Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape.

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

Evaluation of antimalarial activity and toxicity of a new primaquine prodrug.

Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission.