Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ferreira, D. Articles by Yoshida, N. Search for Related Content PubMed PubMed Citation Articles by Ferreira, D. Articles by Yoshida, N.

 Previous Article  |  Next Article 

Infection and Immunity, October 2006, p. 5522-5528, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00518-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Actin Cytoskeleton-Dependent and -Independent Host Cell Invasion by Trypanosoma cruzi Is Mediated by Distinct Parasite Surface Molecules

Daniele Ferreira, Mauro Cortez, Vanessa D. Atayde, and Nobuko Yoshida^*

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, R. Botucatu, 862, 6^o andar, 04023-062, São Paulo, Brazil

Received 29 March 2006/ Returned for modification 29 May 2006/ Accepted 7 July 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The disassembly of host cell actin cytoskeleton as a facilitator of Trypanosoma cruzi invasion has been reported by some authors, while other workers claim that it instead inhibits internalization of the parasite. In this study we aimed at elucidating the basis of this discrepancy. We performed experiments with metacyclic trypomastigotes of T. cruzi strains G and CL, which differ markedly in infectivity and enter target cells by engaging the surface molecules gp35/50 and gp82, respectively, which have signaling activity. Treatment of HeLa cells with the F-actin-disrupting drug cytochalasin D or latrunculin B inhibited the invasion by strain G but not the invasion by strain CL. In contrast to cells penetrated by strain CL, which were previously shown to have a disrupted actin cytoskeleton architecture, no such alteration was observed in HeLa cells invaded by strain G, and parasites were found to be closely associated with target cell actin. Coinfection with enteroinvasive Escherichia coli (EIEC), which recruits host cell actin for internalization, drastically reduced entry of strain CL into HeLa cells but not entry of strain G. In contrast to gp82 in its recombinant form, which induces disruption of F-actin and inhibits EIEC invasion, purified mucin-like gp35/50 molecules promoted an increase in EIEC uptake by HeLa cells. These data, plus the finding that drugs that interfere with mammalian cell signaling differentially affect the internalization of metacyclic forms of strains G and CL, indicate that the host cell invasion mediated by gp35/50 is associated with signaling events that favor actin recruitment, in contrast to gp82-dependent invasion, which triggers the signaling pathways leading to disassembly of F-actin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many pathogenic microorganisms exploit host cell components or even subvert cellular functions for invasion. Host cell actin cytoskeleton rearrangement, for instance, is a strategy used by diverse enteropathogenic bacteria, such as Listeria, Yersinia, Shigella, and Salmonella, for internalization. In order to induce target cell actin remodeling, Listeria and Yersinia engage transmembrane cell adhesion proteins, whereas Shigella and Salmonella use a specialized protein secretion system to inject into host cells virulence factors that trigger a variety of signaling pathways (11, 34).

Trypanosoma cruzi, the agent of Chagas' disease, is an obligate intracellular parasite that infects different mammalian cell types. According to some authors, T. cruzi internalization is independent of the host cell actin cytoskeleton so that treatment of target cells with cytochalasin D, a drug that disrupts actin microfilaments, does not affect or increases parasite invasion (29, 35). On the other hand, other authors have claimed that this drug has an inhibitory effect on T. cruzi entry into diverse cell types, including fibroblasts, skeletal muscle myoblasts, cardiomyocytes, and resident peritoneal macrophages (3, 24). This discrepancy, observed in experiments with tissue culture trypomastigotes (TCT), extends to metacyclic trypomastigotes, the developmental forms that in natural infections are responsible for the first T. cruzi interaction with host cells. Schenkman and Mortara (28) have found that entry of metacyclic forms into HeLa cells is not affected by cytochalasin D, whereas Osuna et al. (20) have shown that the invasion process is significantly inhibited by latrunculin B, a drug that also alters the state of actin polymerization. The basis of these differences is unknown. Here we investigated the possibility that target cell actin cytoskeleton-dependent or -independent invasion of T. cruzi is determined by the parasite surface molecules that are differentially expressed in different strains and, upon binding to the host cell receptors, trigger the signaling routes leading to differential F-actin reorganization.

