Avaliation of the vitro and in vivo antimalarial activity of violacein extrated from Chromobacteriuim violaceum

Orientador: Fabio Trindade Maranhão CostaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A violaceína é um pigmento violeta extraído da bactéria Gram-negativa Chromobacterium violaceum. Diversos trabalhos atribuíram à violaceína diferentes atividades biológicas tais como bactericida, antiviral, fungicida e antitumoral. Os efeitos da violaceína também foram observados contra protozoários patogênicos, como Leishmania sp e Trypanossoma sp. Neste trabalho, avaliamos a atividade antimalárica da violaceína in vitro e in vivo contra Plasmodium humano e murino, respectivamente. Neste sentido, a violaceína se mostrou tão eficiente quanto o quinino em eliminar P. falciparum (3D7), mas três vezes menos eficiente que a cloroquina. Além disso, não encontramos diferença na sua atividade contra formas jovens e maduras, mostrando uma atuação similar nos diferentes estágios de desenvolvimento do parasita. Os experimentos in vivo utilizando camundongos infectados com PcchAS tratados por 11 dias consecutivos (0-10) revelaram uma potente atividade desta droga, inibindo o desenvolvimento parasitário em até 86% no pico da parasitemia. Também foi encontrado 60% de inibição quando o tratamento iniciou-se 5 dias após o estabelecimento da infecção. Quando administrada em animais infectados com uma cepa murina letal (PcchAJ), a violaceína protegeu 80% dos animais, em contraste à 100% de mortalidade dos animais não tratados. A comparação do ED50 demonstrou que a violaceína é tão eficiente quanto o artesunato e 2 vezes mais ativa que a cloroquina, sob as mesmas condições de tratamento. Finalmente, animais naive tratados com a violaceína durante 11 dias consecutivos não mostraram alterações na densidade de glóbulos vermelhos e no peso. Também não foi observado nenhum dano morfológico nos cortes histológicos. Coletivamente, estes resultados demonstram claramente o potencial antimalárico da violaceína e abre perspectivas para o entendimento dos mecanismos envolvidos na inibição parasitária por este composto.Abstract: Violacein is a violet pigment extracted from the bacteria Gram-negative Chromobacterium violaceum. Growing bodies of evidences have implicated violacein as an antimicrobial, antifungal, antitumoral, and a moderate trypanocidal and leishmanial activity has also been observed. Herein, we evaluated the anti-malarial activity of violacein against murine and human-derived Plasmodium. Indeed, violacein showed to be efficient in killing P. falciparum (3D7 strain) as much as quinine, but 3 fold less pronounced than chloroquine. Moreover, this anti-Plasmodial activity detected was direct against both young and mature stages of this human parasite. In vivo experiments in P. c. chabaudi AS (PcchAS) infected mice treated during 10 consecutive days after infection (0-10) revealed a powerful activity of this drug, by inhibiting parasite burden up to 86%. Also, 60% of inhibition was noticed when violacein was administrated 5 days after infection establishment. When administrated in mice infected with a lethal strain of murine-derived Plasmodium (PcchAJ) violacein protected 80% of these mice, in contrast to 100% of mortality reported to the non-treated animals. ED50 comparisons demonstrated that violacein was efficient as much as artesunate and twice more active than chloroquine. Finally, naïve mice inject with violacein during 10 consecutive days did not display alterations on red blood cell density; weight and neither morphological damages were noticed in spleen and liver histological sections. Collectively, these data clearly demonstrated the anti-malarial effect of violacein and open perspectives to understating the mechanisms involved in killing this parasite by this compound.MestradoImunologiaMestre em Genética e Biologia Molecula

Violacein treatment modulates acute and chronic inflammation through the suppression of cytokine production and induction of regulatory T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOInflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 mu g of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naive mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+ Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacteriumChromobacterium vio105116FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP [2011/17965-3]CNPq [471066/2012-5]FAPESP [2014/02631-0, 2011/23664-6, 2012/01892-0]2011/17965-3; 471066/2012-5; 2014/02631-0; 2011/23664-6; 2012/01892-0sem informaçã

