TcI/TcII co-infection can enhance Trypanosoma cruzi growth in Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is an obligate haematophagous insect and one of the most important vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. T. cruzi is a highly variable parasite which is not transmitted in the same efficiency by the different triatomine vectors. Because different T. cruzi genotypes are aetiopathologically divergent, further elucidation of the transmission abilities of different Chagas disease vectors is extremely important. FINDINGS: In the present study, the growth behaviour of two T. cruzi isolates, MDID/BR/1993/C45 (TcI) and TBRA/BR/1999/JCA3 (TcII), sharing the same microhabitat (intestinal tract) in single and mixed infections, was examined. The distribution patterns and parasite population densities were evaluated at 7, 14 and 21 days after feeding (daf) by quantification of parasites using Neubauer haemocytometric measurements and mini-exon PCR to identify TcI and TcII subpopulations. Parasitic colonization in the small intestine was more successful in the mixed infection model than the single infection models at 21 daf. In the rectal lumen and wall, the growth behaviour of the mixed infection was similar to that of the TcI group, although the total parasite number was lower. In the TcII group, no metacyclic trypomastigote forms were found. PCR analysis of the contents of each dissected region showed different genotype fractions in the mixed infection model, in which TcI seemed to be the predominant isolate. CONCLUSION: The different growth behaviour of the TcI and TcII isolates in single and mixed infection models demonstrated that possibly an intraspecific factor modulates parasitic development in the intestine of R. prolixus.

Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization.

Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.

Cathepsin L of Triatoma brasiliensis (Reduviidae, Triatominae): sequence characterization, expression pattern and zymography.

Triatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects.

Trypanosoma cruzi infection modulates the expression of Triatoma brasiliensis def1 in the midgut.

Antimicrobial peptides are an essential component of the insect immune system. One of the most ubiquitous is defensin, which has been identified in all examined insect orders. Triatoma brasiliensis (Heteroptera, Triatominae), the main Trypanosoma cruzi vector in semi-arid regions of north-eastern Brazil, expresses def1, a defensin encoding gene, predominantly in the anterior region (cardia and stomach) of the midgut. In the present study, we compared the transcript abundance of T. brasiliensis def1 in the anterior (stomach) and posterior midgut (small intestine) regions of naïve bugs with those infected with a familiar T. cruzi isolate. In the stomach, only slight differences between the two insect groups were visible, whereas in the small intestine wide differences (up to 9.6-fold) between infected and noninfected bugs become apparent. The highly increased def1 transcript abundance at 20 days after the infective blood meal might be a response to the T. cruzi infection, suggesting a potential function of intestinal defensin in the parasite population control.

An overview of Chagas disease and the role of triatomines on its distribution in Brazil.

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi and affects about 15 million people in the Americas. In the Brazilian Amazon Basin, the disease is enzootic, with bugs from the genus Rhodnius serving as the main vectors, while in the northeastern region, mainly the genus Triatoma is naturally infected with T. cruzi. Oral infections appear sporadically in some regions of Brazil. Even though the illness is typically present in Latin America, autochthonous cases have been reported in the United States, mainly in the south of the country. The Triatominae subfamily comprises a large number of insect species that are potential vectors of Chagas disease. Triatomines are hematophagous insects, ingesting blood in all life stages. Since the insects play an important role in parasite selection, they also influence the geographical distribution of T. cruzi. The globalization process is one of the most important elements influencing the outcome of Chagas diseases in Brazil. This article outlines the status of reemerging Chagas disease in different regions of Brazil, the distribution of its vectors, and the consequences of global climate changes with respect to interactions of the pathogen with triatomines.

Two novel defensin-encoding genes of the Chagas disease vector Triatoma brasiliensis (Reduviidae, Triatominae): gene expression and peptide-structure modeling.

Defensins are cysteine-rich peptides involved in the innate immunity of insects and many other organisms. In the present study, two novel defensin-encoding cDNAs and the respective genomic DNAs (def3 and def4) of Triatoma brasiliensis were identified and their tissue-specific and temporal expression was characterized. Both of the deduced mature peptides consisted of 43 amino acid residues and were highly similar to previously identified triatomine defensins (81.4-100%). Semi-quantitative RT-PCR data showed that def3 was constitutively expressed in the fat body and was induced in salivary glands and the small intestine at 5 and 3 days after feeding (daf), respectively. The def4 mRNA level was highly up-regulated in the stomach and fat-body tissues at 5 and 3 daf, respectively. The three-dimensional structures of these defensins were predicted using a homology modeling approach with Def-AAA, the defensin from Anopheles gambiae, as template (62-74% identity). A map of the electrostatic potential of these models revealed that, despite their similar folding patterns, mature Def2 and Def4 have a more cationic structure than is the case for Def1 and Def3. Such differences may orient the antimicrobial profile of these defensins against distinct targets in different organs of the insect.

