Secreted Proteins On Pathogenesis Of Cryptococcus Neoformans And Antifungal Effects Of New Drugs

Objects Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, studies in Part I were designed to investigate those secreted proteins that mediate biofilm formation by C. neoformans. In Part II, given that expression of CnMPR1 in Saccharomyces cerevisiae was solely responsible for promoting the attachment and migration of Sc across the BBB, we have harnessed this gain of function to identify surface proteins of the BBB that specifically promote a permeable BBB and thus facilitate the transmigration of fungal cells. Recently, a class of new core-shell nanostructures, Pd@Ag nanosheets(Pd@Ag NSs), were created using deposition techniques and demonstrated excellent inhibitory effects on various bacteria in vitro. In this study(Part III), we expored the antifungal activity of Pd@Ag NSs against common invasive fungal pathogens.Methods In Part I we generated deletion mutants of these biofilm associated genes to test biofilm formation and their virolence in vivo and in vitro. In Part II surface exposed proteins from hCMEC/D3 cells(human brain endothelial cell line) was labeled with NHS-biotin and then isolated via magnetic beads attached to a biotin antibody. The labled BBB proteins were incubated with the Sc-expressing Mpr1 strain(or wild type cells(no Mpr1) to serve as a control). Proteins were eluted and identified by MALDI-TOF MS/MS. Part III Pd@Ag NSs with different sizes were synthesized and tested for antifungal activity against a panel of invasive fungal pathogens.Results Part I Compared to other two mutants, a vid?(Vacuolar import and degradation protein) mutant exhibited a significant reduction of biofilm formation and attenuated virulence. Part II In the Sc-expressing Mpr1 strain group, 79 proteins were identified by MALDI-TOF MS/MS. Part III Among tested organisms, Cryptococcus neoformans complex species was most susceptible to Pd@Ag NSs, which exhibited potent antifungal activity against various molecular types or sources of cryptococcal strains including fluconazole-resistant isolates. The anticryptococcal activity of Pd@Ag NSs was significantly greater than fluconazole and similar to that of amphotericin B(AmB). At relatively high concentrations, Pd@Ag NSs exhibited fungicidal activity against Cryptococcus spp., which can likely be attributed to the disruption of cell integrity, intracellular protein synthesis, and energy metabolism. Intriguingly, Pd@Ag NSs also exhibited strong synergistic cryptococcacidal effects at low concentrations in combination with AmB but exhibited much better safety in erythrocytes than AmB, even at the minimal fungicidal concentration.Conclusion Part I Vid is essential for the biofilm formation and virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host. Part II Those proteins identified suggesting that Mpr1 up-regulates a complex web of factors that mediate cytoskeleton-plasa membrane reorganization and endosomal trafficking. Part III Pd@Ag NSs may be a promising adjunctive agent for treating cryptococcosis, and further investigation for clinical applications is required.