Miercio E.A. Pereira
Tufts Medical Center
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Featured researches published by Miercio E.A. Pereira.
Cell | 1991
Eduardo Ortega-Barria; Miercio E.A. Pereira
T. cruzi invades mammalian cells in various organs after migrating through the ECM. These activities appear to be mediated by a unique 60 kd protein exposed on the T. cruzi surface, which promotes selective adhesion of trypomastigotes to three ECM components: heparin, heparan sulfate, and collagen. The affinity-purified protein binds to host fibroblasts in a saturable and glycosaminoglycan- and collagen-inhibitable manner. When adsorbed to plastic, it promotes adhesion and spreading of fibroblasts, as does the recombinant protein expressed in E. coli. The endogenous protein, and reactive ECM proteins, are very effective in preventing T. cruzi invasion of culture cells. The recombinant protein localizes on the E. coli surface and induces the bacteria that express it to adhere to and penetrate nonphagocytic Vero cells in a proteoglycan- and collagen-inhibitable manner. Therefore, the protein, named penetrin, could play a critical role in T. cruzi binding to the ECM and to cells, and in host cell invasion.
Cell | 1995
Mao Ming; Mark E. Ewen; Miercio E.A. Pereira
Trypanosoma cruzi invades most nucleated mammalian cells by as yet unknown mechanisms. We report here that while T. cruzi attaches to epithelial cells lacking signaling transforming growth factor beta (TGF beta) receptor I or II, the adherent parasites cannot penetrate and replicate inside the mutant cells, as they do in parental cells. Invasion of the mutants is restored by transfection with the TGF beta receptor genes, as are biological responses to TGF beta. Similar rescue of both TGF beta antiproliferative response and T. cruzi invasion was demonstrated in a hybrid of TGF beta-resistant bladder and colon carcinoma cells. In addition, T. cruzi did not efficiently invade epithelial cells with dysfunction of the intracellular signaling cascade caused by the constitutive expression of the cyclin-dependent kinase cdk4 or of the oncogene H-ras. Treatment with TGF beta, but not with other antiproliferative agents of non-phagocytic cells, greatly enhances T. cruzi invasion. Moreover, infective, but not noninfective, trypanosomes strongly induce a TGF beta-responsive reporter gene in TGF beta-sensitive, but not in TGF beta-insensitive, cell lines. Thus, T. cruzi itself may directly trigger activation of the TGF beta signaling pathway required for parasite entry into the mammalian cells.
Molecular and Biochemical Parasitology | 1993
Mao Ming; Marina V. Chuenkova; Eduardo Ortega-Barria; Miercio E.A. Pereira
Trypanosoma cruzi attaches and invades a large variety of mammalian cells. The nature of the cell receptors and of the corresponding parasite counter-receptors that mediate T. cruzi-host cell interaction are not known. Three sialic acid-deficient mutants of Chinese hamster ovary (CHO) cells were used to probe the role of host sialyl residues in T. cruzi infection. All three mutants supported adhesion and infection to a much lower extent than the parental CHO cells. One of the mutants, Lec2, contains sugar chains terminating in non-reducing beta Gal residues, which are acceptors for sialylation by the T. cruzi trans-sialidase. Re-sialylation of Lec2 cells restored T. cruzi adhesion and invasion to about the same extent as wild-type cells. Digestion of wild-type cells with bacterial sialidase reduced T. cruzi interaction but after re-sialylation, the cells were almost as good as control, naturally sialylated parental cells. These results suggest that T. cruzi recognizes sialyl residues on the surface of host cells during invasion. On the other hand, affinity-purified trans-sialidase blocked T. cruzi adherence and invasion of sialylated cells, and had no effect on parasite interaction with sialic acid-deficient Lec2 mutant. Furthermore, 2,3-sialyllactose, a substrate for the trans-sialidase, competitively inhibited T. cruzi invasion of sialylated parental K1 cells, but 2,6-sialyllactose, which does not react with the trans-sialidase, was without effect, as were other sugars that do not contain alpha 2,3 sialyl residues. These results suggest that the trans-sialidase functions as a counter-receptor for trypomastigote binding to alpha 2,3-sialyl receptors on host cells as a prelude to T. cruzi invasion.
