Iván Neira

Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells

ABSTRACT Upon oral infection, Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by metacyclic forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of metacyclic forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.

Diterpenoids from Azorella yareta and their trichomonicidal activities

Diterpenoids with trichomonicidal activity were isolated from the aerial parts of Azorella yareta Hauman. One was 13beta-hydroxyazorellane, together with the known constituents mulinolic acid, mulin-11,13-dien-20-oic acid, azorellanol and 13alpha-hydroxyazorellane. Their structures were determined by spectroscopic and chemical methods.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 M, these compounds displayed a strong lytic activity. It ranged from 88.4 0.6 to 99.0 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 M and 41-87 M, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 M. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 M. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

Infection by Trypanosoma cruzi Metacyclic Forms Deficient in gp82 but Expressing a Related Surface Molecule, gp30

ABSTRACT Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium after oral infection. In this study we analyzed the process of infection by T. cruzi isolates deficient in the expression of gp82, the metacyclic stage-specific surface glycoprotein implicated in target cell entry in vitro and in promoting mucosal infection in mice after oral challenge. Mice infected by the oral route with metacyclic forms of gp82-deficient isolate 569 or 588 developed patent parasitemia but at greatly reduced levels compared to those infected with the gp82-expressing isolate CL. Metacyclic forms of both isolates expressed gp30, a surface glycoprotein detectable by monoclonal antibody (MAb) 3F6 directed to gp82. Otherwise, the gp82-deficient isolates displayed a surface profile similar to that of the CL isolate and also entered epithelial HeLa cells in a manner inhibitable by MAb 3F6 and dependent on the parasite signal transduction that involved the activation of protein tyrosine kinase and Ca2+ mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca2+ response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of metacyclic forms of isolates 569 and 588 by ∼90 and ∼70%, respectively. A cell invasion assay performed in the presence of gastric mucin, mimicking the in vivo infection, showed an inhibition of 70 to 75% in the internalization of gp82-deficient isolates but not of the CL isolate. The recombinant gp82 exhibited an adhesive capacity toward gastric mucin much higher than that of gp30. Taken together, our findings indicate that target cell entry of metacyclic trypomastigotes can be mediated either by gp82 or gp30 but that efficient mucosal infection depends on the expression of gp82.

Cell invasion by Trypanosoma cruzi amastigotes of distinct infectivities: studies on signaling pathways

Trypanosoma cruzi metacyclic trypomastigotes of the major phylogenetic lineages use specific signaling pathways to invade host cells. Using a panel of drugs, we studied if the differences in the ability of extracellular amastigotes (EA) from G (T. cruzi I) and CL (T. cruzi II) strains to invade host cells could be associated to activation of specific signaling routes. Sonicated extracts from G or CL strain EA induced transient raises in HeLa cell intracellular Ca2+ levels in a dose-dependent manner. Treatment of EA with drugs that affect Ca2+ release from inositol-1,4,5-triphosphate-sensitive stores did not significantly affect the infectivity of either strain, whereas EA of both strains treated with ionomycin plus NH4Cl or nigericin that release Ca2+ from acidocalcisomes had their infectivity reduced. Treatment of parasites with adenylate cyclase activator forskolin increased the infectivity of both strains towards HeLa cells. These data, taken together, suggest that, for host cell invasion, G and CL strain EA engage signaling pathways that lead to an increase of cyclic adenosine monophosphate and Ca2+ mobilization from acidocalcisomes. Moreover, treatment of EA with genistein reduced by ∼45% the invasion of HeLa cells by G but not by CL strain, implicating a protein tyrosine kinase in the process. In line with this, HeLa cell extracts contained a protein tyrosine kinase activity that mediated the phosphorylation of 87- and 175-kDa polypeptides of EA from G but not from CL strain. Regarding the target cell response, the activation of host PI3 kinase appears to be required for invasion by either strain as treatment of HeLa cells with wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process involving metacyclic trypomastigotes.

Cell Adhesion and Ca2+ Signaling Activity in Stably Transfected Trypanosoma cruzi Epimastigotes Expressing the Metacyclic Stage-Specific Surface Molecule gp82

ABSTRACT Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca2+ mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca2+ response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca2+ response in HeLa cells, in the same manner as the metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in metacyclic forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca2+ concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca2+-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.

AZORELLANE DITERPENOIDS FROM LARETIA ACAULIS, AND ITS TOXOPLASMACIDAL ACTIVITY

Desde de las partes aereas de Laretia acaulis (Cav.) Gill et Hook hemos aislado azorellanol y un nuevo diterpenoide 7-desacetilazorellanol. Su estructura esta basada en la comparacion de sus datos espectroscopicos con los de azorellanol y transformaciones quimicas. Azorellanol y 7-desacetilzorellanol muestran una marcada actividad inhibitoria frente a los trofozoitos del protozoo parasito Toxoplasma gondii. La dosis inhibitoria 50 (DI50) de azorellanol es 54 mM y para 7-desacetylazorellanol 42 mM

Nuevos casos de infección humana por Diphyllobothrium pacificum (Nybelin, 1931) Margolis, 1956 en Chile y su probable relación con el fenómeno de El Niño, 1975-2000

The effect of El Nino/ENSO on terrestrial atmosphere appears to be extremelly clear. However there are outstanding evidences showing its effect on humans and their activities. In fact, prevalence of some parasitic infections have increased during El Nino phenomenon. The reasons for that are the migrations of sylvatic mammals, fishes and birds as well as by environmental contamination. In this report, we show evidence respect of new cases of human infection by Diphyllobothrium pacificum clearly associated with a cyclic manifestation of El Nino in the Chilean Pacific coast during 1975-2000

Serum antibodies to Trypanosoma cruzi antigens in Atacameños patients from highland of northern Chile

In the present work we have investigated the serum antibody spectrum to parasite antigens involved in human T. cruzi infection. Analysis was performed by conventional serology (IHA, IFAT and ELISA), complement-mediated lysis, anti-gal antibody assay and reactivity against recombinant and synthetic peptides and metacyclic antigens by immunowestern-blotting. All the sera showed a significant reactivity in IHA, IFAT and ELISA. We found that 84.2% of the sera showed lytic activity and thirty serum samples (78.9%) which showed a lytic activity higher than 50%, also showed anti-gal antibodies at serum dilutions higher than 1:1,600. Ninety-four percent of sera reacted with one or more of the recombinant DNA clones and 97.3% reacted with one or more of the synthetic peptides. A pool of serum samples with a lytic activity higher than 75% were able to produce 60% to 78% inhibition of cell invasion. Thirty-six of the serum samples (94.7%) were able to react by immunowestern blotting with a T. cruzi metacyclic antigen with molecular size of 70 kDa. The results obtained give preliminary information about the humoral immune response and the possible role of antibodies in protection against T. cruzi infection of chronic patients from the highlands of Chile.