Trypanosoma cruzi and vector interactions
Before considering more details of the vector - parasite interactions, it is important to understand some aspects of the parasite life cycle in the invertebrate host. Points of contact between T. cruzi, the etiologic agent of Chagas disease, and its triatomine vectors begin with ingestion of the infective blood meal. Blood feeding is essential for the parasite growth in all instars and in the adult insect (Kollien and Schaub 1998). Interactions between T. cruzi and its vector insects begin with the arrival of an infected blood meal in the insect gut. During feeding, the trypomastigotes forms from the blood of the infected vertebrate host are ingested by the insect (Garcia and Azambuja 1991, Kollien and Schaub 2000). After a few days in the stomach (anterior part of the midgut) of the insect most of the bloodstream trypomastigotes transform into epimastigotes and some spheromastigotes (Garcia and Azambuja 1991, Kollien and Schaub 2000). Subsequently, mainly in the intestine (posterior part of the midgut), the epimastigotes divide repeatedly by binary division and can attach to the perimicrovillar membranes in the intestinal cells (Gonzalez et al. 1999). At later stages in the rectum, a proportion of the epimastigotes attach to the rectal cuticle and transform into metacyclic trypomastigotes that are eliminated with the feces and urine and are able to infect the vertebrate host (Garcia and Azambuja 1991, Kollien and Schaub 2000).
Since the gut of triatomines is the first environment for the establishment of T. cruzi infection, we studied the possible influence of digestive enzymes on parasite development (Garcia 1987), developed suitable bioassays for specific aspects of triatomine physiology and biochemistry in the gut (for review see Garcia and Azambuja 1997), and analyzed a variety of factors, such as the clone used and feeding with azadirachtin, influencing the life cycle of T. cruzi in the gut of the vector, R. prolixus (Garcia and Azambuja 1991).
Results showed that feeding R. prolixus with the inhibitor of acid SH-proteinase, pepstatin, had no effect on rates of T. cruzi infection (Garcia and Gilliam 1980). Also, trypomastigotes from single-cell-isolates of two clones of T. cruzi produced different rates of growth because of different population doubling times of the parasites and only one clone underwent epimastigote to metacyclic trypomastigote transformation (Garcia and Dvorak 1982). Results therefore indicate that not only the kinetics of T. cruzi epimastigote multiplication but also the transformation to metacyclic trypomastigotes depend on the strains and clones of the infecting parasite (Garcia et al. 1984b). In addition, the passage of homogeneous strains of T. cruzi through the invertebrate host gut was identical with the host was unable to modify any biological, biochemical or genetic characteristics of the parasite (Garcia et al. 1986b).
Although many factors are thought to be important to the establishment of T. cruzi infection in the gut of the vector, only a few molecules have been implicated, such as a stomach lytic factor (Azambuja et al. 1983, 1989), lectins (Pereira et al. 1981, Mello et al. 1996) and hemoglobin fragments (Garcia et al. 1995), all of which have been tested directly against the parasite. The work of Pereira et al. (1981) reported lectins in the digestive tube and hemolymph of R. prolixus and suggested that these molecules could be involved in the development of T. cruzi in the triatomine bug. Mello et al. (1996) demonstrated differences in the development of three strains of T. cruzi in the gut of R. prolixus and related the infectivity of these to the ability of the digestive tube extract to agglutinate these parasitesin vitro. Interestingly, lectins agglutinated the Dm28c clone of T. cruzi which achieved high infectivity levels while, in contrast, the Y strain of the parasite which was not agglutinated, was lysed and failed to develop in the vector gut (Azambuja et al. 1989, Mello et al. 1996, 1999).
