Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

Host cell invasion mediated by Trypanosoma cruzi surface molecule gp82 is associated with F-actin disassembly and is inhibited by enteroinvasive Escherichia coli

The target cell F-actin disassembly, induced by a Ca2+-signaling Trypanosoma cruzi factor of unknown molecular identity, has been reported to promote parasite invasion. We investigated whether the metacyclic trypomastigote stage-specific surface molecule gp82, a Ca2+-signal-inducing molecule implicated in host cell invasion, displayed the ability to induce actin cytoskeleton disruption, using a recombinant protein (J18) containing the full-length gp82 sequence fused to GST. J18, but not GST, induced F-actin disassembly in HeLa cells, significantly reducing the number as well as the length of stress fibers. The number of cells with typical stress fibers scored approximately 70% in untreated and GST-treated cells, as opposed to approximately 30% in J18-treated samples, which also showed decreased F-actin content. J18, but not GST, inhibited approximately 6-fold the HeLa cell entry of enteroinvasive Escherichia coli (EIEC), which depends on actin cytoskeleton. Not only were fewer cells infected with bacteria in the presence of J18, there were also fewer bacteria per cell. The inhibitory activity of J18 was Ca2+ dependent. In co-infection experiments, preincubation of HeLa cells with EIEC drastically reduced gp82-dependent internalization of T. cruzi metacyclic forms. All these data, plus the finding that gp82-mediated penetration of metacyclic forms was associated with disrupted HeLa cell cytoskeletal architecture, indicate that gp82 promotes parasite invasion by disassembling the cortical actin cytoskeleton.     

Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells.

Trypanosoma cruzi invades most nucleated cells by a mechanism distinct from classical phagocytosis. Although parasites enter at the lysosome-poor peripheral cell margins, lysosomal markers are immediately incorporated into the parasitophorous vacuole. No accumulation of polymerized actin was detected around recently internalized parasites, and disruption of microfilaments significantly facilitated invasion. Lysosomes were observed to aggregate at the sites of trypanosome attachment and to fuse with the vacuole at early stages of its formation. Experimentally induced, microtubule-dependent movement of lysosomes from the perinuclear area to the cell periphery enhanced entry. Conditions that deplete cells of peripheral lysosomes or interfere with lysosomal fusion capacity inhibited invasion. These observations reveal a novel mechanism for cell invasion:recruitment of lysosomes for fusion at the site of parasite internalization.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.     

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii .     

Evidence supporting the existence of a host cell surface receptor for Trypanosoma cruzi

Membrane fragments from trypomastigote forms of Trypanosoma cruzi inhibited the association of intact trypomastigotes with rat heart myoblasts whereas a similar preparation from non-invasive epimastigotes did not. Furthermore, killed trypomastigotes bound to the host cell surface and prevented the attachment of living organisms. Conversely, the extent of association of killed parasites with the host cells was reduced by the presence of living flagellates. These results suggest the presence of a distinct structure(s) on the surface of rat heart myoblasts to which infective forms of T. cruzi can bind.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

Interaction of Trypanosoma cruzi with macrophages in vitro: dissociation of the attachment and internalization phases by low temperature and cytochalasin B

Chicken macrophages, obtained by cultivation of blood monocytes, were infected with epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi strain Y. The percentage of macrophages containing parasites within parasitophorous vacuoles and of flagellates attached to cell surfaces was determined. By incubation of the macrophages at 4 degrees C or in the presence of cytochalasin B it was possible to dissociate the attachment from the internalization phases in the process of infection of macrophages. Both treatments had a marked effect on the internalization of epimastigote and trypomastigote forms. Cytochalasin B treatment and placement of the macrophages at 4 degrees C before infection inhibited this process by about 99 and 96%, respectively. These results suggest that endocytosis is the principal mechanism of internalization of T. cruzi by macrophages. They show also that epimastigote and trypomastigote forms of T. cruzi have a different rate of adhesion to the macrophage surface.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.     

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37 masculineC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.     

Enforcing host cell polarity: an apicomplexan parasite strategy towards dissemination

The propagation of apicomplexan parasites through transmitting vectors is dependent on effective dissemination of parasites inside the mammalian host. Intracellular Toxoplasma and Theileria parasites face the challenge that their spread inside the host depends in part on the motile capacities of their host cells. In response, these parasites influence the efficiency of dissemination by altering adhesive and/or motile properties of their host cells. Theileria parasites do so by targeting signalling pathways that control host cell actin dynamics. The resulting enforced polar host cell morphology facilitates motility and invasiveness, by establishing focal adhesion and invasion structures at the leading edge of the infected cell. This parasite strategy highlights mechanisms of motility regulation that are also likely relevant for immune or cancer cell motility.     

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca(2+)-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.     

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.     

Signalling mechanisms involved in apical organelle discharge during host cell invasion by apicomplexan parasites

Malaria is caused by Plasmodium parasites, which belong to the phylum apicomplexa. The characteristic feature of apicomplexan parasites is the presence of apical organelles, referred to as micronemes and rhoptries, in the invasive stages of the parasite life cycle. Survival of these obligate intracellular parasites depends on successful invasion of host cells, which is mediated by specific molecular interactions between host receptors and parasite ligands that are commonly stored in these apical organelles. The timely release of these ligands from apical organelles to the parasite surface is crucial for receptor engagement and invasion. This article is a broad overview of the signalling mechanisms that control the regulated secretion of apical organelles during host cell invasion by apicomplexan parasites.     

Alterations in the surface charge of heart muscle cells during interaction withTrypanosoma cruzi

The surface charge of heart muscle cells (HMC) andTrypanosoma cruzi trypomastigotes was estimated during their interaction by means of zeta potential (ZP). Metacyclic and bloodstream trypomastigote, but not amastigote forms, are able to decrease the surface charge of HMC as well as other nonphagocytic cells. However, no alteration could be detected onT. cruzi-infected macrophage cell line. Trypomastigote forms collected from the supernatant after 20 h of contact with HMC also have their ZP value decreased. The analysis of the surface components of both the parasite and HMC involved in such interaction was also carried out. Assays concerning the kinetics of the cell-parasite interaction demonstrated the influence of parasite surface anionogenicity during its interaction with HMC. The binding of bloodstream forms to HMC was enhanced after their incubation with cationized ferritin (CF), whereas phospholipase C and neuraminidase treatments improved and trypsin treatment inhibited parasite uptake in HMC. Conversely, the incubation of HMC with phospholipase C impaired, and with trypsin enhanced, the interiorization of the parasites. These results suggest that trypomastigote forms ofT. cruzi may process the surface of HMC and its own surface either by removing molecules or by exposing ligands for their internalization.     

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.     

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.     

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP₂ and PIP₃ to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.