Pedro Henrique Nascimento Aguiar

Background: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. Methods: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD⁺/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. Results: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearsonʼs correlations (0.48–0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. Conclusions: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified.

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9^−/− mice were vaccinated with rfd phages expressing the OVA_257–264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8⁺ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9^−/− animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8⁺ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

SUMMARY: Trypanosoma cruziis exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably,T. cruzi’s antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, aT. cruzicell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H₂O₂) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H₂O₂led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels inT. cruzi, resulting in higher levels of residual H₂O₂after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates ofT. cruziinRhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase inT. cruzihas played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.

Schistosoma mansoni is a trematode parasite that causes schistosomiasis, one of the most prevalent neglected tropical diseases, leading to the loss of 2.6 million disability-adjusted life years. Praziquantel is the only drug available, and new drugs are required. The most common strategy in schistosomiasis drug discovery is the use of the schistosomula larval-stage for a pre-screen in drug sensitivity assays. However, assessing schistosomula viability by microscopy has always been a limitation to the throughput of such assays. Hence, the development of validated, robust high-throughput in vitro assays for Schistosoma with simple readouts is needed. Here, we present a simple and affordable alternative to assess schistosomula viability. The method employed is based on the hydrosoluble tetrazolium salt XTT which has been widely used in other organisms but has never been used to drug screen in schistosomes. We showed that schistosomula reduce XTT salt to a coloured formazan product and that absorbance levels reflected the viability and parasites number. This XTT viability assay was validated for high throughput screening of compounds in schistosomula, and dose-response curves of compounds could be reproduced. We conclude that the XTT viability assay could be applied for the screening of large compounds collections in S. mansoni and accelerate the identification of novel antischistosomal compounds.

During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress. In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91^phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo. Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells. The parasite Trypanosoma cruzi is the causative agent of Chagas’ disease, which affects 10 million people, mainly in Latin American. Macrophages are one of the first cellular actors facing the invasion of pathogens and during T. cruzi infection, produce reactive oxygen species (ROS). To deal with oxidative stress, T. cruzi has an antioxidant machinery and, to repair DNA damage triggered by ROS, this parasite possesses enzymes of the oxidized guanine DNA repair system. The understanding of the role of ROS in the infection by T. cruzi can provide us with good insights on T. cruzi biology and virulence. While some studies suggest that ROS is related to parasite control, others have demonstrated that ROS is important for proliferation of this parasite. To investigate the contribution of ROS in T. cruzi infection, we utilized mice deficient in the production of ROS (Phox KO) and parasites that overexpress the enzymes related to DNA repair. Our results show that ROS is not only important for the battle against pathogens, but suggest that ROS can also work as a signal that contributes to the growth of this parasite.

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability. The parasite Trypanosoma cruzi is the causative agent of Chagas disease, a malady endemic throughout Latin America. Studying the DNA repair machinery of this parasite could provide us with good insights about T. cruzi biology and virulence. We focused on the 8-oxoguanine (8-oxoG) DNA lesion and its repair system. This lesion is considered particularly deleterious because it can generate DNA double strand breaks if inserted during the DNA replication. Our approach to investigating the importance of the 8-oxoG repair system in T. cruzi was to generate a parasite population expressing the Escherichia coli MutT enzyme, which is responsible for removing 8-oxo-dGTP from the nucleotide pool. Different parameters such as growth curves, cell infection experiments, antioxidants, enzymes expression, and DNA lesion quantification were used to study this modified parasite in comparison with a control WT population. We also characterized a gene in T. cruzi that has functional homology with the E. coli MutT gene. The overexpression of this gene in T. cruzi caused the same phenotypes observed when we expressed the heterologous gene. Overall, the results indicate the importance of this DNA repair enzyme for T. cruzi resistance to oxidative stress and improving its proliferative ability in the vertebrate host.

A wide variety of DNA lesions arise due to environmental agents, normal cellular metabolism, or intrinsic weaknesses in the chemical bonds of DNA. Diverse cellular mechanisms have evolved to maintain genome stability, including mechanisms to repair damaged DNA, to avoid the incorporation of modified nucleotides, and to tolerate lesions (translesion synthesis). Studies of the mechanisms related to DNA metabolism in trypanosomatids have been very limited. Together with recent experimental studies, the genome sequencing of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, has revealed interesting features of the DNA repair mechanism in these protozoan parasites, which will be reviewed here.