Development of a Trypanosoma cruzi (TcI) isolate in the digestive tract of an unfamiliar vector, Triatoma brasiliensis (Hemiptera, Reduviidae).

Triatoma brasiliensis is an important vector of Trypanosoma cruzi, commonly found in semi-arid areas of north-eastern Brazil. T. cruzi (TcI) is a widely distributed genotype in all biomes of Brazil. To evaluate selective pressures exerted by a vector species on the development of TcI derived from a different biome (Atlantic Rainforest), T. brasiliensis larvae were infected with the MDID/BR/1994/C48 isolate. Parasite densities of T. cruzi were determined in three regions of the gut at 3, 5 and 10 days after feeding. Percentages of the different stages of the flagellate were identified in Giemsa stained smears. The TcI isolate possessed always significantly higher densities in the rectum than in the small intestine. Epimastigotes reached their highest percentage at 3 days after feeding in the small intestine and trypomastigotes at 10 days after feeding in the rectal wall. Additionally, high metacyclogenesis rates in the T. brasiliensis gut showed competence of this TcI strain to complete its life cycle in this unfamiliar vector species.

Growth behaviour of two Trypanosoma cruzi strains in single and mixed infections: In vitro and in the intestinal tract of the blood-sucking bug, Triatoma brasiliensis.

Competition and cooperation are well-recognized biological phenomena, even among parasites. Co-infection of parasites in a single host leads to several outcomes, one being competition for a limited resource. Here, the behaviour of mixed infection was evaluated using two isolates of Trypanosoma cruzi, previously typed as belonging to genotypes TcI and TcII. The growth in vitro and in the different compartments of the gut of Triatoma brasiliensis was studied. In vitro growth showed that MDID/BR/1999/M1 (TcI) has a doubling time of 19.5h and MIDID/BR/1999/JCPD4 (TcII) of 9.6h, while the mixed infection group presented a doubling time of 13.9h. In vivo, three groups of infection were done: M1/TcI, JCPD4/TcII and mixed infection (50% of each strain), respectively. All comparisons among the groups were done using the Kruskal-Wallis non-parametric test. The data showed that the in vitro culture of mixed populations has a similar pattern to the growth of M1/TcI, apparently suggesting a positively selection for M1/TcI strain, in axenic culture. In the gut of the insects, M1/TcI isolate and mixed infections colonized predominantly the rectal wall and rectal lumen, in contrast to the JCPD4/TcII isolate, which was found mainly colonizing the small intestine. According to the isolates investigated, it could be concluded that the doubling time was not determinant factor for the final composition of a co-infection. Moreover, mixed infections resulted in a homogenous distribution of the parasites, comparing to the isolates studied separately. Apparently, in the gut of the bugs, the simultaneous presence of JCPD4/TcII isolate resulted in an improvement of the number of parasites from M1/TcI.

Synthetic derivatives of curcumin and their activity against Leishmania amazonensis.

In a previous work, the in vitro and in vivo activity of a series of diarylheptanoid derivatives against Leishmania amazonensis has been described. Based on the promising results, ten new compounds belonging to the same chemical class were synthesized and have been investigated in relation to their leishmanicidal activity. The compounds were obtained through several chemical modifications on the basic structure of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) in an attempt to increase its effectiveness and decrease the potential toxic effects. The drugs were assayed in vitro against L. amazonensis promastigotes and using pentamidine isethionate as reference drug. The results showed that the most effective compound is 1,7-bis-(4-propargyl-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, which is about ten times more efficient than the original curcumin. Nevertheless, these results did not allow us to make any correlation between the leishmanicidal activity and the chemical structure of the compounds.

Studies on the effectiveness of diarylheptanoids derivatives against Leishmania amazonensis

In a previous work we demonstrated that diarylheptanoids extracted from Centrolobium sclerophyllum are very active against Leishmania amazonensis promastigotes. In order to continue our studies with these class of compounds, we decided to evaluate the activity of several diarylheptanoids derived from curcumin (diferuloyl methane) against the extracellular form (promastigotes) of L. amazonensis. Furthermore, an experiment against the intracellular form of the parasite (amastigotes) was carried out, comparing the most active compound among the curcumin derivatives (the methylcurcumin) with des-O-methylcentrolobine, the most active diarylheptanoid derived from C. sclerophyllum