Trypanosoma brucei interaction with host: Mechanism of VSG release as target for drug discovery for african trypanosomiasis

This research was funded by GHTM-UID/multi/04413/2013 (FCT-Portugal) and Grant number 019/2013 (Capes-Brazil).The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.publishersversionpublishe

Vlp-based vaccines as a suitable technology to target trypanosomatid diseases

Funding Information: This research was funded by Global Health and Tropical Medicine (Grant number IHMT UID/multi/04413/2013 and Grant number PTDC/CVT-CVT/28908/2017), Funda??o para Ci?ncia e Tecnologia (FCT), Portugal; Grant GIU18/172 Grupos de Investigaci?n de la UPV/EHU, and Grant from CSIC Programa I-COOP+2020 (ICOOPB20503), Spain.A.M.V.Q., J.W.d.F.O., and C.J.M. thanks to the financial support (PhD scholar-ships) provided by CNPq and CAPES, Brazil. M.S.S. thanks to CNPq for the Research Grant (Bolsa de Produtividade em Pesquisa). We are also grateful to Paulo Fanado and Laysa Ohanna for editing this manuscript. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite–host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.publishersversionpublishe

Bisphosphonate-based molecules as potential new antiparasitic drugs

Neglected tropical diseases such as Chagas disease and leishmaniasis affect millions of people around the world. Both diseases affect various parts of the globe and drugs traditionally used in therapy against these diseases have limitations, especially with regard to low efficacy and high toxicity. In this context, the class of bisphosphonate-based compounds has made significant advances regarding the chemical synthesis process as well as the pharmacological properties attributed to these compounds. Among this spectrum of pharmacological activity, bisphosphonate compounds with antiparasitic activity stand out, especially in the treatment of Chagas disease and leishmaniasis caused by Trypanosoma cruzi and Leishmania spp., respectively. Some bisphosphonate compounds can inhibit the mevalonate pathway, an essential metabolic pathway, by interfering with the synthesis of ergosterol, a sterol responsible for the growth and viability of these parasites. Therefore, this review aims to present the information about the importance of these compounds as antiparasitic agents and as potential new drugs to treat Chagas disease and leishmaniasis.publishersversionpublishe

Cell Culture and Maintenance of the Evolutionary Forms of <em>Trypanosoma cruzi</em> for Studies of Parasitic Biology

This chapter aims to present and discuss the main cell culture techniques used for the growth and maintenance of the different evolutionary forms of the protozoan Trypanosoma cruzi, the etiologic agent of the Chagas disease. Chagas disease is a neglected tropical disease endemic in several Latin American countries. Here, we intend to present the main difficulties, advantages, and disadvantages of using Trypanosoma cruzi cell culture in parasitic biology. Finally, we present the main research opportunities in the context of Trypanosoma cruzi parasitic biology using cell culture techniques, such as drug development, characterization of pharmacological targets, molecular markers for diagnosis, structural biology, and many other biomedical applications

Insights of antiparasitic activity of sodium diethyldithiocarbamate against different strains of Trypanosoma cruzi

Funding Information: This research was funded by Global Health and Tropical Medicine: Grant number IHMT-UID/multi/04413/2013 and Grant number PTDC/CVT-CVT/28908/2017, FCT-Portugal. Funding Information: We would like to thank Prof. João Aristeu da Rosa and Dr. Aline Rimodi Rimeiro at UNESP (Universidade Estadual de São Paulo) Araraquara (Brazil) for offering four different strains of T. cruzi. JWFO, CJGM, and BAC thanks to the financial support (PhD and Post-doctoral fellowships) provided by Capes/Brazil; MSS and HAOR thanks to CNPq/Brazil for the Research Grant (Bolsa de Produtividade). We also would like to thank the Department of Materials Engineering at UFRN for allowing the use of their scanning electron microscope, and the Department of Biochemistry at UFRN for allowing the use of their Flow Cytometer. We are also grateful to Paulo Fanado for editing this manuscript. Publisher Copyright: © 2021, The Author(s).Chagas disease is caused by Trypanosoma cruzi and affects thousands of people. Drugs currently used in therapy are toxic and have therapeutic limitations. In addition, the genetic diversity of T. cruzi represents an important variable and challenge in treatment. Sodium diethyldithiocarbamate (DETC) is a compound with pharmacological versatility acting as metal chelators and ROS generation. Thus, the objective was to characterize the antiparasitic action of DETC against different strains and forms of T. cruzi and their mechanism. The different strains of T. cruzi were grown in LIT medium. To evaluate the antiparasitic activity of DETC, epimastigote and trypomastigote forms of T. cruzi were used by resazurin reduction methods and by counting. Different response patterns were obtained between the strains and an IC50 of DETC ranging from 9.44 ± 3,181 to 60.49 ± 7.62 µM. Cell cytotoxicity against 3T3 and RAW cell lines and evaluated by MTT, demonstrated that DETC in high concentration (2222.00 µM) presents low toxicity. Yet, DETC causes mitochondrial damage in T. cruzi, as well as disruption in parasite membrane. DETC has antiparasitic activity against different genotypes and forms of T. cruzi, therefore, representing a promising molecule as a drug for the treatment of Chagas disease.publishersversionpublishe