Antibody recognition of plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infectionsthis presents an intriguing and underexplored area of research. In addition to the rapid access of infected p1095598601FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/17114-3576128/2008-2To Wolfgang Fischer and Márcio Yamamoto, for sequencing of DBLα tag sequences, and to Drs Mauro Shugiro Tada and Tony Katsuragawa, for help with data and sample collection at the site

Amazonian plant natural products:perspectives for discovery of new antimalarial drug leads

Plasmodium falciparum and P. vivax malaria parasites are now resistant, or showing signs of resistance, to most drugs used in therapy. Novel chemical entities that exhibit new mechanisms of antiplasmodial action are needed. New antimalarials that block transmission of Plasmodium spp. from humans to Anopheles mosquito vectors are key to malaria eradication efforts. Although P. vivax causes a considerable number of malaria cases, its importance has for long been neglected. Vivax malaria can cause severe manifestations and death; hence there is a need for P. vivax-directed research. Plants used in traditional medicine, namely Artemisia annua and Cinchona spp. are the sources of the antimalarial natural products artemisinin and quinine, respectively. Based on these compounds, semi-synthetic artemisinin-derivatives and synthetic quinoline antimalarials have been developed and are the most important drugs in the current therapeutic arsenal for combating malaria. In the Amazon region, where P. vivax predominates, there is a local tradition of using plant-derived preparations to treat malaria. Here, we review the current P. falciparum and P. vivax drug-sensitivity assays, focusing on challenges and perspectives of drug discovery for P. vivax, including tests against hypnozoites. We also present the latest findings of our group and others on the antiplasmodial and antimalarial chemical components from Amazonian plants that may be potential drug leads against malaria

The activity of methylene blue against asexual and sexual stages of Plasmodium vivax

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 µM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment

Total parasite biomass but not peripheral parasitaemia is associated with endothelial and haematological perturbations in Plasmodium vivax patients

Plasmodium vivax is the major cause of human malaria in the Americas. How P. vivax infection can lead to poor clinical outcomes, despite low peripheral parasitaemia remains a matter of intense debate. Estimation of total P. vivax biomass based on circulating markers indicates existence of a predominant parasite population outside of circulation. In this study we investigate associations between both peripheral and total parasite biomass and host response in vivax malaria. We analysed parasite and host signatures in a cohort of uncomplicated vivax malaria patients from Manaus, Brazil, combining clinical and parasite parameters, multiplexed analysis of host responses and ex vivo assays. Patterns of clinical features, parasite burden and host signatures measured in plasma across the patient cohort were highly heterogenous. Further data deconvolution revealed two patient clusters, here termed Vivaxlow and Vivaxhigh. These patient subgroups were defined based on differences in total parasite biomass but not peripheral parasitaemia. Overall Vivaxlow patients clustered with healthy donors and Vivaxhigh patients showed more profound alterations in haematological parameters, endothelial cell (EC) activation and glycocalyx breakdown and levels of cytokines regulating different haematopoiesis pathways compared to Vivaxlow. Vivaxhigh patients presented more severe thrombocytopenia and lymphopenia, along with enrichment of neutrophils in the peripheral blood and increased neutrophil-to-lymphocyte ratio (NLCR). When patients' signatures were combined, high association of total parasite biomass with a subset of markers of EC activation, thrombocytopenia and lymphopenia severity was observed. Finally, machine learning models defined a combination of host parameters measured in the circulation that could predict the extent of parasite infection outside of circulation. Altogether, our data show that total parasite biomass is a better predictor of perturbations in host homeostasis in P. vivax patients than peripheral parasitaemia. This supports the emerging paradigm of a P. vivax tissue reservoir, in particular in the hematopoietic niche of bone marrow and spleen

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation - a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.publishersversionpublishe