Metacyclic forms of T. cruzi strains G and CL, which belong to highly divergent genetic groups (7) and differ markedly in infectivity in vitro and in vivo (25, 40), invade host cells by engaging the cell adhesion glycoproteins gp35/50 and gp82 (22, 43), respectively, which are structurally diverse molecules. gp35/50 molecules are highly glycosylated mucin-like molecules with glycans O linked to threonine residues in the protein core through N-acetylglucosamine (1, 30). Strain G gp35/50 binds to target cells through the carbohydrate portion containing a ß-galactofuranose residue. Strain CL expresses a variant form of mucin lacking ß-galactofuranose (41). gp82 is a glycoprotein containing N-linked oligosaccharide (22), and the carbohydrate portion is not involved in cell adhesion and invasion (25). Its cell binding site is a peptide sequence, possibly formed by juxtaposition of two charged sequences separated by a hydrophobic stretch (18). Although gp82 is expressed in both strains, evidence shows that strain G metacyclic forms preferentially use gp35/50 to interact with the host cell, possibly because the cell adhesion capacity of gp35/50 is higher than that of gp82 (25). Both gp35/50 and gp82 are signaling molecules that induce host cell Ca²⁺ mobilization (25). We presume that the binding of gp35/50 and gp82 to their respective receptors, which so far have not been identified, leads to activation of distinct signaling pathways. Recently, we have found that the recombinant form of gp82 induces F-actin depolymerization in HeLa cells and inhibits the uptake of enteroinvasive Escherichia coli (EIEC), a process that depends on actin cytoskeleton (10). As metacyclic forms of strain G rely predominantly on gp35/50 mucins to enter target cells, in contrast to strain CL parasites that invade cells in a gp82-mediated manner (42, 43), they could use distinct mechanisms, associated with recruitment of F-actin. A series of experiments were performed to validate this hypothesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parasites, mammalian cell culture, and invasion assays. T. cruzi strains G (40) and CL (6) were maintained cyclically in mice and in liver infusion tryptose (LIT) medium. The conditions used for differentiation of epimastigotes into metacyclic trypomastigotes were different for strains G and CL. In LIT medium cultures containing 5% fetal bovine serum, about 30 to 40% of the strain G parasites differentiated into metacyclic forms. To obtain a similar rate of differentiation, strain CL was grown for one passage in Grace's medium by seeding 5 ml of a LIT medium culture into 35 ml of medium with less than 1% serum. Metacyclic forms from cultures in the stationary growth phase were purified by passage through a DEAE-cellulose column, as described previously (36). HeLa cells, which are human carcinoma-derived epithelial cells, were grown at 37°C in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum, streptomycin (100 µg/ml), and penicillin (100 U/ml) in a humidified 5% CO₂ atmosphere. Cell invasion assays were carried out as described elsewhere (43) by seeding the parasites into each well of 24-well plates containing 13-mm-diameter round glass coverslips coated with 1.5 x 10⁵ HeLa cells. After 1 h of incubation with parasites, the duplicate coverslips were washed in phosphate-buffered saline (PBS), fixed in methanol, and stained with Giemsa stain, and the intracellular parasites in at least 500 stained cells were counted.

Visualization of HeLa cell actin fibers. HeLa cells were seeded onto 13-mm round glass coverslips in 24-well plates and grown for 20 h at 37°C. After washing in PBS, HeLa cells were fixed with acetone at –20°C for 5 min and then incubated with Alexa Fluor 488 phalloidin (Molecular Probes) in PBS containing 1% bovine serum albumin at room temperature for 30 min before microscopic visualization. Images of stress fibers were obtained with a confocal system (Zeiss Axiovert 100 M) using a 63x 1.3 oil objective and the LSM 510 Expert Mode SP2 software.

Assays for detection of colocalized T. cruzi and host cell actin. HeLa cells were infected with metacyclic trypomastigotes, and then the cells were fixed with 3.5% formaldehyde in PBS for 1 h at room temperature, washed in PBS, and treated with 50 mM NH₄Cl for 15 min. After washing with PBS and soaking in PBS containing 0.15% gelatin and 0.05% NaN₃ (PGN solution), cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed in PBS, and treated with PGN solution containing antibodies directed to metacyclic trypomastigote surface molecules, followed by fluorescein isothiocyanate conjugate for visualization of parasites, phalloidin-rhodamine (Sigma-Aldrich) for F-actin localization, and DAPI (4',6'-diamino-2-phenylindole dihydrochloride) (Molecular Probes) for nuclear staining. Images were acquired with a Nikon E600 fluorescence microscope coupled to a Nikon DXM 1200F digital camera using the ACT-1 software.

Purification of native gp35/50 and recombinant gp82. gp35/50 was obtained from strain G metacyclic forms as previously described (42). The recombinant protein containing the full-length T. cruzi gp82 sequence in frame with glutathione S-transferase (GST), designated J18 (GenBank database accession number L14824), was produced in E. coli DH5- transformed with a pGEX-3 construct containing the gp82 gene, as described previously (27). J18 and GST were purified by a procedure described elsewhere (10).

Bacterial invasion assays. Enteroinvasive E. coli O28ac was used for bacterial invasion assays. Experiments were performed in 24-well plates containing 13-mm-diameter round glass coverslips coated with 1.5 x 10⁵ HeLa cells, using a multiplicity of infection of 100. After 1 h of incubation at 37°C, the cells were washed with PBS, and the preparations were incubated for another 1 h in the presence of 100 µg/ml gentamicin to kill extracellular bacteria. Following washes in PBS, the coverslips were stained with Giemsa stain. EIEC internalization was also ascertained in parallel assays in which viable intracellular bacteria were quantified by plating infected cell lysates in LB agar plates. Briefly, upon cell lysis with Triton X-100 and plating in LB agar plates supplemented with 40 µg/ml kanamycin, followed by overnight incubation at 37°C, the CFU were counted.

