Enhanced Antifungal Effects By Nicotinamide And Its Molecular Mechanism

Invasive candidiasis remains a significant burden due to the high morbidity and mortality,in which Candida albicans(C.albicans)is the leading pathogen of candidemia worldwide.Available antifungal agents used in clinical practice are limited,which also have some unwanted effects.Besides,the frequent evolvement of resistance in drug-sensitive fungal pathogens and clinical treatment failures are of concern.Thus,it calls for alternative strategies to improve the therapeutic effects and relieve the significant side effects of current employed antifungal drugs,in which synergistic drug combination has been proved to be pragmatic and effectiveNicotinamide(NAM)is the amide form of vitamin B3,known for many years to cure pellagra.Recent phase III clinical trials proved that NAM is useful in the amelioration of frataxin deficiency in Friedreich's ataxia and the prevention of nonmelanoma skin cancer.Besides,NAM was also reported to execute antifungal activity against various kinds of fungi in vitro.Thus,in order to elleviate the clinical burden,we determined the effect of NAM on the commonly employed agents against C.albicans in the present study.With drug susceptibility test we found that C.albicans grown in the presence of NAM became more susceptible to shikonin(SK),fluconazole(FCZ),Amphotericin B(AmB)and caspofungin(CaS),NAM could also inhibit the trailing growth of C.albicans when treated with azoles.With checkerboard microdilution assay,NAM was found to enhance the antifungal activities of antifungals against clinical-resistant C.albicans isolats,CaS-resistant C.albicans isolats,Candida tropicalis as well as Cryptococcus neoformans.Besides,NAM could also enhance the effects of antifungals against C.albicans hypae.The murine invasive Candidiasis model was employed to evaluate the enhanced effect of NAM on FCZ,AmB or CaS in vivo.The result showed that the combined used of NAM and antifungals could greatly prolong the survival rate of mice.And the tissue damage and hyphae invasion were remarkably elleviate when treated murine with the combination of NAM and FCZ or CaS.The enhanced effects of CaS by NAM exhibited a dose-dependent manner.The result of Western-Blot demonstrated that SK is a compound reported to execute its antifungal activity via directly boosting the excessive acetylation of histone H3 on lysine 56(H3K56)level mediated by the inhibition of histone deacetylation process.However,FCZ,AmB and CaS could not induce the excessive level of histone H3 on lysine 56,9 or 18.With Flow Cytometer we confirmed that NAM could induce certain apoptosis in C.albicans as well as remarkably changed the cell cycle,leading to the arrest of G1 or S period,which might help enhance the antifungal activities of commonly employed antifungal agents.We also determined the cell wall disturbance by laser scanning confocal microscope,transmission electron microscopy,flocculation test and cell surface hydrophobicity test,in which lower mannose content,increased inner chitin layer,the exposure of ?-(1,3)-glucan,loose cell wall,shrinking cytomembrane,the indistinct edge of endochylema organelles,enhanced flocculation and cell surface hydrophobicity in C.albicans were detected.The disturbance of cell wall was due to the function of HST3 and GIN4.Furthermore,various metabolism pathways were found to be changed by NAM by employing metabonomics.Specially,the levels of putrescine and spermine were dramatically increased,and the addition of polyamines or polyamine biosynthesis inhibitors could remarkably suppressed the antifungal activity of NAM against C.albicans,but neither arginine nor ornithine influenced the activity of NAM,which suggests the essential role of Ornithine decarboxylase or its related gene on the enhanced antifungal effects of antifungal agents by NAM.To sum up,NAM serves as a potent synergist of antimycotic drugs in vitro and in vivo,which provides a novel therapeutic strategy curing invasive candidiasis and reverse drug resistance of C.albicans.It also opens a path to develop antifungal agents or synergist with potent activity and low side effects.