Depletion of Na+/H+ Exchanger Isoform 1 Increases the Host Cell Resistance to Trypanosoma cruzi Invasion.

Na+/H+ exchanger isoform 1 (NHE1), a member of a large family of integral membrane proteins, plays a role in regulating the cortical actin cytoskeleton. Trypanosoma cruzi, the agent of Chagas disease, depends on F-actin rearrangement and lysosome mobilization to invade host cells. To determine the involvement of NHE1 in T. cruzi metacyclic trypomastigote (MT) internalization, the effect of treatment in cells with NHE1 inhibitor amiloride or of NHE1 depletion was examined in human epithelial cells. MT invasion decreased in amiloride-treated and NHE1-depleted cells. The phosphorylation profile of diverse protein kinases, whose activation is associated with remodeling of actin fibers, was analyzed in amiloride-treated and NHE1-depleted cells. In amiloride-treated cells, the phosphorylation levels of protein kinase C (PKC), focal adhesion kinase (FAK) and Akt were similar to those of untreated cells, whereas those of extracellular signal-regulated protein kinases (ERK1/2) increased. In NHE1-deficient cells, with marked alteration in the actin cytoskeleton architecture and in lysosome distribution, the levels of phospho-PKC and phospho-FAK decreased, whereas those of phospho-Akt and phospho-ERK1/2 increased. These data indicate that NHE1 plays a role in MT invasion, by maintaining the activation status of diverse protein kinases in check and preventing the inappropriate F-actin arrangement that affects lysosome distribution.

Shedding of Trypanosoma cruzi Surface Molecules That Regulate Host Cell Invasion Involves Phospholipase C and Increases Upon Sterol Depletion.

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-ß-cyclodextrin (MßCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MßCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MßCD-treated parasites. CM from untreated and MßCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MßCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.

Interaction of Trypanosoma cruzi Gp82 With Host Cell LAMP2 Induces Protein Kinase C Activation and Promotes Invasion.

The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.

Depletion of Host Cell Focal Adhesion Kinase Increases the Susceptibility to Invasion by Trypanosoma cruzi Metacyclic Forms.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.

Oral infection of mice and host cell invasion by Trypanosoma cruzi strains from Mexico.

Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.

Host cell protein LAMP-2 is the receptor for Trypanosoma cruzi surface molecule gp82 that mediates invasion.

Host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is mediated by MT-specific surface molecule gp82, which binds to a still unidentified receptor, inducing lysosome spreading and exocytosis required for the parasitophorous vacuole formation. We examined the involvement of the major lysosome membrane-associated LAMP proteins in MT invasion. First, human epithelial HeLa cells were incubated with MT in the presence of antibody to LAMP-1 or LAMP-2. Antibody to LAMP-2, but not to LAMP-1, significantly reduced MT invasion. Next, HeLa cells depleted in LAMP-1 or LAMP-2 were generated. Cells deficient in LAMP-2, but not in LAMP-1, were significantly more resistant to MT invasion than wild-type controls. The possibility that LAMP-2 might be the receptor for gp82 was examined by co-immunoprecipitation assays. Protein A/G magnetic beads cross-linked with antibody directed to LAMP-1 or LAMP-2 were incubated with HeLa cell and MT detergent extracts. Gp82 bound to LAMP-2 but not to LAMP-1. Binding of the recombinant gp82 protein to wild-type and LAMP-1-deficient cells, which was dose dependent and saturable, had a similar profile and was much higher as compared with LAMP-2-depleted cells. These data indicate that MT invasion is accomplished through recognition of gp82 by its receptor LAMP-2.

Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion.

Successful infection by Trypanosoma cruzi, the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role.

S. mansoni-T. cruzi co-infection modulates arginase-1/iNOS expression, liver and heart disease in mice.

Although Schistosoma species and Trypanosoma cruzi share common endemic areas, co-infections by these parasites remains overlooked. By using a murine model of S. mansoni and T. cruzi co-infection, we investigated if and to what extent these infections might interact to change the pathological outcomes typically observed when the host is infected by a single parasite species. Swiss mice were randomized into four groups: uninfected (NI) and those infected by S. mansoni (SM), T. cruzi (TC) or co-infected (SM + TC). After 120 days of S. mansoni infection, T. cruzi was concurrently inoculated and the infection occurred for 30 days. Taken together, we identified that the overlap of Th2 (schistosomiasis) and Th1 (Chagas disease) immunological patterns changes the host resistance against both pathogens. Beyond impairing the control of granulomatous inflammation, T. cruzi parasitemia and parasitism in co-infected animals, the Th2 inflammatory response against S. mansoni elicits the activation of the arginase-1 pathway to the detriment of inducible oxide nitric synthase (iNOS) expression and nitric oxide (NO) production, contributing to the liver damage, with minor effects on heart pathology.

Beta-adrenergic antagonist propranolol inhibits mammalian cell lysosome spreading and invasion by Trypanosoma cruzi metacyclic forms.

The involvement of ß-adrenergic receptor (ß-AR) in host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is not known. We examined whether isoproterenol, an agonist of ß-AR, or nonselective ß-blocker propranolol affected MT internalization mediated the stage-specific surface molecule gp82. Treatment of HeLa cells with propranolol significantly inhibited MT invasion whereas isoproterenol had no effect. Propranolol, but not isoproterenol, also inhibited the lysosome spreading required for gp82-dependent MT invasion. The effect of propranolol in inhibiting MT internalization was not due to the prevention of gp82 interaction with ß-AR. It was mainly associated with its ability to impair lysosome spreading.

Curcumin Enhances the Anti-Trypanosoma cruzi Activity of Benznidazole-Based Chemotherapy in Acute Experimental Chagas Disease.

Although curcumin can increase the effectiveness of drugs against malaria, combination therapies using the molecule have never been investigated in Chagas disease (ChD). Therefore, we evaluated the efficacy of curcumin as a complementary strategy to benznidazole (Bz)-based chemotherapy in mice acutely infected with Trypanosoma cruzi Eighty-four 12-week-old Swiss mice were equally randomized into seven groups: uninfected (NI), T. cruzi infected and untreated (INF), infected and treated with 100 mg/kg of body weight Bz (B100), 50 mg/kg Bz (B50), 100 mg/kg curcumin (C100), 100 mg/kg Bz plus 100 mg/kg curcumin (B100 plus C100), and 50 mg/kg Bz plus 100 mg/kg curcumin (B50 plus C100). After microscopic identification of blood trypomastigotes (4 days after inoculation), both drugs were administered by gavage once a day for 20 days. Curcumin showed limited antiparasitic, anti-inflammatory, and antioxidant effects when administered alone. When curcumin and Bz were combined, there was a drastic reduction in parasitemia, parasite load, mortality, anti-T. cruzi IgG reactivity, circulating levels of cytokines (gamma interferon [IFN-γ], interleukin 4 [IL-4], and MIP1-α), myocardial inflammation, and morphological and oxidative cardiac injury; these results exceeded the isolated effects of Bz. The combination of Bz and curcumin was also effective at mitigating liver toxicity triggered by Bz, increasing the parasitological cure rate, and preventing infection recrudescence in noncured animals, even when the animals were treated with 50% of the recommended therapeutic dose of Bz. By limiting the toxic effects of Bz and enhancing its antiparasitic efficiency, the combination of the drug with curcumin may be a relevant therapeutic strategy that is possibly better tolerated in ChD treatment than Bz-based monotherapy.