Decyl Gallate as a Possible Inhibitor of N-Glycosylation Process in Paracoccidioides lutzii.

The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and broad-spectrum antifungal activity. Previously, our group demonstrated the promising anti-Paracoccidioides activity of G14. In this work, to evaluate the antifungal characteristics of G14 for Paracoccidioides lutzii, a chemical-genetic interaction analysis was conducted on a Saccharomyces cerevisiae model. N-glycosylation and/or the unfolded protein response pathway was identified as a high-confidence process for drug target prediction. The overactivation of unfolded protein response (UPR) signaling was confirmed using this model with IRE1/ATF6/PERK genes tagged with green fluorescent protein (GFP). In P. lutzii, this prediction was confirmed by the low activity of glycosylated enzymes [α-(1,3)-glucanase, N-acetyl-ß-d-glucosaminidase (NAGase), and α-(1,4)-amylase], by hyperexpression of genes involved with the UPR and glycosylated enzymes, and by the reduction in the amounts of glycosylated proteins and chitin. All of these components are involved in fungal cell wall integrity and are dependent on the N-glycosylation process. This loss of integrity was confirmed by the reduction in mitochondrial activity, impaired budding, enhancement of wall permeability, and a decrease in viability. These events led to a reduction of the ability of fungi to adhere on human lung epithelial cells (A549) in vitro Therefore, G14 may have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering with the microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti-Paracoccidioides compound.

Understanding the mechanism of action of peptide (p-BthTX-I)2 derived from C-terminal region of phospholipase A2 (PLA2)-like bothropstoxin-I on Gram-positive and Gram-negative bacteria.

Based on the antimicrobial activity of bothropstoxin-I (BthTX-I) and on the premise that a C-terminal peptide of Lys49 myotoxin can reproduce the antimicrobial activity of the parent protein, we aimed to study the mechanism of action of a peptide derived from the C-terminal region of the myotoxin BthTX-I [(p-BthTX-I)2, sequence: KKYRYHLKPFCKK, disulfide-linked dimer] against Gram-positive and Gram-negative bacteria. Fluorescence quenching technique showed that the carboxyfluorescein labeled-peptide [CF-(p-BthTX-I)2] when incubated with E. coli displayed a superior penetration activity than when incubated with S. aureus. Cell death induced by the peptide (p-BthTX-I)2 showed a loss of membrane integrity in E. coli and S. aureus; however, the mechanisms of cell death were different, characterized by the presence of necrosis-like and apoptosis-like deaths, respectively. Scanning electron microscopy studies in E. coli and S. aureus showed morphological changes in the cells, with superficial deformities, appearance of wrinkles and bubbles, and formation of vesicles. Our results demonstrate that the mechanism of action of the peptide (p-BthTX-I)2 is different in Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Knowledge of the mechanism of action of these peptides is important, since they are promising prototypes for new antimicrobial drugs.