Quantum Biochemistry Screening and In Vitro Evaluation of Leishmania Metalloproteinase Inhibitors

Funding Information: Global Health and Tropical Medicine funded this research: Grant number IHMT-UID/multi/04413/2013 and Grant number PTDC/CVT-CVT/28908/2017, FCT-Portugal. Funding Information: We would like to thank to CNPq/Brazil, CAPES/Brazil and FCT/Portugal for grants and fellowships. C.J.G.M., J.W.d.F.O., T.K.d.B.P., F.L.d.S.J. and B.A.-C. thank the financial support (PhD and Post-doctoral fellowships) provided by Capes/Brazil. M.S.S. and H.A.O.R. thank CNPq/Brazil for the Research Grant (Bolsa de Produtividade em Pesquisa). We also would like to thank the Department of Materials Engineering at UFRN for allowing the use of their scanning electron microscope, and the Department of Biochemistry at UFRN for allowing the use of their flow cytometer. Publisher Copyright: © 2022 by the authors.Leishmanolysin, also known as major promastigote protease (PSP) or gp63, is the most abundant surface glycoprotein of Leishmania spp., and has been extensively studied and recognized as the main parasite virulence factor. Characterized as a metalloprotease, gp63 can be powerfully inactivated in the presence of a metal chelator. In this study, we first used the structural parameters of a 7-hydroxycoumarin derivative, L1 compound, to evaluate the theoretical–computational experiments against gp63, comparing it with an available metal chelator already described. The methodology followed was (i) analysis of the three-dimensional structure of gp63 as well as its active site, and searching the literature and molecular databases for possible inhibitors; (ii) molecular docking simulations and investigation of the interactions in the generated protein–ligand complexes; and (iii) the individual energy of the gp63 amino acids that interacted most with the ligands of interest was quantified by ab initio calculations using Molecular Fraction with Conjugated Caps (MFCC). MFCC still allowed the final quantum balance calculations of the protein interaction to be obtained with each inhibitor candidate binder. L1 obtained the best energy quantum balance result with −2 eV, followed by DETC (−1.4 eV), doxycycline (−1.3 eV), and 4-terpineol (−0.6 eV), and showed evidence of covalent binding in the enzyme active site. In vitro experiments confirmed L1 as highly effective against L. amazonensis parasites. The compound also exhibited a low cytotoxicity profile against mammalian RAW and 3T3 cells lines, presenting a selective index of 149.19 and 380.64 µM, respectively. L1 induced promastigote forms’ death by necrosis and the ultrastructural analysis revealed disruption in membrane integrity. Furthermore, leakage of the contents and destruction of the parasite were confirmed by Spectroscopy Dispersion analysis. These results together suggested L1 has a potential effect against L. amazonensis, the etiologic agent of diffuse leishmaniasis, and the only one that currently does not have a satisfactory treatment.publishersversionpublishe

Estabelecimento do modelo murino para a modulação de toxicidade do Benzonidazol durante a terapêutica da Doença de Chagas (Trypanosoma cruzi)