Molecular and Biochemical Parasitology | 1994
Enrique M. Herrera; Mao Ming; Eduardo Ortega-Barria; Miercio E.A. Pereira
Trypanosoma cruzi attaches and invades a large variety of mammalian cells by receptor-mediated interactions, one of them involving the binding of parasite trans-sialidase to host sialyl receptors. Three proteoglycan-deficient mutants of Chinese hamster ovary (CHO) cells were used to probe the role of host heparin and heparan sulfate glycosaminoglycans (GAG) in T. cruzi invasion. All three mutants supported adhesion and infection to a much lower extent than the parental CHO cells. One of the mutants, pgsD-677, did not express heparan sulfate while containing three- to four-fold excess chondroitin sulfate, yet the cell line was a poor substrate for T. cruzi adhesion. Proteoglycan-deficient cells obtained by inhibiting GAG synthesis in parental cells with p-nitrophenyl-beta-D-xyloside, were also poor hosts for T. cruzi invasion. Furthermore, digestion of parental cells with heparinase and heparitinase, two lyases that specifically depolymerize heparin and heparan sulfate, reduced the potential of the cells to support T. cruzi adhesion and growth. Lyases that digested chondroitin sulfate and other GAGs did not affect T. cruzi invasion. These results suggest that heparin/heparan sulfate epitopes are receptors for T. cruzi invasion. The corresponding counter-receptor on T. cruzi appears to be penetrin, a heparin-binding protein that promotes trypanosome penetration into cells. Purified penetrin caused agglutination of red blood cells, and the hemagglutination was exquisitely sensitive to heparin and heparan sulfate. However, sialic acid and sialyl compounds did not inhibit penetrin-induced hemagglutination. Recombinant penetrin competitively inhibited T. cruzi invasion of proteoglycan-containing parental cells, but not of proteoglycan-deficient mutants nor of heparitinase-treated cells. Furthermore, consistent with the sugar specificity of penetrin as a hemagglutinin, recombinant penetrin competed for trypanosome invasion of a CHO cell mutant (Lec2) that expresses heparan sulfate but not sialyl residues. Given that the release of sialic acid from the proteoglycan-deficient mutants further reduced T. cruzi invasion, as did the removal of heparan sulfate from the Lec2 mutant, and given that penetrin does not bind to sialic acid with high affinity, the results indicate that the penetrin-heparan sulfate pathway for T. cruzi invasion is distinct from the trans-sialidase-sialic acid route.
Journal of Immunological Methods | 1983
Miercio E.A. Pereira
A neuraminidase assay based on peanut lectin agglutination is described. Human red blood cells are used both as substrate for the enzyme and as probe for the lectin. The validity of the method is ascertained by measuring the enzyme and lectin activities on erythrocytes whose outer membrane sialic acid was labeled with tritium after oxidation with sodium periodate followed by reduction with sodium borotritiide. The neuraminidases of Trypanosoma cruzi and Vibrio cholera are used as examples; in both cases, a linear relationship is observed between the degree of erythrocyte desialylation and the peanut hemagglutination titer. For the hemagglutination assay, lectin in homogeneous form as well as in crude peanut extracts may be used, and free sialic acid need not be separated from substrate-bound sialic acid. The hemagglutinating activity of peanut lectin is not affected by pre-treatment of the erythrocytes with various proteases. The method is particularly useful in the neuraminidase analysis of multiple samples, such as in gel filtration chromatography and for screening of hybridoma antibodies against neuraminidase.
