The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MHC class I involvement

We have examined the process of Theileria parva sporozoite entry into susceptible bovine lymphocytes and have begun to identify one of the possible molecular interactions involved in the process. The entry process involves a defined series of events and we have used a number of experimental procedures in combination with a method of quantitation to examine various aspects of this process. T. parva sporozoites are nonmotile organisms and the initial sporozoite-lymphocyte interaction is a chance event which can occur at 0-2 degrees C. All subsequent stages in the process are temperature dependent, require the participation of live intact sporozoites and host cells, and involve some cytochalasin-inhibitable rearrangement of the host cell surface membrane or cytoskeleton. Sporozoite entry can be inhibited by antibodies (mAbs) reactive with major histocompatibility complex (MHC) class I molecules (IL-A 19, IL-A 88) and with beta 2 microglobulin (B1G6), whereas mAbs reactive with MHC class II molecules (IL-A 21, J 11), and a common panleucocyte surface antigen, (IL-A 87; a bovine equivalent of CD 11a) have no effect. These results indicate that MHC class I molecules play a role in the process of T. parva sporozoite entry into bovine lymphocytes although as yet the precise role has not been determined. Once internalized within the lymphocyte, a process that takes less than 3 min at 37 degrees C, the sporozoite rapidly escapes from the encapsulating host cell membrane; a process which occurs concurrently with the discharge of the contents of the sporozoite rhoptries and microspheres. The intracytoplasmic parasite is covered by a layer of sporozoite-derived fuzzy material to which host cell microtubules rapidly become associated.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Interaction of sporozoites of Theileria parva with bovine lymphocytes in vitro. I. Early events after invasion

Sporozoites of Theileria parva rapidly enter bovine lymphocytes by a mechanism of passive endocytosis that depends upon progressive circumferential binding of ligands on the parasite to receptors on the host-cell membrane. Within 10 min of entry, the micronemes of the sporozoite discharge their content and the enveloping host-cell membrane is lysed. The host cell responds within 30 min of invasion by polymerization of microtubules arrayed tangential to the sporozoite and converging upon the cytocentrum. Multivesicular bodies and lysosomes are generated and gather around the parasite but are ineffective in the absence of an endocytotic membrane with which they can fuse. Thus Theileria parva can be added to the category of obligate intracellular parasites that ensure their survival by lysis of the parasitophorous vacuole.     

Host cell actin polymerization is required for cellular retention of Trypanosoma cruzi and early association with endosomal/lysosomal compartments

One of the hallmarks of Trypanosoma cruzi invasion of non-professional phagocytes is facilitation of the process by host cell actin depolymerization. Host cell entry by invasive T. cruzi trypomastigotes is accomplished by exploiting a cellular wound repair process involving Ca(2+)-regulated lysosome exocytosis (i.e. lysosome-dependent) or by engaging a recently recognized lysosome-independent pathway. It was originally postulated that cortical actin microfilaments present a barrier to lysosome-plasma membrane fusion and that transient actin depolymerization enhances T. cruzi entry by increasing access to the plasma membrane for lysosome fusion. Here we demonstrate that cytochalasin D treatment of host cells inhibits early lysosome association with invading T. cruzi trypomastigotes by uncoupling the cell penetration step from lysosome recruitment and/or fusion. These findings provide the first indication that lysosome-dependent T. cruzi entry is initiated by plasma membrane invagination similar to that observed for lysosome-independent entry. Furthermore, prolonged disruption of host cell actin microfilaments results in significant loss of internalized parasites from infected host cells. Thus, the ability of internalized trypomastigotes to remain cell-associated and to fuse with host cell lysosomes is critically dependent upon host cell actin reassembly, revealing an unanticipated role for cellular actin remodelling in the T. cruzi invasion process.     

Host microtubule plus‐end binding protein CLASP1 influences sequential steps in the Trypanosoma cruzi infection process

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin‐dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus‐end tracking proteins (+TIPs) in the co‐ordination of T. cruzi trypomastigote internalization and post‐entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium‐regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1‐depleted cells. These post‐entry phenotypes correlate with a generalized impairment of minus‐end directed transport of lysosomes in CLASP1 knock‐down cells and mimic the effects ofdynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1‐dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite‐elicited signalling, host microtubule plus‐end tracking proteins and dynein‐based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells

The in vivo site of influenza virus infection is a polarized epithelium, and it is well established that the virus preferentially enters from the apical surface of polarized epithelial cells; however, many of the molecular events involved during the endocytosis of the virus into polarized epithelia remain unclear. Here we examined the role of actin microfilaments and the myosin VI motor protein during influenza entry into a panel of polarized and non-polarized cells. By treatment of cells with cytochalasin D and jasplakinolide, we show that influenza virus entry into all the polarized epithelial cells tested requires actin dynamics, with a specific role for the actin cytoskeleton in the process of virus internalization from the plasma membrane. In contrast, influenza could still could efficiently enter and infect all non-polarized cells tested after disruption or stabilization of the actin cytoskeleton. To examine the role of the actin motor protein, myosin VI, we expressed a dominant-negative construct in both polarized and non-polarized cells. Influenza virus infectivity in myosin VI tail mutant-transfected cells was significantly decreased in polarized epithelial cells, but not in non-polarized cells. As a whole, our data suggest indispensable roles of a dynamic actin cytoskeleton for influenza virus entry into polarized epithelial cells, a feature not shared with non-polarized cells.     

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.     