Treatment of HeLa cells with drugs. HeLa cells were treated with 2 µg/ml cytochalasin D or 0.2 µg/ml latrunculin B for 15 min or with 250 µM genistein, 10 µM forskolin, 1 µM thapsigargin, or 100 nM wortmannin for 30 min. Other treatments included incubation at 37°C with 100 nM 4-phorbol myristate 13-actetate (PMA) for 20 min or with 1 µM U73122 for 4 min. After treatment, the drug was washed out with PBS before cell invasion assays were performed.

Statistics. Student's t test was used to determine significance in T. cruzi and EIEC cell invasion assays.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Invasion by T. cruzi strains G and CL is differentially affected by treatment of host cells with F-actin-disrupting drugs. To examine the requirement for host cell actin cytoskeleton for metacyclic trypomastigote internalization, HeLa cells were treated with cytochalasin D or latrunculin B. These treatments altered the normal actin cytoskeleton architecture (data not shown). After the drug was washed out, the cells were incubated with parasites for 1 h at 37°C. Following fixation and Giemsa staining, the internalized parasites were counted. As shown in Fig. 1A, infection by strain G was significantly reduced in drug-treated cells, whereas the infectivity of strain CL was unaffected. We also determined the effects of the two F-actin-disrupting drugs on Vero cells. The results were similar to those obtained with HeLa cells. Treatment of Vero cells with cytochalasin D or latrunculin B inhibited the internalization of strain G but not the internalization of strain CL (data not shown).

View larger version (34K): [in this window] [in a new window]

FIG. 1. Requirement of host cell actin cytoskeleton for invasion by metacyclic forms of T. cruzi strain G. (A) HeLa cells pretreated with cytochalasin D (CD) or latrunculin B (LB) were incubated for 1 h with metacyclic forms of strain G or CL. After fixation and Giemsa staining, the internalized parasites were counted. The values are the means ± standard deviations of three independent experiments performed in duplicate. Infection by strain G was significantly inhibited in HeLa cells treated with cytochalasin D (P < 0.0001) or latrunculin B (P < 0.0005). (B) Metacyclic forms were incubated with HeLa cells for 1 h, followed by processing of cells for staining with anti-T. cruzi antibodies and fluorescently conjugated immunoglobulin G (green), with phalloidin-rhodamine for F-actin localization (red), and with DAPI for visualization of nuclei (blue). Note that in the HeLa cell invaded by strain G the parasite penetration is apparently associated with actin recruitment (arrows), whereas in the cell invaded by strain CL the actin cytoskeleton architecture is disrupted, in contrast to that of the neighboring noninfected cell displaying a normal appearance, which has visible stress fibers.

Internalization of metacyclic forms of strain G is associated with host cell actin recruitment. We previously observed a disrupted actin cytoskeletal architecture in HeLa cells invaded by strain CL metacyclic forms (10). The inhibitory effects of cytochalasin D and latruculin B on HeLa cell invasion by strain G (Fig. 1A) indicated that this parasite used a distinct strategy, possibly recruiting actin for internalization, like many enteropathogenic bacteria. To demonstrate that this is indeed the case, two sets of experiments were performed. In one set, strain G metacyclic forms were incubated with HeLa cells for 1 h. After fixation, the cells were processed as described in Materials and Methods for visualization of parasites, F-actin, and cell nuclei. We found that there was a close association between invading parasites and host cell actin. Figure 1B shows a parasite in the process of penetrating HeLa cells, with its internalized portion colocalized with actin. By contrast, confirming our previous findings (10), a disorganized actin cytoskeleton was seen in HeLa cells invaded by strain CL metacyclic forms (Fig. 1B).

In another set of assays, HeLa cells were coinfected with T. cruzi metacyclic trypomastigotes and enteroinvasive EIEC. This bacterium has an invasion plasmid similar to that harbored by Shigella and the same mode of host cell infection (17, 26), and its uptake by nonphagocytic mammalian cells depends on the target cell actin cytoskeleton. We expected that, in contrast to invasion by strain CL metacyclic forms, which is greatly diminished when HeLa cells are coinfected with EIEC (10), strain G internalization would be either unaltered or enhanced by EIEC. HeLa cells were incubated with EIEC for 15 min before coincubation with metacyclic forms for 1 h. Following gentamicin treatment for 1 h, fixation, and staining, the intracellular parasites were counted. Coinfection with EIEC did not significantly affect HeLa cell entry by strain G (Fig. 2A). We also examined the effect of coinfection on the rate of bacterial uptake by adding T. cruzi to HeLa cells 15 min before EIEC was added. Compared to the controls incubated with EIEC alone, a significant increase in the number of EIEC-infected cells was observed in the presence of strain G, whereas strain CL had an inhibitory effect (Fig. 2B).

