Ethanolic Extract of the Fungus Trichoderma asperelloides Induces Ultrastructural Effects and Death on Leishmania amazonensis.

The Trichoderma genus comprises several species of fungi whose diversity of secondary metabolites represents a source of potential molecules with medical application. Because of increased pathogen resistance and demand for lower production costs, the search for new pharmacologically active molecules effective against pathogens has become more intense. This is particularly evident in the case of American cutaneous leishmaniasis due to the high toxicity of current treatments, parenteral administration, and increasing rate of refractory cases. We have previously shown that a fungus from genus Trichoderma can be used for treating cerebral malaria in mouse models and inhibit biofilm formation. Here, we evaluated the effect of the ethanolic extract of Trichoderma asperelloides (Ext-Ta) and its fractions on promastigotes and amastigotes of Leishmania amazonensis, a major causative agent of cutaneous leishmaniasis in the New World. Ext-Ta displayed leishmanicidal action on L. amazonensis parasites, and its pharmacological activity was associated with the low-molecular-weight fraction (LMWF) of Ext-Ta. Ultrastructural analysis demonstrated morphological alterations in the mitochondria and the flagellar pocket of promastigotes, with increased lipid body and acidocalcisome formation, microtubule disorganization of the cytoplasm, and intense vacuolization of the cytoplasm when amastigotes were present. We suggest the antiparasitic activity of Trichoderma fungi as a promising tool for developing chemotherapeutic leishmanicidal agents.

Toxoplasma gondii induces extracellular traps release in cat neutrophils.

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite.

Leishmania amazonensis hijacks host cell lysosomes involved in plasma membrane repair to induce invasion in fibroblasts.

Intracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector, which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting, but overlooked finding, is that other cell types and even non-phagocytic cells have been found to be infected by Leishmania spp. Nevertheless, the mechanisms by which Leishmania invades such cells had not been previously studied. Here, we show that L. amazonensis can induce their own entry into fibroblasts independently of actin cytoskeleton activity, and, thus, through a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions, such as Ca2+ signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair.This article has an associated First Person interview with the first author of the paper.

Effects of a novel ß-lapachone derivative on Trypanosoma cruzi: Parasite death involving apoptosis, autophagy and necrosis.

Natural products comprise valuable sources for new antiparasitic drugs. Here we tested the effects of a novel ß-lapachone derivative on Trypanosoma cruzi parasite survival and proliferation and used microscopy and cytometry techniques to approach the mechanism(s) underlying parasite death. The selectivity index determination indicate that the compound trypanocidal activity was over ten-fold more cytotoxic to epimastigotes than to macrophages or splenocytes. Scanning electron microscopy analysis revealed that the R72 ß-lapachone derivative affected the T. cruzi morphology and surface topography. General plasma membrane waving and blebbing particularly on the cytostome region were observed in the R72-treated parasites. Transmission electron microscopy observations confirmed the surface damage at the cytostome opening vicinity. We also observed ultrastructural evidence of the autophagic mechanism termed macroautophagy. Some of the autophagosomes involved large portions of the parasite cytoplasm and their fusion/confluence may lead to necrotic parasite death. The remarkably enhanced frequency of autophagy triggering was confirmed by quantitating monodansylcadaverine labeling. Some cells displayed evidence of chromatin pycnosis and nuclear fragmentation were detected. This latter phenomenon was also indicated by DAPI staining of R72-treated cells. The apoptotis induction was suggested to take place in circa one-third of the parasites assessed by annexin V labeling measured by flow cytometry. TUNEL staining corroborated the apoptosis induction. Propidium iodide labeling indicate that at least 10% of the R72-treated parasites suffered necrosis within 24 h. The present data indicate that the ß-lapachone derivative R72 selectively triggers T. cruzi cell death, involving both apoptosis and autophagy-induced necrosis.