Project description:A systematic approach allowing the identification of the molecular way-of-action of novel potential drugs represents the golden-tool for drug-discovery. While high-throughput screening technologies of large libraries is now well established, the assessment of the drug targets and mechanism of action is still under development. Taking advantage of the yeast model Saccharomyces cerevisiae, we herein applied BarSeq, a Next Generation Sequencing-based method to the analysis of both haploinsufficiency and homozygous fitness effects of a novel antifungal drug ('089') compared to the well-known antifungal ketoconazole. '089' was a novel compound identified in during a screen for antifungal drugs, as it was showing fungicidal effects, and able to affect the yeast fitness at the mitochondrial level (Stefanini et al., 2010. (Dissection of the Effects of Small Bicyclic Peptidomimetics on a Panel of Saccharomyces cerevisiae Mutants;.J Biol Chem, 285: 23477-23485.) Integrative bioinformatic analysis of BarSeq, whole genome expression analysis and classical biological assays identified the target and cell pathways affected by the novel antifungal. Confirmation of the effects observed in the yeast model and in pathogenic fungi further demonstrated the reliability of the multi-sided approach and the novelty of the targets and way-of-action of the new class of molecules studied representing a valuable source of novel antifungals.
Project description:In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach and the biological activity of crude venoms against three Candida species was established.
Project description:Collimonas is a genus of soil bacteria which comprises three recognized species: C. fungivorans, C. pratensis and C. arenae. The bacteria belonging to this genus share the ability to lyse chitin (chitinolysis) and feed on living fungal hyphae (mycophagy), but they differ in colony morphology, physiological properties and antifungal activity. In order to gain a better insight into the genetic background underlying this phenotypic variability of collimonads, we investigated the variability in the genomic content of five strains representing the three formally recognized Collimonas species. The genomic content of four test strains was hybridized on an array representing the reference strain C. fungivorans Ter331.
Project description:In the light of the increasing occurrence of antifungal resistance, there is an urgent need to search for new therapeutic strategies to overcome this phenomenon. One of the applied approaches is the synthesis of small-molecule compounds showing antifungal properties. Here we present a continuation of the research on the recently discovered anti-Candida albicans agent 4-AN. Using next generation sequencing and transcriptional analysis, we revealed that the treatment of C. albicans with 4-AN can change the expression profile of a large number of genes. The highest up-regulation was observed in the case of genes involved in cell stress, while the highest down-regulation was shown for genes coding sugar transporters. Real-time PCR analysis revealed 4-AN mediated reduction of the relative expression of genes engaged in fungal virulence (ALS1, ALS3, BCR1, CPH1, ECE1, EFG1, HWP1, HYR1, and SAP1). The determination of the fractional inhibitory concentration index (FICI) showed that the combination of 4-AN with Amphotericin B is synergistic. Finally, flow cytometry analysis revealed that the compound induces mainly necrosis in Candida albicans cells.
Project description:Cryptococcus neoformans is a human fungal pathogen responsible for fatal infections, especially in patients with a depressed immune system. Overexposure to antifungal drugs due to prolonged treatment regimens and structure-similar applications in agriculture have weakened the efficacy of current antifungals in the clinic. The rapid evolution of antifungal resistance urges the discovery of new compounds that inhibit fungal virulence factors, rather than directly killing the pathogen as alternative strategies to overcome disease and reduce selective pressure towards resistance. Recent studies, including our own, have highlighted the antimicrobial properties of natural sources, such as invertebrates, against human fungal pathogens, including C. neoformans, through virulence factor impairment. Here, we evaluated the efficacy of freshwater mussel extracts (crude and clarified) against virulence factor production (i.e., thermotolerance, melanin, capsule, and biofilm) in C. neoformans. Similarly, we demonstrated the critical potential of these extracts to increase the susceptibility of the pathogen to fluconazole across resistant strains, overcoming a globally devastating problem. Additionally, we measured the inhibitory activity of the extracts against peptidases related to fungal virulence and drug resistance. Furthermore, we integrated these phenotypic findings with quantitative proteomics profiling to define distinct signatures of each treatment and validated a new mechanism of anti-virulence action for a selected extract. By understanding the mechanisms driving the antifungal activity of mussels, we may develop innovative treatments for fungal infections lacking susceptibility to conventional drugs.
Project description:Candida albicans were treated with a sublethal concentration of the antifungal Jagaricin for either a short time (30 min) or until an OD of 0.5 (indicating log growth) was reached. Controls were grown without any antifungal to determine cellular reactions to the compound.