View larger version (12K): [in this window] [in a new window]

FIG. 2. Coinfection of HeLa cells with T. cruzi and EIEC. (A) HeLa cells were incubated with or without EIEC for 15 min before addition of metacyclic trypomastigotes. (B) HeLa cells were incubated with or without T. cruzi for 15 min before addition of EIEC. In both cases, after 1 h of incubation at 37°C, the cells were washed, treated with gentamicin for 1 h, fixed, and stained with Giemsa stain. The numbers of intracellular parasites in the EIEC experiments (A) and the numbers of EIEC-infected cells in the T. cruzi experiments (B) were determined by counting at least 500 cells. The values are the means ± standard deviations of four independent assays performed in duplicate. In the EIEC experiments (A), infection by T. cruzi strain CL was significantly inhibited by EIEC (P < 0.0001). In the T. cruzi experiments (B), EIEC uptake was significantly increased by coinfection with strain G (P < 0.05) and was inhibited by strain CL (P < 0.05).

T. cruzi gp35/50 mucins and J18 differentially affect EIEC uptake by HeLa cells. We presumed that the differential effects on EIEC uptake by HeLa cells observed in coinfection experiments with T. cruzi strains (Fig. 2B) were determined by gp35/50 and gp82, the surface molecules with signal-inducing activity that mediate host cell invasion by strains G and CL, respectively (25). Binding of these molecules to their receptors would trigger the signaling process, leading to actin microfilament rearrangements. This hypothesis was tested by analyzing the effects of purified molecules on EIEC entry into HeLa cells. We used native gp35/50 mucins and J18, a recombinant protein corresponding to full-length gp82 fused to GST, which exhibits properties similar to those of the native molecule (25). After we checked the purity and identity of gp35/50 and J18 (Fig. 3A and B), that gp35/50 and J18 bound to HeLa cells in a dose-dependent and saturable manner (Fig. 3C), and that only J18 had an actin cytoskeleton-disrupting effect (Fig. 3D), EIEC invasion assays were performed.

View larger version (73K): [in this window] [in a new window]

FIG. 3. Purity and properties of gp35/50 and J18. (A) Schiff staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel containing gp35/50 purified from strain G metacyclic forms and immunoblot revealed with specific monoclonal antibody 10D8 (MAb 10D8). (B) Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel containing J18, a recombinant protein corresponding to full-length gp82 fused to GST, and immunoblot revealed with monoclonal antibody 3F6 (MAb 3F6). (C) HeLa cells were fixed with 4% paraformaldehyde in PBS, blocked with PBS containing 10% fetal calf serum, and incubated for 1 h with gp35/50, J18, or GST. The reaction was revealed by sequential incubation with specific antibodies and anti-mouse immunoglobulin G conjugated to peroxidase. O.D.492, optical density at 492 nm. (D) HeLa cells were incubated for 15 min in the absence or in the presence of gp35/50 or J18 at a concentration of 20 µg/ml. After acetone fixation and staining with Alexa Fluor 488 pahlloidin, confocal images of actin fibers were obtained with a confocal system (Zeiss Axiovert 100 M). Note the disruption of stress fibers in cells treated with J18. Scale bars, 20 µm.

gp35/50 and J18 had opposite effects on bacterial uptake by HeLa cells. While gp35/50 mucins significantly increased EIEC internalization, J18 greatly inhibited bacterial invasion compared to untreated or GST-treated controls (Fig. 4A). gp35/50 was also capable of promoting the uptake of noninvasive EIEC (data not shown). To further ascertain that EIEC internalization was impaired by J18, viable intracellular bacteria were also quantified by plating infected cell lysates on LB agar plates and counting the CFU after overnight incubation at 37°C. Significantly reduced bacterial growth was observed in cells treated with J18 compared to the growth in untreated or GST-treated controls, whereas bacterial growth was increased in gp35/50-treated cells (Fig. 4B). In the presence of J18, not only were fewer cells infected but also fewer bacteria per cell were found (Fig. 4C). We also tried to quantify the bacterial invasion by determining the number of internalized bacteria. However, as this is extremely difficult unless the number of bacteria per cell is small, we counted the cells containing up to 16 bacteria/cell for 200 EIEC-infected cells and found 20 to 24% such cells in untreated and GST-treated cells and 14% and 56% such cells in gp35/50- and J18-treated cells, respectively.

