A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

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BackgroundAngomonas deanei is a trypanosomatid parasite of insects that has a bacterial endosymbiont, which supplies amino acids and other nutrients to its host. Bacterium loss induced by antibiotic treatment of the protozoan leads to an aposymbiotic strain with increased need for amino acids and results in increased production of extracellular peptidases. In this work, a more detailed examination of A. deanei was conducted to determine the effects of endosymbiont loss on the host calpain-like proteins (CALPs), followed by testing of different calpain inhibitors on parasite proliferation.ResultsWestern blotting showed the presence of different protein bands reactive to antibodies against calpain from Drosophila melanogaster (anti-Dm-calpain), lobster calpain (anti-CDPIIb) and cytoskeleton-associated calpain from Trypanosoma brucei (anti-CAP5.5), suggesting a possible modulation of CALPs influenced by the endosymbiont. In the cell-free culture supernatant of A. deanei wild type and aposymbiotic strains, a protein of 80 kDa cross-reacted with the anti-Dm-calpain antibody; however, no cross-reactivity was found with anti-CAP5.5 and anti-CDPIIb antibodies. A search in A. deanei genome for homologues of D. melanogaster calpain, T. brucei CAP5.5 and lobster CDPIIb calpain revealed the presence of hits with at least one calpain conserved domain and also with theoretical molecular mass consistent with the recognition by each antibody. No significant hit was observed in the endosymbiont genome, indicating that calpain molecules might be absent from the symbiont. Flow cytometry analysis of cells treated with the anti-calpain antibodies showed that a larger amount of reactive epitopes was located intracellularly. The reversible calpain inhibitor MDL28170 displayed a much higher efficacy in diminishing the growth of both strains compared to the non-competitive calpain inhibitor PD150606, while the irreversible calpain inhibitor V only marginally diminished the proliferation.ConclusionsAltogether, these results indicate that distinct calpain-like molecules are expressed by A. deanei, with a possible modulation in the expression influenced by the endosymbiont. In addition, treatment with MDL28170 affects the growth rate of both strains, as previously determined in the human pathogenic species Leishmania amazonensis and Trypanosoma cruzi, with whom A. deanei shares immunological and biochemical relationships.

Introdução: Apesar dos avanços tecnológicos, as doenças transmitidas por alimentos (DTA) continuam sendo um problema de saúde pública global. Nesse contexto, o manipulador de alimentos tem sido associado a muitos surtos, principalmente em função de falhas na higiene pessoal e na manipulação inadequada dos alimentos. Objetivo: Avaliar o conhecimento em Boas Práticas de Manipulação (BPM), a percepção de risco em DTA e a existência do fenômeno conhecido como viés otimista em manipuladores de alimentos de um hospital público. Método: Foi realizado um estudo de caso exploratório, utilizando um questionário estruturado dividido em três blocos de perguntas (1 - perfil sociodemográfico; 2 - avaliação do conhecimento de Boas Práticas de Manipulação; e 3 - percepção de risco em Doenças Transmitidas por Alimentos e viés otimista) com 50 respondentes. Os dados foram analisados por estatística descritiva, utilizando os testes Qui-quadrado, para identificar associações, e t-Student, para análise de similaridade entre variáveis. Resultados: O perfil da amostra foi de ≥ 41 anos (70%), sexo feminino (82%), ensino médio completo (46%) e experiência ≥ 6 anos na função (64%), com participação em treinamento há menos de três meses (84%). A média do grau de conhecimento em BPM foi satisfatória. Com relação à percepção de risco em DTA, os respondentes demonstraram alta percepção para identificar fatores de risco sanitário, não sendo observado viés otimista nas respostas. Conclusão: Apesar de os manipuladores apresentarem conhecimentos satisfatórios em BPM, algumas lacunas foram observadas, sendo importante ainda analisar se o conhecimento se transforma em atitudes e práticas adequadas e seguras.