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Leishmanicidal and trypanocidal activity of seventeen lipophilic diamines was evaluated in vitro against Leishmania braziliensis, L. chagasi, and Trypanosoma cruzi. Twelve compounds presented anti-Leishmania and six showed anti-T. cruzi amastigote activity. Compound 14 (N-tetradecyl-1,4-butanediamine) was the most active against both L. braziliensis (IC50 = 2.6 μm) and L. chagasi (IC50 = 3.0 μm) which showed a selectivity index (SI) >100. N-decyl-1,6-hexanediamine (compound 9) presented an IC50 = 1.6 μm and SI >187 and was over six times more potent than the reference drug benznidazole against T. cruzi. Treatment of infected or uninfected macrophages with compounds 9 and 14 did not induce significant TNFα and NO production. Four compounds (15, 16, 22, and 23) inhibited 78.9%, 77.7%, 83.7%, and 70.1% of rTRLb activity, respectively, and compound 23 inhibited 73.3% of rTRTc activity at 100 μm. A concentration-dependent effect on mitochondrial membrane depolarization was observed in T. cruzi epimastigotes treated with compound 9, suggesting this mechanism may be involved in the trypanocidal effect. On the contrary, in L. braziliensis promastigotes treated with compound 14, no mitochondrial depolarization was observed. Our results demonstrate that N-decyl-1,6-hexanediamine and N-tetradecyl-1,4-butanediamine are promising molecules for the development of novel leading compounds against T. cruzi and Leishmania spp., particularly given a possible alternative mechanism of action.

Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

Background Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins. Results Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex. Conclusion Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoanTrypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway ofT. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinantT. cruzicruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes ofT. cruzi.

Background Although known to be highly endemic in the Amazon regions of Brazil, the presence of cutaneous leishmaniasis (CL) in the subtropical southern part of the country has largely been ignored. This study was conducted to demonstrate CL is emerging in the Brazilian state of Santa Catarina, as well as to characterize the epidemiological profile and Leishmania species involved. Methodology/Principal Findings For this cross-sectional study, data from all CL cases from Santa Catarina, Brazil, reported to the Brazilian National Notifiable Diseases Information System from 2001 to 2009 were investigated. Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR-RFLP) was conducted to screen for Leishmania species present in patient biopsy. Overall, 542 CL cases were reported, with majority resulting from autochthonous transmission (n = 401, 73.99%) and occurring in urban zones (n = 422, 77.86%). Age, gender, zone of residence, origin of case, clinical form and case outcome were found to differ significantly by region. Imported cases were over seven times more likely to relapse (95% CI 2.56–21.09). Mapping of cases revealed new endemic areas in northeastern Santa Catarina with two species present. With the exception of three L. (Leishmania) amazonensis cases (1.20%), majority of PCR positive samples were found to be L. (Viannia) braziliensis (n = 248, 98.80%). Conclusions/Significance CL is now endemic in the state of Santa Catarina, Brazil, with case profiles varying significantly by region. L. (V.) braziliensis has been identified as the predominant species in the region.

A study of the natural infection of phlebotomine sand flies by Leishmania (Viannia) was conducted in a focus of cutaneous leishmaniasis in Picarras, on the northeastern coast of the Brazilian state of Santa Catarina. In total, 562 female Nyssomyia neivai were collected by miniature light traps near houses, separated in 61 pools and examined by PCR and Southern blot hybridization. Eight pools, four of them from the same light trap/night, were positive. This is the first finding of natural infection by Le. braziliensis of adequately identified Ny. neivai in Brazil. In this preliminary observation we observed the abundance and predominance of Ny. neivai among the captured phlebotomine species (98.5%), indicating that Ny. neivai may be the dominant vector of Leishmania in the subgenus Viannia in this area.

Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil / UCLA School of Medicine. Department of Microbiology and Immunology. Los Angeles, CA, USA. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade de S?o Paulo. Instituto de Ci?ncias Biol?gicas. Departamento de Parasitologia. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Universidade Federal do Rio de Janeiro. Centro de Ci?ncias da Sa?de. Departamento de An?lises Cl??nicas e Toxicol?gicas. Rio de Janeiro, RJ, Brazil. Universidade Federal de Santa Catarina. Centro de Ci?ncias Biol?gicas. Departamento de Microbiologia e Parasitologia. Florian?polis, SC, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Laborat?rio de Parasitologia Celular e Molecular. Belo Horizonte, MG, Brazil. UCLA School of Medicine. Department of Microbiology and Immunology. Los Angeles, CA, USA. Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.

Background American Trypanosomiasis or Chagas disease is caused by Trypanosoma cruzi which currently infects approximately 16 million people in the Americas causing high morbidity and mortality. Diagnosis of American trypanosomiasis relies on serology, primarily using indirect immunofluorescence assay (IFA) with T. cruzi epimastigote forms. The closely related but nonpathogenic Trypanosoma rangeli has a sympatric distribution with T. cruzi and is carried by the same vectors. As a result false positives are frequently generated. This confounding factor leads to increased diagnostic test costs and where false positives are not caught, endangers human health due to the toxicity of the drugs used to treat Chagas disease. Results In the present study, serologic cross-reactivity between the two species was compared for the currently used epimastigote form and the more pathologically relevant trypomastigote form, using IFA and immunoblotting (IB) assays. Our results reveal an important decrease in cross reactivity when T. rangeli culture-derived trypomastigotes are used in IFA based diagnosis of Chagas disease. Western blot results using sera from both acute and chronic chagasic patients presenting with cardiac, indeterminate or digestive disease revealed similar, but not identical, antigenic profiles. Conclusion This is the first study addressing the serological cross-reactivity between distinct forms and strains of T. rangeli and T. cruzi using sera from distinct phases of the Chagasic infection. Several T. rangeli-specific proteins were detected, which may have potential as diagnostic tools.

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.

Ten chalcones were synthesized and tested as potential leishmanicidal and trypanocidal agents. All tested compounds caused concentration-dependent inhibition of the in vitro growth ofLeishmania braziliensisandTrypanosoma cruziwith no significant toxic effect towards host macrophages. Our results show that the positions of the substituents seem to be critical for their antiprotozoal activities.