View larger version (66K): [in this window] [in a new window]

FIG. 4. Differential effects of gp35/50 and J18 on EIEC entry into HeLa cells. (A) Purified gp35/50 at a concentration of 15 µg/ml and J18 or GST at a concentration of 20 µg/ml were added to HeLa cells 15 min before seeding of EIEC and maintained throughout incubation for 1 h at 37°C. After an additional 1 h of incubation in the presence of gentamicin and staining with Giemsa stain, the numbers of cells containing bacteria were determined for at least 500 cells. The values are the means ± standard deviations of three experiments performed in duplicate. There was a significant difference between the untreated control and samples treated with gp35/50 (P < 0.05) or J18 (P < 0.001). (B) Quantification of internalized bacteria, performed by plating infected cell lysates on LB agar plates and, after overnight incubation, counting the CFU. The values are the means ± standard deviations of three experiments. There was a significant difference between the untreated control and samples treated with gp35/50 (P < 0.01) or J18 (P < 0.0001). (C) Giemsa-stained preparations showing that there were fewer bacteria per cell in J18-treated samples than in untreated or gp35/50-treated samples.

Distinct signaling routes are induced in host cells by T. cruzi strains G and CL. Metacyclic forms of strains G and CL, as well as the purified native gp35/50 and gp82 molecules, can trigger Ca²⁺ signaling in HeLa cells (25). Whether this Ca²⁺ response results from activation of different signaling pathways during invasion by these T. cruzi strains is unknown. Our finding that EIEC entry into HeLa cells is differentially affected by coinfection with strains G and CL (Fig. 2B), as well as by purified gp35/50 and J18 (Fig. 4), suggested that distinct signaling cascades are triggered by these parasites. To address this question, we performed assays in which HeLa cells were treated with drugs that interfere with cell signaling before incubation with parasites. Pretreatment of HeLa cells with forskolin, an activator of adenylate cyclase (31), increased invasion by strain G but not invasion by strain CL (Fig. 5A). On the other hand, invasion by strain CL but not invasion by strain G was significantly reduced by pretreatment of HeLa cells with PMA, which can downregulate protein kinase C (PKC) and inhibit diverse cell processes (14, 15, 19), with the lipid kinase inhibitor wortmannin, or with thapsigargin (Fig. 5B to D). Genistein, a specific inhibitor of protein tyrosine kinase (2), and U73122, a specific inhibitor of phospholipase C (5), had no significant effect on internalization of strain G or CL (data not shown).

View larger version (26K): [in this window] [in a new window]

FIG. 5. Effects of treatment of HeLa cells with different drugs on invasion by T. cruzi strains G and CL. In all assays (A to D), HeLa cells were treated with a drug, which was washed out before a 1-h incubation with parasites. The values are the means ± standard deviations of four independent assays. The difference between forskolin-treated cells and untreated controls was significant (P < 0.01) for strain G invasion. For strain CL, there was a significant difference between untreated cells and cells treated with PMA (P < 0.0001), wortmannin (P < 0.05), or thapsigargin (P < 0.01).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data indicate that metacyclic trypomastigotes of T. cruzi strains G and CL invade host cells predominantly through actin cytoskeleton-dependent and -independent routes, respectively, by engaging distinct surface molecules for attachment and triggering distinct signal transduction pathways.

Host cell invasion by strain CL metacyclic forms, mainly mediated by the surface molecule gp82, is associated with F-actin disassembly (10). In contrast to this mode of entry, gp35/50-mediated invasion by strain G metacyclic forms depends on target cell actin cytoskeleton (Fig. 1), as suggested by several observations. Treatment of HeLa cells with cytochalasin D or latrunculin B inhibited the internalization of strain G (Fig. 1A), and HeLa cell entry by EIEC, a process that involves actin recruitment (10), was increased by purified gp35/50 molecules (Fig. 4). Coinfection with EIEC and strain G augmented the bacterial uptake by HeLa cells (Fig. 2B). In addition, invading metacyclic forms of strain G were found in close association with host cell actin (Fig. 1B).

It has been reported that host cell F-actin is disassembled during invasion by TCT of T. cruzi as a direct consequence of increases in Ca²⁺ levels (23). Ca²⁺ is mobilized in mammalian cells upon activation of signal transduction pathways induced by T. cruzi, and this is a requirement for parasite internalization (9, 13, 41). Metacyclic trypomastigotes of both strains G and CL, as well as purified native gp35/50 and gp82 molecules, trigger an increase in the cytosolic Ca²⁺ concentration in HeLa cells (25). The Ca²⁺ dependence of host cell actin cytoskeleton disruption has been observed during gp82-dependent penetration by strain CL metacyclic forms (10).

Although both metacyclic trypomastigotes and TCT rely on Ca²⁺ signaling for cell invasion, presumably there are differences in the infection processes of these parasite forms. TCT do not express the gp35/50 or gp82 molecule; they do express larger mucin-like glycoproteins (70 to 200 kDa), as well members of the gp85/sialidase family to which the metacyclic molecule gp82 also belongs, but it is not known whether these TCT molecules have Ca²⁺ signal-inducing activity (41). The sole TCT factor reported to induce host cell Ca²⁺ mobilization by itself and to promote parasite internalization is a soluble compound whose identity is unknown (23). It is also not known whether there is any difference in infectivity between TCT of strains G and CL.

