The Functional Study Of ARE2 Gene And Antifungal Mechanisms Of Two Natural Products

In recent years,the infection caused by Candida species has threatened human health,especially in the immunocompromised population.Currently,azoles,polyenes,and echinocandins are widely used to treat fungal infection.Despite the use of antifungal therapies,the battle against candidiasis has remained largely unsuccessful because there are a limited number of antifungal drugs available and many cases of resistant candidiasis.Therefore,development of new antifungal agents and effective strategies to overcome fungal infection are highly necessary.The pathogenic yeast Candida albicans is part of the healthy human normal typical flora that can be isolated from the gastrointestinal tract,vagina,and oral cavity.Nevertheless,when the anatomical barrier is disrupted or when host immunity is compromised,C.albicans can behave as a pathogen,causing superficial infections or life-threatening systemic diseases.The ability of C.albicans to infect such diverse host niches is supported by a wide range of virulence factors.Thus,targeting virulence factors is a promising antifungal strategy.As previous reported,in the yeast S.cerevisiae,Arel and Are2 are explained as acyl-CoA:sterol acyltransferase(ASAT),and CaAre2p has acyl-CoA:sterol acyltransferase(ASAT)activity in fungi.Here,we report a novel role of ARE2 in C.albicans.In this work,are2 ?/? cells,not only showing growth defects in vitro,but also disturb the synthesis of sterol.Interesting,the level of total ergosterol is decreased in are2?/? mutant strain than the wide-type strain,and the content of squalene is significantly increased in mutant strain.Sterol is the main component of the cell membrane and closely related to the fluidity and permeability of the cell membrane.We used DPH staining to indicate the membrane dynamics,and we found inactivity of ARE2 can change the flexible of plasma membrane.The plasma membrane plays important roles in physiological processes and redox state.In this study,are2 mutant strain has a high state of ROS levels,and low levels of GSH.In addition,the mutant was hypersensitivity to temperature(45?),oxidative stresses(H2O2),cell wall stress agents(CFW,Congo red,SDS)and antifungal agents(AMB,CAS,FLC).The increased sensitivity to cell wall stress agents was suggested that cell wall integrity in are2 mutant was compromised.The cell wall of are2?/? null mutant was thicker than the wild type and the reconstituted strain as observed by TEM.We used ConA-FITC and anti-?-(l,3)-glucan antibody,to indicate mannan,and P-(l,3)-glucan,and concluded that deletion of ARE2 resulted in decreased the content of mannan and enhanced ?-glucan exposure,which could result in enhanced innate immune recognition of C.albicans by macrophages and neutrophils,and induced a stronger proinflammatory cytokine response,driven through the C-type lectin-like receptor,Dectin-1.There are strong evidence to point that lipids are crucial for the development of hyphae and development of biofilms in C.albicans.In this study,our data indicates that the absence of ARE2 gene causes a severe defect in hyphal development.Here,the ability of hyphal formation in are2 mutant strain might be affected by the accumulation of farnesol in are2 mutant strain,which could reduce the count of cAMP,and regulate the Rasl-cAMP-Efgl pathway.Besides,this study showed that are2 ?/? displays the inability of adhesion,cell invasion and biofilm formation,suggesting that Are2 function has a role in maintaining virulence in vitro.The higher ROS state,enhanced ?-glucan exposure,and defection of hyphal formation,may affect phagocytosed by macrophages.Furthermore,blocking ARE2 also altered the virulence of C.albicans in invasive infection in a murine model of systemic candidiasis.Mice infected with are2 mutant strain increased the survival compared with those infected with the wide-type,and less inflammatory influx and tissue necrosis of the kidneys.In this study,we identified for the first time that C.albicans strains lacking CaAre2 are avirulent,suggesting that Are2 function has a role in maintaining virulence.Therefore,there has been much interest in identifying the molecules targeting Are2 as potential drugs.Natural products have long been regarded as ample sources for discovery of novel drugs,with a broad spectrum of biological and pharmacological properties.From 1981 to 2014,nearly half of all new drugs approved by the FDA were either natural products or derived thereof.Bioactive compounds with antifungal activity have been identified;many of them are potent against C.albicans.Our lab isolated a series of phenolic compound derivative and p-terphenyl pigment derivative.Among them,Biatriosporin D(BD),a small phenolic compound isolated from an endolichenic fungus Biatriospora sp.,and Floricolin C(FC),a p-terphenyl pigment isolated from an endolichenic fungus Floricola striata,showed antifungal activity against Candida species.In this thesis,we investigated the antifungal mechanisms of these two compounds.Hyphae are more invasive and better at tissue penetration than the yeast form.In addition,biofilms formation aggravated the drug-resistance and repeated infections.Our previous research found this compound could render clinically derived FLC-resistant strains sensitized to FLC.This study,we found that BD displayed anti-virulence activity by inhibiting adhesion,hyphal morphogenesis and biofilm formation of C.albicans.Of note is the high efficacy of BD in preventing filamentation with a much lower dose than its MIC value.Furthermore,BD prolonged the survival of worms infected by C.albicans in vivo.To uncover the underlying molecular basis of inhibited hyphal formaiton by BD,a series of experiments was applied.QPCR,exogenous cAMP rescue experiments and intracellular cAMP measurements revealed that BD regulates the Rasl-cAMP-Efgl pathway by reducing cAMP levels to inhibit the hyphal formation.Further investigation showed that BD could upregulate Dpp3 to synthesize much more farnesol,which could inhibit the activity of Cdc35 and reduce the generation of cAMP and thereby disrupting the morphologic transition and attenuating the virulence of C.albicans.Our study uncovers the underlying mechanism of BD as a prodrug in fighting against pathogenic C.albicans and provides a potential application of BD in fighting clinically relevant fungal infections by targeting fungal virulence.The rise of fungal infection and drug-resistance demands the development of novel antifungal agents.In previous study in our lab,FC exhibited potent antifungal activity against C.albicans via disruption of the cytoplasmic membrane.In this study,we observed that FC killed C.albicans cells at planktonic state or within biofilms through reactive oxygen species(ROS)accumulation.Further test revealed that FC could directly damage the mitochondria to cause ROS accumulation.In addition,FC can quench thiol-based agents through a Michael reaction involving the a,p-unsaturated carbonyl group,whose effect may chelate intracellular thiol-based molecules or proteins in C.albicans,resulting in imbalance of redox homeostasis.Increased ROS generation led to mitochondrial dysfunction,nuclear dispersion and consequently cell death.In this study,in the presence of FC,mitochondrial membrane potential and ATP count were significantly changed,and the release of Cyt c from mitochondria was increased,suggesting the dysfunction of mitochondrial.Besides,the low dose of FC induced more apoptotic cells than necrotic ones,suggesting that FC causes both apoptosis and necrosis of C.albicans cells.We further demonstrated that FC could prevent biofilm formation of other Candida species and eradicate their pre-formed biofilms.In vivo study demonstrated that FC prolonged the survival of C.albicans-infected Caenorhabditis elegans.In this thesis,we identified for the first time that C.albicans strains lacking CaAre2 are avirulent,and investigation of the function of ARE2 gene in the pathogenesis of Candida albicans,which increase our understanding of its pathogenic mechanisms,and provide a new method to overcome fungal infection.Besides,the investigation of mechanism of BD and FC compounds provides a basis for the application of these compounds to combat Candida infections.