be twisted around its basal part. The formation of invasomes was found to be inhibited by cytochalasin D but virtually unaffected by nocodazole, colchicine or taxol, indicating that invasome-mediated invasion is an actin-dependent and microtubuli-independent process. Bacterial internalisation via the invasome was consistently observed with several clinical isolates of B. henselae, while a spontaneous mutant obtained from one of these isolates was impaired in invasome-mediated invasion. Instead, this mutant showed increased uptake of bacteria into perinuclear localising phagosomes, suggesting that invasome-formation may interfere with this alternative mechanism of bacterial internalisation. Internalisation via the invasome represents a novel paradigm for the invasion of bacteria into host cells which may serve as a cellular colonisation mechanism in vivo, e.g. on proliferating and migrating endothelial cells during Bartonella-induced vaso-proliferative tumour growth.

Characterization of HEp-2 cell projection formation induced by diffusely adherent Escherichia coli.

Diffusely adherent Escherichia coli (DAEC) are diarrheagenic E. coli whose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Preincubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be "embedded' within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon TnphoA mutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.