The hypothesis that different signaling routes are activated in HeLa cells during invasion by strains G and CL is supported by the finding that pretreatment of HeLa cells with a panel of drugs that interfere with cell signaling differentially affects strain G and CL internalization (Fig. 5). For instance, invasion by metacyclic forms of strain CL, but not invasion by metacyclic forms of strain G, was inhibited by thapsigargin (Fig. 5D), a drug that depletes Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺ ATPase (37). The Ca²⁺ required for strain CL internalization is probably released from thapsigargin-sensitive stores in a process independent of inositol 1,4,5-triphosphate (InsP₃), provided that the treatment of HeLa cells with a specific inhibitor of phospholipase C, which mediates InsP₃ production (4), has no effect on invasion. The InsP₃-sensitive Ca²⁺ pool is a subset of the thapsigargin-sensitive Ca²⁺ pool (39). Thapsigargin can release Ca²⁺ from an intracellular store by a mechanism that is independent of hydrolysis of phosphoinositides and the concomitant activation of protein kinase C (16). In this regard, it is noteworthy that downregulation of HeLa cell PKC by PMA inhibited invasion by strain CL metacyclic forms (Fig. 5B). A key function of the PKC family in many systems is regulation of ion channel activity (32), including Ca²⁺ influx through Ca²⁺ channels in different cell types (21, 33). In addition to PKC, the lipid kinase phoshoinositide 3-kinase appears to be part of the signaling cascade triggered by strain CL but not by strain G (Fig. 5C). Phoshoinositide 3-kinase may be activated upstream of PKC (38) and exert its effect through PKC (12). In contrast to strain CL invasion, HeLa cell entry by strain G is stimulated by the adenylate cyclase activator forskolin (Fig. 5A), indicating that there is involvement of cAMP, which converges with Ca²⁺ signaling pathways at important molecular loci in nonexcitable cells (8). What remains to be determined is whether the distinct signaling pathways triggered by strains G and CL are linked to F-actin disruption or to the actin recruitment process.

It is noteworthy that upon treatment of target cells with drugs that inhibit internalization of strain CL metacyclic forms (Fig. 5), the rate of host cell invasion was roughly comparable to the rate of host cell invasion by the strain G parasites, raising the possibility that the baseline invasion may be due a mechanism similar to that used by strain G and could involve the strain CL mucin-like gp35/50 molecules lacking ß-galactofuranose. This mechanism, plus the highly effective gp82-mediated invasion process, could contribute to the higher infectivity of strain CL than of strain G.

 
This work was supported by Fundação de Amparo á Pesquisa do Estado de São Paulo (FAPESP) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

We thank Sergio Schenkman for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862, 6^o andar, 04023-062, São Paulo, S.P. Brasil. Phone: 55-11-5576-4532. Fax: 55-11-5571-1095. E-mail: nyoshida{at}ecb.epm.br.