Frainderaich et al. (1993) demonstrated that metacyclogenesis of T. cruzi is promoted in vitro by an aD-globin-derived peptide present in hemoglobin corresponding to residue 1-40 from the amino terminus found in the gut of Triatoma infestans. Synthetic peptides having the amino terminal globin sequences and containing conserved domains spanning amino acid residues 30 to 40 are recognized by a surface receptor in epimastigote cells and stimulate T. cruzi adenylyl cyclase (Frainderaich et al. (1993). Garcia et al. (1995) also studied in vivo in R. prolixus, the effects of hemoglobin and synthetic peptides carrying aD-globin fragments on both the growth and transformation of T. cruzi epimastigotes into metacyclic trypomastigotes. This differentiation in the insect gut occurred when hemoglobin and synthetic peptides corresponding to residues 30-49 and 35-73 of the aD-globin were added to the plasma diet. However, synthetic peptide 41-73 did not induce differentiation of epimastigotes even in the presence of the two former peptides so that peptide 41-73 appeared to block the action of these stimulatory peptides. In addition, the whole hemoglobin molecule was shown to be a very important blood component for the growth of parasites (Garcia et al. 1995). These data identified an unusual molecular mechanism, which modulates the dynamics of transformation of epimastigotes into metacyclic trypomastigotes in the triatomine vectors gut.
Recently, Azambuja et al. (2004) opened an exciting new research area by studying the effects of resident bacteria in the stomach of R. prolixus on erythrocyte lysis and T. cruzi infection. Following feeding, bacteria rapidly multiplied and the number of surviving Y strain of T. cruzi in the stomach declined drastically, while infection with Dm28c clone remained stable. Hemolytic bacteria were isolated and identified as Serratia marcescens biotype A1a (referenced as RPH), which produces the pigment, prodigiosin. In vitro experiments, comparing incubation of RPH or S. marcescens SM365, a prodigiosin pigment producer, or S. marcescens DB11, a none pigmented variant, as a control, with erythrocytes and T. cruzi demonstrated that: (i) at 0°C or 30°C, both SM365 and RPH reduced the populations of Y strain, but not of the DM28c clone, and DB11 was unable to lyse either T. cruzi strain; and (ii) all three strains of S. marcescens were able to lyse erythrocytes (Azambuja et al. 2004). These results suggest that S. marcescens trypanolytic activity from the SM365 and RPH strains is distinct from the hemolytic activity and that prodigiosin is an important factor for the trypanolytic action of the bacteria in the gut of the vector. The study of bacteria in the gut of invertebrate hosts may be important and provide new tools to block the development of parasites in the insect vector.
One of the more interesting outcomes of theT. cruzi - vector interactions resulted from the investigations made with the compound azadirachtin, a natural growth inhibitor from the neem tree (Azadirachta indica A. Juss), which strongly interferes with the neuroendocrine regulation of the insect hormone titers (Garcia and Rembold 1984, Garcia et al. 1984a, 1986a, 1990). This compound not only affects the development of triatomines but also the establishment of T. cruzi infection in the gut of different species of triatomines (Garcia et al. 1989, Gonzalez and Garcia 1992, Gonzalez et al. 1999, Kollien et al. 1998).
Nogueira et al. (1997) analyzed the process of interaction of epimastigotes and trypomastigotes of T. cruzi (Dm 28c clone and Y strain) with the intestinal epithelium of R. prolixus in vitro. Their observations showed that both parasite developmental stages move towards the epithelial surface. However, while trypomastigotes did not attach to the epithelial surface, all epimastigotes strongly attached through the flagellum, preferentially at the periphery of the cells. When the R. prolixus larvae were decapitated or treated with azadirachtin, the T. cruzi epimastigotes were unable to adhere to the stomach or intestinal epithelium (Gonzalez et al. 1999). This may result from the effect of these treatments on factors present in the perimicrovillar membrane, which interact with glycoinositol phospholipids (GIPLs) molecules abundant on the epimastigote plasma membrane and involved in the process of parasite insect host-interaction (Garcia and Azambuja, unpublished data). Thus, manipulation of the physiological condition of the vector host may affect the development of T. cruzi, with decapitation, head transplantation, azadirachtin and ecdysone therapy consequently all influencing the parasite development. Insects that received these treatments showed a distinct effect characterized by ultrastructural disorganization of the midgut epithelial cells of R. prolixus and indicated that the prothoracicotropic hormone (PTTH)-ecdysone pathway interferes with T. cruzi survival and development in its vectors (Gonzalez et al. 1999). These results provide the first clear evidence showing the importance of the insect endocrine system in establishing T. cruzi infection in the vector.
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