Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.

Plasmodium chabaudi Merozoites Obtained through a Simpler Method Do Not Survive in Classically Activated Macrophages.

Malaria is caused by apicomplexan parasites of the Plasmodium genus. Plasmodium chabaudi is an excellent animal model for the study of human malaria caused by P. falciparum. Merozoites invade erythrocytes but are also found in other host cells including macrophages from the spleen and liver. Methodologies for obtaining merozoites usually involve treatment with protease inhibitors. However, merozoites obtained in this way may have their enzymatic profile altered and, therefore, are not ideal for cell-interaction assays. We report the obtainment of P. chabaudi merozoites naturally egressed from a synchronous erythrocyte population infected with schizonts forms. Merozoites had their infectivity and ultrastructure analyzed. Interaction assays were performed with mice erythrocytes and classically activated mice peritoneal macrophages, a very well-established classic model. Obtained merozoites were able to kill mice and efficiently infect erythrocytes. Interestingly, a lower merozoite:erythrocyte ratio resulted in a higher percentage of infected erythrocytes. We describe a simpler method for obtaining viable and infective merozoites. Classically activated macrophages killed merozoites, suggesting that these host cells may not serve as reservoirs for these parasites. These findings have implications for our understanding of P. chabaudi merozoite biology and may improve the comprehension of their host-parasite relationship.

Quantitative Risk Assessment of Oocyst Versus Bradyzoite Foodborne Transmission of Toxoplasma gondii in Brazil.

Toxoplasma gondii is a globally distributed zoonotic protozoan parasite. Infection with T. gondii can cause congenital toxoplasmosis in developing fetuses and acute outbreaks in the general population, and the disease burden is especially high in South America. Prior studies found that the environmental stage of T. gondii, oocysts, is an important source of infection in Brazil; however, no studies have quantified this risk relative to other parasite stages. We developed a Bayesian quantitative risk assessment (QRA) to estimate the relative attribution of the two primary parasite stages (bradyzoite and oocyst) that can be transmitted in foods to people in Brazil. Oocyst contamination in fruits and greens contributed significantly more to overall estimated T. gondii infections than bradyzoite-contaminated foods (beef, pork, poultry). In sensitivity analysis, treatment, i.e., cooking temperature for meat and washing efficiency for produce, most strongly affected the estimated toxoplasmosis incidence rate. Due to the lack of regional food contamination prevalence data and the high level of uncertainty in many model parameters, this analysis provides an initial estimate of the relative importance of food products. Important knowledge gaps for oocyst-borne infections were identified and can drive future studies to improve risk assessments and effective policy actions to reduce human toxoplasmosis in Brazil.

Bacteroides Fragilis induces conformational alterations in Trichomonas Vaginalis ultrastructure in an in vitro interaction

Bacteroides genus are commonly found on mucous membranes, including the female genital tract, acting as agents for several site infections. Anaerobic infections are usually polymicrobial and endogenous. Trichomonas vaginalis, the trichomoniasis etiologic agent, is a facultative anaerobic flagellated parasite spread worldwide. The purpose of this study was to explore the association between vaginal bacteria and T. vaginalis, as well as to understand factors that may favour the infection of T. vaginalis. We have, therefore, used T. vaginalis trophozoites and the species Bacteroides fragilis, which is considered the most important in its genus, once it is the most commonly isolated bacteria from endogenous infections. The parasite-bacteria interaction was performed in different proportions in periods varying from 1 to 12 hours applying viability tests. The data were analyzed to compare the parasite viability in vitro in the presence and absence of B. fragilis. The results indicate that in the 1:100 proportion postinteraction analysis, ultrastructural alterations were noticeable after 6 hours. After 8 hours, T. vaginalis viability decreased, and after 12 hours of interaction no viable trophozoites were found. These data suggest that the parasite can deal with B. fragilis in short interaction periods. However, in longer interaction periods the trophozoites collapse, indicating that B. fragilis may produce toxic metabolites against T. vaginalis activity.