Trypanosoma cruzi TcSMUG L-surface mucins promote development and infectivity in the triatomine vector Rhodnius prolixus.

BACKGROUND: TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. CONCLUSION AND SIGNIFICANCE: Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.

Involvement of sulfated glycosaminoglycans on the development and attachment of Trypanosoma cruzi to the luminal midgut surface in the vector, Rhodnius prolixus.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.

Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prolixus.

Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of the hematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 days after blood meal, providing the compartmentalization of the enzymatic processes in the intestinal microenvironment. Using an enzyme cytochemical approach, Mg2+-ATPase and ouabain-sensitive Na+K+-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cells and in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritin and colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM. Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of alpha-d-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-d-galactosamine and N-acetyl-galactosamine-alpha-1,3-galactose were detected on the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neoglycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM) recognizing N-acetyl-d-galactosamine, alpha-d-mannose, alpha-l-fucose and alpha-d-glucose in the posterior midgut epithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These results have led the authors to suggest that for some components the PMM resembles the MM lining the midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to the luminal space.

Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus.

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 microM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50-100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal beta-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.

The haemoxisome: a haem-iron containing structure in the Rhodnius prolixus midgut cells.

Rhodnius prolixus midgut was analysed using transmission electron microscopy and electron spectroscopic imaging in order to localize the cellular structures involved in haem metabolism. In the posterior midgut, special cellular electron-dense structures were observed. These structures are here designated haemoxisomes. Haemoxisomes are present in the epithelial cells at various time points after a blood meal. Several days after the blood meal, some of them become less electron-dense. By electron spectroscopic imaging, large amounts of iron and oxygen were detected in these cellular structures. The iron is probably bound to the porphyrin ring as an iron-protoporphyrin IX complex, as detected using the diaminobenzidine technique. An interesting observation was the presence of endoplasmic reticulum surrounding the haemoxisomes during some special periods. Iron content was monitored in the posterior midgut epithelium and was found to be constant at the initial days after a blood meal, but slightly higher at the end of the digestive process (from 13th up to 20th day). These results are in agreement with the observation that the appearance of the haemoxisomes changes at the end of the digestive process. The ability to degrade haem seems to depend on the presence of endoplasmic reticulum as observed using a haem degradation assay in the presence of an endoplasmic reticulum-enriched fraction. Taken together these results suggest that haemoxisomes may play a role in intracellular haem detoxification.