Ligantes de miócitos cardíacos para a glicoproteína de 85kda. (tc-85) de Trypanosoma cruzi

O Trypanosoma cruzi expressa um grupo de glicoprotcinas de superfície, denominadas Tc-85, que pertencem à superfumília gêmca das gp85/traus-sialidases. Nosso laboratório clonou e caracterizou um membro da fumília Tc85 (Tc85-11), cuja região carboxila tenninal (clone Tc85-1) adere em laminina e em células de mamífero. Usando peptídeos sintéticos, correspondendo em seqüência à Tc85-1, caracterizou-se o motivo mais conservado da superfamilia gênica das gp85/trans-sialidases (VTVxNVFLYNR), o qual não adere em laminina. Esse motivo foi chamado peptídeo J. Por cromatografia de extratos de membrana de cardiomiócitos em coluna de afmidade contendo peptídeo J, foi isolada uma molécula de 30kDa identificada como sendo a subunidade β3 da Na+, K+ ATPase. A porção extracelular da subunidade β3 da Na+, K+ ATPase foi clonada e a interação in vitro desta proteína com peptídeo J foi observada. Deste modo, é sugerido aqui que a subunidade β3 da Na+, K+ ATPase pode ter um papel importante na interação do parasita com a célula hospedeira.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell in vitro

Megazol, nifurtimox, benznidazol and allopurinol were investigated, by light and electron µscopy, for their action on T. cruzi. Both the direct effect upon amastigote and trypomastigote forms and the effect upon the interaction of heart muscle cells (HMC) with bloodstream trypomastigotes were studied. The proliferation of amastigotes in Warren medium was inhibited in a dose-dependent manner by megazol, nifurtimox and benznidazol. Treatment of amastigotes (25-50 µM/24 h) and trypomastigotes (25 µM/24h) led to several ultrastructural alterations in the parasites. These three drugs also had a potent effect on the treatment of infected heart muscle cells when added at the beginning of the interaction or after one or three days of infection. The interiorized parasites showed a similar pattern of ultrastructural alterations as observed by the direct effect on the amastigotes. The primary heart muscle cell culture proved to be a suitable model for the study of drugs on intracellular parasites. Likewise, the amastigote proliferation in axenic medium was shown to be an adequate assay for an initial trial of drugs. These parameters seem very reliable to us for a systematic investigation of the mechanism of action of new drugs.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and,to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxity between anti-trypanossomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypothesis for the mechanism of action of phenothiazines are discussed.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Sialoglycoconjugates in Trypanosoma cruzi-host cell interaction: possible biological model - a review

A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.

Interaction between Didelphis albiventris and Triatoma infestans in relation to Trypanosoma cruzi transmission

This paper attempts to prove if a high Trypanosoma cruzi prevalence of opossums might be reached with few potential infective contacts. One non-infected Didelphis albiventris to T. cruzi and 10 infected nymphs of Triatoma infestans were left together during 23 hr in a device that simulated a natural opossum burrow. Twenty-six replicates were perfomed using marsupials and triatomines only once. Potentially infective contacts occurred in all the trials. From the 26 opossums used in trials, 54% did not eat any bug. Of the 260 bugs used, 21% were predated. In the 25 trials involving 205 surving bugs, 36 % of them did not feed. In 15/25 cases, maior ou igual a 60% of the triatomines were able to feed. The parasitological follow-up of 24 opossums showed that among 10 that had eaten bugs, 4 turned out infected and among the 14 that had not predate, 3 (21%) became positive. In sum, 7/24 (29%) of the marsupials acquired the infection after the experiment. This infection rate was similar to the prevalences found for the opossum population of Santiago del Estero, Argentina, suggesting that the prevalences observed in the field might be reached if each marsupial would encounter infected bugs just once in its lifetime.

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

Plasminogen interaction with Trypanosoma cruzi

The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This interaction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator.The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.