Após um século da sua descoberta, a doença de Chagas causada por protozoário Trypanosoma cruzi representa uma das principais doenças tropicais negligenciadas em vários países da América Latina. O Benzonidazol (BNZ) é o fármaco de primeira escolha para o tratamento da doença, contudo apresenta uma eficácia terapêutica variável na fase crónica da doença. Além disso, em muitas situações os pacientes apresentam efeitos adversos graves, que conduzem ao abandono do tratamento. A eficácia antiparasitária do fármaco pode estar diretamente relacionada com à heterogeneidade de T. cruzi, bem como as propriedades farmacocinéticas desfavoráveis, a baixa solubilidade aquosa e imponderável biodisponibilidade do BNZ. Deste modo, este estudo teve como objetivos principais a determinação da toxicidade aguda do BNZ e das nanoformulações do BNZ em modelo murino através do ensaio da determinação da dose máxima tolerada e quantificação dos biomarcadores da função hepática. Paralelamente, realizaram-se o estudo da suscetibilidade in vitro ao BNZ das estirpes de T. cruzi (Y, Bolívia e QMM5) e o estudo da infeciosidade das mesmas estirpes em modelo murino. Os resultados indicaram que os excipientes e as nanoformulações do BNZ apresentaram um bom perfil de tolerância deste modo podem ser uma alternativa para a terapêutica da doença de Chagas. As três estirpes apresentaram suscetibilidade in vitro ao BNZ e a análise dos soros dos murganhos Mus musculus (Balb/c) inoculados com as estirpes do parasita demostram a presença anticorpos anti- T. cruzi. A resposta humoral foi poliisotípica, com predomínio da produção dos anticorpos IgG e IgM anti-T. cruzi bem como algumas subclasses de anticorpo, a IgG1 e IgG2a e IG3. A produção do NO e da citocina IL-4 aumentou durante a fase inicial da infeção com diminuição na fase mais avançada da infeção, setenta dias pós infeção sendo sugestivo da ação imunomodulatória na infeção por T. cruzi. Estes resultados reforçam a compreensão da fisiopatologia da doença de chagas e permitem auxiliar futuramente na compreensão da eficácia das nanoformulações do BNZ durante o tratamento da doença de chagas no modelo murino.After a century of its discovery, Chagas disease caused by protozoan Trypanosoma cruzi is one of the major neglected tropical diseases in several Latin American countries. The benznidazole (BNZ) is the drug of choice for the treatment of disease, but has a variable therapeutic efficacy in chronic phase of the disease. Furthermore, in many situations the patients have serious side effects that leading to abandonment of the treatment. The antiparasitic drug efficacy can be directly related to the heterogeneity of T. cruzi population in addition with the unfavorable pharmacokinetic properties of BNZ, the low aqueous solubility and bioavailability of imponderable. Thus, this study had as main objectives the determination of the acute toxicity of BNZ and nanoformulations of BNZ in mice through the test for determining the maximum dose tolerated and quantification of iomarkers of liver function. Meanwhile, there have been the study of in vitro susceptibility to BNZ strains of T. cruzi (Y, Bolivia and QMM5) and study the infectivity of the same strains in mice. The results indicate that excipients and nanoformulations of BNZ showed a good tolerability profile and can be an alternative for the treatment of Chagas disease. The three strains showed susceptibility to the BNZ in vitro and analysis of the sera of Mus musculus (Balb/c) inoculated with strains of the parasite demonstrate the presence of anti- T. cruzi. The humoral response was poliisotípica, predominantly the production of IgG and IgM anti-T.cruzi well as some antibody subclasses, IgG1 and IgG2a and IG3. NO production and cytokine IL-4 increased during the initial phase of infection with decrease in the later stage of infection seventy days after infection being suggestive of immunomodulatory action in infection by T. cruzi. These results reinforcing aspects of the pathophysiology of Chagas disease and will allow understanding of the effectiveness of these nanoformulations of BNZ for the treatment of Chagas disease in the murine model in the future

Variable Surface Glycoprotein from Trypanosoma brucei Undergoes Cleavage by Matrix Metalloproteinases: An in silico Approach

In order to survive as extracellular parasites in the mammalian host environment, Trypanosoma brucei has developed efficient mechanisms of immune system evasion, which include the abundant expression of a variable surface glycoprotein (VSG) coat. VSGs are anchored in the parasite membrane by covalent C-terminal binding to glycosylphosphatidylinositol and may be periodically removed by a phospholipase C (PLC) and a major surface protein (TbMSP). VSG molecules show extraordinary antigenic diversity and a comparative analysis of protein sequences suggests that conserved elements may be a suitable target against African trypanosomiasis. However, the cleavage mechanisms of these molecules remain unclear. Moreover, in protozoan infections, including those caused by Trypanosoma brucei, it is possible to observe an increased expression of the matrix metalloproteinases (MMPs). To address the cleavage mechanism of VSGs, the PROSPER server was used for the identification of VSG sequence cleavage sites. After data compilation, it was observed that 64 VSG consensus sequences showed a high conservation of hydrophobic residues, such as valine (V), methionine (M), leucine (L) and isoleucine (I) in the fifth position&mdash;the exact location of the cleavage site. In addition, the PROSPER server identified conserved cleavage site portions of VSG proteins recognized by three matrix metalloproteases (gelatinases: MMP-2, MMP-3 and MMP-9). However, further biological studies are needed in order to analyze and confirm this prediction