Editor: W. A. Petri, Jr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Acosta, A. S., S. Schenkman, N. Yoshida, A. Mehler, J. M. Richardson, and M. A. J. Ferguson. 1995. The lipid structure of the GPI-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J. Biol. Chem. 270:27244-27253.[Abstract/Free Full Text]
2
2. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592-5595.[Abstract/Free Full Text]
3
3. Barbosa, H. S., and M. N. Merielles. 1995. Evidence of participation of cytoskeleton of heart muscle cells during the invasion of Trypanosoma cruzi. Cell Struct. Funct. 20:275-284.[Medline]
4
4. Beridge, M. J. 1993. Inositol triphosphate and calcium signaling. Nature 361:315-325.[CrossRef][Medline]
5
5. Bleasdale, J. E., R. Thakur, R. S. Gremban, G. L. Bundy, F. A. Fitzpatrick, R. J. Smith, and S. Bunting. 1990. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. Exp. Therap. 255:756-768.
6
6. Brener, Z., and E. Chiari. 1963. Variações morfológicas observadas em diferentes amostras de Trypanosoma cruzi. Rev. Inst. Med. Trop. Sao Paulo 5:220-224.
7
7. Briones, M. R. S., R. P. Souto, B. S. Stolf, and B. Zingales. 1999. The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol. Biochem. Parasitol. 104:219-232.[CrossRef][Medline]
8
8. Bruce, J. I. E., S. V. Straub, and D. I. Yule. 2003. Crosstalk between cAMP and Ca²⁺ signaling in non-excitable cells. Cell Calcium 34:431-444.[CrossRef][Medline]
9
9. Burleigh B. A., and N. W. Andrews. 1998. Signaling and host cell invasion by Trypanosoma cruzi. Curr. Opin. Microbiol. 1:451-465.
10
10. Cortez, M., V. Atayde, and N. Yoshida. 2006. Host cell invasion mediated by Trypanosoma cruzi surface molecule gp82 is associated with F-actin disassembly and is inhibited by enteroinvasive Escherichia coli. Microbes Infect. 8:1502-1512.[CrossRef][Medline]
11
11. Cossart, P., and P. J. Sansonetti. 2004. Bacterial invasion: the paradigm of enteroinvasive pathogens. Science 304:242-248.[Abstract/Free Full Text]
12
12. Derman, M. P., A. Toker, J. H. Hartwig, K. Spokes, J. R. Falck, C. Chen, L. C. Cantely, and L. G. Cantely. 1997. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J. Biol. Chem. 272:6465-6470.[Abstract/Free Full Text]
13
13. Docampo, R., and S. J. Moreno. 1996. The role of Ca²⁺ in the process of cell invasion by intracellular parasites. Parasitol. Today 12:61-65.[CrossRef][Medline]
14
14. Goldstein, R. H., A. Fine, L. J. Farnsworth, C. Poliks, and P. Polgar. 1990. Phorbol ester-induced inhibition of collagen accumulation by human lung fibroblasts. J. Biol. Chem. 265:13623-13628.[Abstract/Free Full Text]
15
15. Hu, K. Q., J. M. Backer, G. Sahagian, E. P. Feener, and G. L. King. 1990. Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C. J. Biol. Chem. 265:13864-13870.[Abstract/Free Full Text]
16
16. Jackson, T. R., S. I. Patterson, O. Thastrup, and M. R. Hanley. 1988. A novel tumor promoter, thapsigargin, transiently increases cytoplasmic free Ca²⁺ without generation of inositol phosphates in NG115-4011 neuronal cells. Biochem. J. 253:81-86.[Medline]
17
17. Lan, R., B. Lumb, D. Ryan, and P. R. Reeves. 2001. Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive E. coli. Infect. Immun. 69:6303-6309.[Abstract/Free Full Text]
18
18. Manque, P. M., D. Eichinger, M. A. Juliano, L. Juliano, J. Araya, and N. Yoshida. 2000. Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage surface glycoprotein gp82. Infect. Immun. 68:478-484.[Abstract/Free Full Text]
19
19. Nishimura, T., S. J. Burakoff, and S. H. Herrmann. 1989. Inhibition of lymphokine-activated killer cell-mediated cytotoxity by phorbol ester. J. Immunol. 142:2155-2161.[Abstract]
20
20. Osuna, A., N. Rodriguez-Cabezas, M. Boy, S. Castanys, and F. Gamarro. 1993. The invasion mechanism of the metacyclic forms of Trypanosoma cruzi in nonphagocytic host cells. Biol. Res. 26:19-26.[Medline]
21
21. Platano, D., A. Pollo, E. Carbone, and G. Aicardi. 1996. Up-regulation of L- and non-L, non-N-type Ca²⁺ channels by basal and stimulated protein kinase C activation in insulin-secreting RINm5F cells. FEBS Lett. 391:189-194.[CrossRef][Medline]
22
22. Ramirez, M. I., R. C. Ruiz, J. E. Araya, J. Franco da Silveira, and N. Yoshida. 1993. Involvement of the stage-specific 82-kilodalton adhesion molecule of Trypanosoma cruzi metacyclic trypomastigotes in host cell invasion. Infect. Immun. 61:3636-3641.[Abstract/Free Full Text]
23
23. Rodriguez, A., M. G. Rioult, A. Ora, and N. Andrews. 1995. A trypanosome-soluble factor induces IP₃ formation, intracellular Ca²⁺ mobilization and microfilament rearrangement in host cells. J. Cell Biol. 129:1263-1273.[Abstract/Free Full Text]
24
24. Rosestolato, C. T. F., J. M. F. Dutra, W. Souza, and T. M. U. Carvalho. 2002. Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells. Cell Struct. Funct. 27:91-98.[CrossRef][Medline]
25
