Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell.

Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis.

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis.

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 µM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 µM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process.

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.

Role of small GTPases in Trypanosoma cruzi invasion in MDCK cell lines.

Trypanosoma cruzi can modulate a large number of host intracellular responses during its invasion. GTPases such as RhoA, Rac1 and Cdc42 are examples of molecules that could be activated at this moment and trigger changes in the pattern of F-actin cytoskeleton leading to the formation of structures like stress fibers, lamellipodium and fillopodium, respectively. Here we investigate the role of these GTPases in the cytoskeletal rearrangement of MDCK cell transfectants expressing variants of RhoA, Rac1 and Cdc42 during T. cruzi infection. The adhesion, internalization and the survival rate were determined. Rac1 mutants showed the higher adhesion and internalization indexes but the lower survival index after 48 h of infection. Confocal laser scanning microscopy showed changes in the pattern of F-actin distribution and reorganization at the site of trypomastigote invasion. These observations suggest that these GTPases act in the signaling mechanisms that affect the F-actin cytoskeleton during T. cruzi invasion.

Protein phosphorylation during the process of interaction of Toxoplasma gondii with host cells.

Previous studies have shown that tachyzoites of Toxoplasma gondii were able to penetrate into macrophages using two mechanisms: phagocytosis and active penetration. We show here that previous incubation of the macrophages or the parasites with staurosporine, a wide range inhibitor of protein kinases, tyrfostin and genistein, specific inhibitors of tyrosine kinases, significantly interfered with the process of parasite-macrophage interaction. Staurosporine treatment induced the formation of many filopodium-like surface projections of the macrophages and markedly increased the attachment of the tachyzoites to the cell surface. Genistein inhibited about 50% penetration of T. gondii into macrophages. Previous incubation of tachyzoites with genistein also significantly inhibited their attachment to and penetration into the macrophages. Confocal laser scanning microscopy was used to locate phosphoproteins in macrophages interacting with tachyzoites. Antiphosphotyrosine antibodies labeled the surface of macrophages, but not L929 cells, incubated in presence of T. gondii, even those cells did not show associated parasites. Anti phosphotyrosine, phosphothreonine and phosphoserine antibodies labeled the region surrounding the parasitophorous vacuoles. These observations suggest that protein phosphorylation is a key event in the process of T. gondii-host cell interaction.

Behaviour of microtubules in cells infected with Toxoplasma gondii

Toxoplasma gondii proliferates within the parasitophorous vacuole of the host cell. Simultaneously with parasite division and vacuolar development, lipids traffic and change in the spatial distribution of organelles of the host cell cytoplasm occur. Using fluorescence microscopy, and antibodies recognizing tubulin, we showed that microtubules change their distribution during host cell infection by tachyzoites of T. gondii. In addition, transmission electron microscopy of thin sections and replicas of partially extracted cells showed that host cell microtubules concentrate around the parasitophorous vacuole. Such microtubules distribution was evident in early infection times and was more prominent after 24 h of infection, when parasitophorous vacuole was completely surrounded by microtubules. However, the meshwork microtubule filaments became slack or absent after 72 h of infection of host cell. Colchicine and taxol treatment altered the shape of the parasitophorous vacuole containing tachyzoites. These observations suggest a close association between microtubules and intravacuolar development of parasites.

Behaviour of microtubules in cells infected with Toxoplasma gondii

Toxoplasma gondii proliferates within the parasitophorous vacuole of the host cell. Simultaneously with parasite division and vacuolar development, lipids traffic and change in the spatial distribution of organelles of the host cell cytoplasm occur. Using fluorescence microscopy, and antibodies recognizing tubulin, we showed that microtubules change their distribution during host cell infection by tachyzoites of T. gondii. In addition, transmission electron microscopy of thin sections and replicas of partially extracted cells showed that host cell microtubules concentrate around the parasitophorous vacuole. Such microtubules distribution was evident in early infection times and was more prominent after 24 h of infection, when parasitophorous vacuole was completely surrounded by microtubules. However, the meshwork microtubule filaments became slack or absent after 72 h of infection of host cell. Colchicine and taxol treatment altered the shape of the parasitophorous vacuole containing tachyzoites. These observations suggest a close association between microtubules and intravacuolar development of parasites.(AU)

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§C 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.