Molecular and Biochemical Parasitology | 1991
Ian Rosenberg; Reginaldo P. Prioli; Eduardo Ortega-Barria; Miercio E.A. Pereira
Several studies indicate that the neuraminidase from Trypanosoma cruzi (TCNA) [1 ] plays an important role in infection. Trypomastigotes remove sialic acid from rat myocardial and human endothelial cells in vitro [2], and from mouse [1] and human [3] erythrocytes during infection with T. cruzi in vivo. The extent of desialylation of host cells correlates with the number of parasites and TCNA activity [3]. Inhibition of enzyme activity by polyclonal [4] and monoclonal [5] antibodies and by high [6] and low-density lipoproteins [7] augment invasion of host cells by trypomastigotes suggesting that the enzyme exerts a negative control on T. cruzi infection (reviewed in ref. 8). We have previously shown that TCNA is spontaneously released into condition medium when tissue culture trypomastigotes are incubated at 4°C [4] or 37°C [5] for several hours or days, suggesting that the enzyme is anchored to the cell surface by a glycophosphatidylinositol (GPI) linkage. Release of membrane-bound proteins by PI-PLC is now regarded as convincing evidence for the involvement of GPI in membrane anchoring [9]. We therefore tested whether TCNA could be specifically removed from the surface of the Silvio X10/4
Molecular and Biochemical Parasitology | 1986
Miercio E.A. Pereira; Rodney Hoff
Various strains, stocks, and clones of Trypanosoma cruzi were analyzed for neuraminidase (NA) activity using fetuin and human erythrocytes as substrate. In all cases the activity was found to be developmentally regulated. Zymodeme type I strains, which are histotropic for skeletal muscle, had greater NA activity than zymodeme type II strains which are histotropic for either macrophages or cardiac muscle cells. Heterogeneity of NA expression within strains is suggested by the finding that one Silvio X10 clone had greater NA activity than another clone of the same stock. The differences observed were more pronounced when human erythrocytes and not fetuin were used as substrate. Trypomastigotes of the high producing strains reared in bovine artery smooth muscle cells had enhanced expression compared to trypomastigotes reared in 3T3 or human fibroblast cells. The first harvest of trypomastigotes from cell cultures had greater NA activity than trypomastigotes harvested on subsequent days.
Molecular and Biochemical Parasitology | 1990
Reginaldo P. Prioli; Ian Rosenberg; Miercio E.A. Pereira
Trypanosoma cruzi exhibits a developmentally regulated neuraminidase activity that is inhibited by high-density lipoprotein (HDL). We report here that the infection of culture cells by T. cruzi trypomastigotes is enhanced by HDL in a dose-dependent manner. The enhanced infection is prevented by Vibrio cholerae neuraminidase, an enzyme whose activity is not inhibited by HDL, suggesting that sialic acid is involved in T. cruzi-host interaction. Similar enhancement of infection is also produced by low-density lipoprotein (LDL), which inhibits T. cruzi neuraminidase as well as HDL. Further evidence that the enhancement is due to lipoproteins is provided by the fact that infection of host cells in lipoprotein-deficient medium is less than in normal medium; it can be restored to the higher level by the addition of HDL, LDL or both to the lipoprotein-deficient medium. In view of these results, we propose that HDL and LDL regulate T. cruzi infection in mammalian hosts by inhibiting the parasite neuraminidase activity.
Molecular and Biochemical Parasitology | 1988
Reginaldo P. Prioli; Ian Rosenberg; Sumati Shivakumar; Miercio E.A. Pereira
Binding of high density lipoprotein (HDL) to Trypanosoma cruzi was examined because of its ability to specifically inhibit the parasites neuraminidase. 125I-Labeled HDL bound to live and glutaraldehyde-fixed parasites equally well either at 37 degrees C or at 4 degrees C. Binding was saturable and inhibited by unlabeled HDL but not by unrelated plasma proteins. Specificity of the T. cruzi-HDL interaction was confirmed using fluorescein labeled HDL which bound to T. cruzi but not to T. rangeli, a species whose neuraminidase is not inhibited by HDL. Binding of HDL to T. cruzi paralleled the neuraminidase activity exhibited by the parasites different stages and strains. In agreement with this finding, Steck and Wallach analysis of the binding data showed that the number of HDL binding sites was greater in infective trypomastigotes and on strains with high neuraminidase activity. However, the association constant of the binding did not change within the various developmental forms and strains of T. cruzi, suggesting that HDL bound to the same receptor, presumably having neuraminidase activity.
Molecular and Biochemical Parasitology | 1985
Miercio E.A. Pereira; Deborah Moss
Supernatants of cultures and extracts of Trypanosoma rangeli readily release N-acetyl neuraminic acid from a variety of substrates. The activity in both supernatant and cell extract is precipitated between 30 and 50% ethanol, and between 40 and 70% ammonium sulfate. Fractionation of the culture supernatant by gel exclusion gives a single peak of neuraminidase activity of molecular weight 48 000. The culture supernatant releases sialic acid at different rates from the following substrates:fetuin, sialyllactose and orosomucoid but not from bovine submaxillary mucin and ovomucoid. The enzyme in the culture supernatant is also active against human erythrocytes of all ABO types. The enzyme showed an optimum pH of 5.0 for sialyllactose and erythrocyte substrates. Large amounts of the enzyme are preferentially secreted during growth in vitro.