A Peptide From GAPDH Serves As A Promising Antifungal Agent Against Cryptococcus Neoformans

The Cryptococcus is a major pathogenic fungus that can cause infections in immunocompromised patients.When a Cryptococcus cell enters the human body,it can infect multiple organs and even cause death.Central nervous system infections are the most serious and account for approximately 80%of all infections.Currently,the limited availability of drugs against cryptococcal infections,the fact that the Cryptococcus has developed resistance to most existing drugs and the toxicity of some drugs(such as amphotericin B)to animal cells have seriously hampered the effectiveness of existing drugs.Since the rate of cryptococcal infection is increasing yearly and approximately 600,000 people die of cryptococcal infection every year,it is urgent to develop new antimicrobial drugs.A cationic antimicrobial peptide SP1 derived from the N-terminus of glyceraldehyde-3-phosphate dehydrogenase of S.cerevisiae was found in our laboratory;SP1 has an α-helix structure.SP1 had a strong antifungal effect against C.neoformans JEC21 and H99,and the minimum inhibitory concentrations(MICs)were 4 μm and 8 μm,respectively.Our previous work suggested that SP1 can induce leakage of liposomes,which contain ergosterols,at the minimum inhibitory concentration and has no hemolytic toxicity to HeLa cells or erythrocytes of Gallus domesticus.Further research showed that SP1 can kill clinical strains of C.neoformans and C.gattii,with a MIC of 4-32 μM.SP1 does not cause loss of selectivity of C.neoformans H99 protoplasts,indicating that SP1 is different from typical α-helical cationic antimicrobial peptides and has a unique antifungal mechanism.Four drug-resistant mutants(skn7Δ cep3Δ,fzc1Δ,and fzc38Δ)were obtained by screening a transcription factor knockout library(155 strains)of C.neoformans H99 with SP1.Among these mutants,skn7Δ exhibited effects mainly related to oxidative stress,cell response to stress and cell signal transduction,a finding worthy of further study.RNA-seq analysis comparing the transcriptome differences between the skn7Δand H99 strains revealed that the enhancement of the cell wall synthesis pathway and mitochondria-independent energy metabolism pathway led to SP1 tolerance.C.neoformans is an aerobic fungus.The enhanced mitochondria-independent energy metabolism pathway of the SP1-resistant mutant skn7Δ suggests that SP1 may cause abnormalities in mitochondria and drive cryptococcal cells to rely on anaerobic metabolism for energy.The skn7Δ strain was resistant to fluconazole,which indicated that the ergosterol synthesis pathway has a clear influence on the antifungal efficacy of SP1.A series of filipin Ⅲ staining experiments showed that SP1 targeted ergosterol in the cell membrane.SP1 can target ergosterol in cell membranes and bind to it,and SP1 can then enter cells through endocytosis and accumulate in vacuoles.Further RNA-seq analysis showed that SP1 can enhance the mitochondrial electron transport chain,reduce the antioxidant capacity of mitochondria,and lead to the accumulation of ROS.In addition,the enrichment of SP1 in vacuoles can convert chelated calcium ions to the free state and result in their influx into the cytoplasm,from which they can be transferred to mitochondria and cause mitochondrial membrane potential loss.These phenomena eventually lead to mitochondrial abnormalities and the release of EndoG,which triggers non-caspase-dependent apoptosis.In addition,caspase-1-mediated apoptosis was found to be directly induced by calcium accumulation in the cytoplasm.Finally,we can conclude that SP1 can kill C.neoformans by inducing apoptosis.RNA-seq analysis showed that SP1 can also affect the transcriptome of C.neoformans.In addition to oxidative stress-related genes,SP1 can also reduce the expression of other resistance-related genes,including laccase,indicating that SP1 has the potential to be used in combination with other antifungal drugs.SP1 can kill C.neoformans synergistically with caspofungin or amphotericin B,and SP1 can be combined with other antifungal agents to reduce the needed concentrations of these drugs.The results of this study show that SP1 has the potential to be used in combination with other drugs and has the potential to be used to develop new drugs for treating cryptococcal infections.