25. Ruiz, R. C., S. Favoreto, M. L. Dorta, M. E. M. Oshiro, A. T. Ferreira, P. M. Manque, and N. Yoshida. 1998. Infectivity of Trypanosoma cruzi strains is associated with differential expression of surface glycoproteins with differential Ca²⁺ signaling activity. Biochem. J. 330:505-511.
26
26. Sansonetti, P. J., H. d'Hautevite, C. Echobicon, and C. Pourcel. 1983. Molecular comparison of virulence plasmids of Shigella and enteroinvasive E. coli. Ann. Microbiol. 134A:295-318.
27
27. Santori, F. R., M. L. Dorta, L. Juliano, M. A. Juliano, J. Franco da Silveira, R. C. Ruiz, and N. Yoshida. 1996. Identification of a domain of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 required for attachment and invasion of mammalian cells. Mol. Biochem. Parasitol. 78:209-216.[CrossRef][Medline]
28
28. Schenkman, S., and R. A. Mortara. 1992. HeLa cells extend and internalize pseudopodia during active invasion by Trypanosoma cruzi trypomastigotes. J. Cell Sci. 101:895-905.[Abstract/Free Full Text]
29
29. Schenkman, S., E. Robins, and V. Nussenzweig. 1991. Attachment of Trypanosoma cruzi mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton. Infect. Immun. 59:645-654.[Abstract/Free Full Text]
30
30. Schenkman, S., M. A. J. Ferguson, N. Heise, M. L. Cardoso de Almeida, R. A. Mortara, and N. Yoshida. 1993. Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalysed by trans-sialidase in metacyclic forms of Trypanosoma cruzi. Mol. Biochem. Parasitol. 59:293-304.[CrossRef][Medline]
31
31. Seamon, K. B., W. Padgett, and J. W. Daly. 1981. Forskolin: unique diterpene activatior of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78:3363-3367.[Abstract/Free Full Text]
32
32. Shearman, M. S., K. Sekiguchi, and Y. Nishizuka. 1989. Modulation of ion channel activity: a key function of the protein kinase C enzyme family. Pharmacol. Rev. 41:211-237.[Abstract]
33
33. Shimamura, K., M. Lusaka, and N. Sperelakis. 1994. Protein kinase C stimulates Ca²⁺ current in pregnant rat myometrial cells. Can. J. Physiol. Pharmacol. 72:1304-1307.[Medline]
34
34. Stebbins, G. E., and J. E. Galán. 2001. Structural mimicry in bacterial virulence. Nature 412:701-705.[CrossRef][Medline]
35
35. Tardieux, I., P. Webster, J. Ravesloot, W. Boron, J. A. Lunn, J. E. Heuser, and N. W. Andrews. 1992. Lysosome recruitment and fusion are early events required for Trypanosoma invasion of mammalian cells. Cell 71:1117-1130.[CrossRef][Medline]
36
36. Teixeira, M. M. G., and N. Yoshida. 1986. Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies. Mol. Biochem. Parasitol. 18:271-282.[CrossRef][Medline]
37
37. Thastrup, O., P. J. Cullen, B. K. Drobak, M. R. Hanley, and A. P. Dawson. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca²⁺ stores by specific inhibition of the endoplasmic reticulum Ca²⁺-ATPase. Proc. Natl. Acad. Sci. USA 87:2466-2470.[Abstract/Free Full Text]
38
38. Tong, H., W. Chen, C. Steenbergen, and E. Murphy. 2000. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ. Res. 87:309-315.[Abstract/Free Full Text]
39
39. Verma, A., D. J. Hirsch, M. R. Hanley, O. Thastrup, S. Brogger, and S. H. Snyder. 1990. Inositol triphosphate and thapsigargin discriminate endoplasmic stores of calcium in rat brain. Biochem. Biophys. Res. Commun. 172:811-816.[CrossRef][Medline]
40
40. Yoshida, N. 1983. Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi. Infect. Immun. 40:836-839.[Abstract/Free Full Text]
41
41. Yoshida, N. 2006. Molecular basis of mammalian cell invasion of Trypanosoma cruzi. An. Acad. Bras. Cienc. 78:87-111.[Medline]
42
42. Yoshida, N., M. I. Dorta, A. T. Ferreira, M. E. M. Oshiro, R. A. Mortara, A. Acosta-Serrano, and S. Favoreto, Jr. 1997. Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction. Mol. Biochem. Parasitol. 84:57-67.[CrossRef][Medline]
43
43. Yoshida, N., R. A. Mortara, M. F. Araguth, J. C. Gonzalez, and M. Russo. 1989. Metacyclic neutralizing effect of monoclonal antibody 10D8 directed to the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi. Infect. Immun. 57:1663-1667.[Abstract/Free Full Text]

---

Infection and Immunity, October 2006, p. 5522-5528, Vol. 74, No. 10
0019-9567/06/$08.00+0     doi:10.1128/IAI.00518-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Martins, R. M., Covarrubias, C., Rojas, R. G., Silber, A. M., Yoshida, N. (2009). Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion. Infect. Immun. 77: 3023-3032 [Abstract] [Full Text]  
• Ashton, A. W., Mukherjee, S., Nagajyothi, F., Huang, H., Braunstein, V. L., Desruisseaux, M. S., Factor, S. M., Lopez, L., Berman, J. W., Wittner, M., Scherer, P. E., Capra, V., Coffman, T. M., Serhan, C. N., Gotlinger, K., Wu, K. K., Weiss, L. M., Tanowitz, H. B. (2007). Thromboxane A2 is a key regulator of pathogenesis during Trypanosoma cruzi infection. JEM 204: 929-940 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ferreira, D. Articles by Yoshida, N. Search for Related Content PubMed PubMed Citation Articles by Ferreira, D. Articles by Yoshida, N.