Important but differential roles for actin in trafficking of Epstein-Barr virus in B cells and epithelial cells

Epstein-Barr virus (EBV) uses different virus and cell proteins to enter its two major targets, B lymphocytes and epithelial cells. The routes that the virus takes into the two cell types are also different. To determine if these differences extend to movement from the cell surface to the nucleus, we examined the fate of incoming virus. Essentially all virus that entered a B cell remained stable for at least 8 h. In contrast, up to 80% of virus entering an epithelial cell was degraded in a compartment sensitive to inhibitors of components involved in autophagy. Inhibitors of actin remodeling blocked entry into a B cell but had no effect or enhanced entry into an epithelial cell. Inhibitors of the microtubule network reduced intracellular transport in both cell types, but movement to the nucleus in an epithelial cell also required involvement of the actin cytoskeleton. Deletion of the cytoplasmic tail of CR2, which in an epithelial cell interacts with the actin nucleator FHOS/FHOD when cross-linked by EBV, had no effect on infection. However, inhibitors of downstream signaling by integrins reduced intracellular transport. Cooperation of the microtubule and actin cytoskeletons, possibly activated by interaction with integrin binding proteins in the envelope of EBV, is needed for successful infection of an epithelial cell.     

Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity. We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.     

The invasin protein of Yersinia enterocolitica: internalization of invasin-bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton

Yersinia enterocolitica, a facultative intracellular pathogen of mammals, readily enters (i.e., invades) cultured eukaryotic cells, a process that can be conferred by the cloned inv locus of the species. We have studied the mechanism by which the product of inv, a microbial outer membrane protein termed "invasin," mediates the internalization of bacteria by HEp-2 cells and chicken embryo fibroblasts. Invasin-bearing bacteria initially bound the filopodia and the leading edges of cultured cells. Multiple points of contact between the bacterial surface and the surface of the cell ensued and led to the internalization of the bacterium within an endocytic vacuole; the same multi-step process could be induced by an inert particle coated with invasin-containing membranes. Both adherence and internalization were blocked by an antisera directed against the beta 1 integrin cell-adherence molecule. Ultrastructural studies of detergent-insoluble cytoskeletons from infected cells and immunofluorescence microscopy of phalloidin-labeled cells showed alterations in the structure of the cytoskeleton during the internalization process including the accumulation of polymerized actin around entering bacteria. Bacterial entry was prevented by cytochalasin D indicating that the internalization process requires actin microfilament function. Possible linkages between beta 1 containing integrins and the cytoskeleton were examined during the internalization process through the use of protein-specific antibodies and immunofluorescence microscopy. Like actin, the actin-associated proteins filamin, talin and the beta 1 integrin subunit were also found to accumulate around entering bacteria. These findings suggest that the invasin-mediated internalization process is associated with cytoskeletal reorganization.     

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.     

Phagocytosis of Dictyostelium discoideum studied by the particle-tracking method

Single phagocytic events of cellular slime mold Dictyostelium discoideum were studied by the method of particle tracking. A 2-microm polystyrene bead, which had been covalently coated with folate, was attached to the advancing edge of a Dictyostelium ameba with the aid of an optical trap. The bead was transported backward on the cell surface. Forty-five percent of the transported beads were internalized. The bead motion was analyzed by determining every 33 ms the x-y coordinate of the centroid of the phase-contrast image of the bead. The x(t) and y(t) traces were smoothed over 1 s and the difference between the smoothed (x(t) and y(t)) and the original traces, delta(x) identical with x(t) - x(t) and delta(y) identical with y(t) - y(t), were calculated, which represented relatively rapid components of the bead motion. The plot of delta(2) = (delta(x)(2) + delta(y)(2)) against time could be divided into three phases on the basis of the variance of delta(2). Comparison of the plot with the video sequence indicated that the first phase corresponded to the transport, the second phase to the internalization, and the third phase to the postinternalization process (intracellular movement). Cytochalasin A at 5 microM completely inhibited phagocytosis without affecting the binding of bead to the cell surface, indicating the importance of actin cytoskeleton in all the phases. At 1 microM cytochalasin A the variance of the postinternalization process decreased, and the duration of the transport phase increased. At 0.25 microM cytochalasin A the duration of the internalization phase exhibited a significant increase, but other parameters did not change appreciably. The complex and differential effects of cytochalasin A on the parameters characterizing the three phases in the phagocytic process indicate that various aspects of actin dynamics are involved in the individual process of phagocytosis.     

Src, cortactin and Arp2/3 complex are required for E-cadherin-mediated internalization of Listeria into cells

Listeria monocytogenes is a food-borne pathogen able to invade non-phagocytic cells. InlA, a L. monocytogenes surface protein, interacts with human E-cadherin to promote bacterial entry. L. monocytogenes internalization is a dynamic process involving co-ordinated actin cytoskeleton rearrangements and host cell membrane remodelling at the site of bacterial attachment. Interaction between E-cadherin and catenins is required to promote Listeria entry, and for the establishment of adherens junctions in epithelial cells. Although several molecular factors promoting E-cadherin-mediated Listeria internalization have been identified, the proteins regulating the transient actin polymerization required at the bacterial entry site are unknown. Here we show that the Arp2/3 complex acts as an actin nucleator during the InlA/E-cadherin-dependent internalization. Using a variety of approaches including siRNA, expression of dominant negative derivatives and pharmacological inhibitors, we demonstrate the crucial role of cortactin in the activation of the Arp2/3 complex during InlA-mediated entry. We also show the requirement of the small GTPase Rac1 and that of Src-tyrosine kinase activity to promote Listeria internalization. Together, these data suggest a model in which Src tyrosine kinase and Rac1 promote recruitment of cortactin and activation of Arp2/3 at Listeria entry site, mimicking events that occur during adherens junction formation.     

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.     

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.