The Role Of SDH2 In The Virulence Of Candida Albicans

Candida albicans is a common human pathogenic fungi.A morphogenesis between yeast and hyphal cells happens when C.albicansinvade host organization,so the morphogenesis is the recent focus of study on pathogenic mechanism of C.albicans.Our primary study used Caenorhabditis elegans(C.elegans)model to screen C.albicans cell wall protein mutant strains library trying to find key gene for virulence,and discovered SDH2 mutant lost its virulence completely on the C.elegans model with an obvious defect in filament formation.SDH2 gene encoding protein plays roles inboth tricarboxylic acid(TCA)cycle and mitochondrial electron transport chain(ETC).It encodes the iron-sulfur subunit of succinic dehydrogenase(SDH),which is also the complex II in mitochondrial electron transport chain(ETC),and plays an important role in the process of aerobic respiration.We rebuilt the SDH2 mutant sdh2?/? by SAT1 flipping method,which use nourseothricin resistance marker to screen positive clones,the marker gene can be completely removed in the follow-up experiment,avoiding the interference of nutritional deficiencies sign geneon the pathogenicity.SDH2 mutant exhibited a defect of pathogenicity and filamentation on C.elegans model and mice model.Moreover,SDH2 mutant had filamentation defect on a variety of hyphae inducing mediums,and failed to grow on nonfermentative carbon sources such as glycerol,ethanol,citric acid.One thing to be noted is that in thehyphae inducingmedium containing fermentable carbon sources glucose,sdh2?/? still cannot form solid hyphae.We further explored the mechanisms of how SDH2 affects filamentation.First we explored the relationship between filamentation defect of sdh2?/? and ethanol,since the growth of sdh2?/? largely depended on fermental carbon source.We speculated the deletion of SDH2 impaires the process of aerobic respiration of C.albicans,resulting in a metabolism shift from aerobic respiration to glycolysis.Ethanol produced during glycolysis probably cumulated in the cellswhen growing on the solid hyphae inducing medium.By detection we found sdh2?/? did have an accumulation of intracellular ethanol content.We further studied the effects of ethanol on C.albicansfilamentation by three methods:(1)add ethanol to solid medium for solid hyphae experiment;(2)investigate the effect of intracellular ethanol on solid hyphae formation of C.albicans by using TCA cycle inhibitor malonate(succinic acid analogues,competitively inhibit succinate dehydrogenase to block the TCA cycle)to inhibit aerobic respiration;(3)explored the ADH1(coding ethanol dehydrogenation enzyme,catalytic reduction of acetaldehyde to ethanol and the reverse reaction)mutant adh1?/? to further verify the contacts between ethanol and filamentation.We found thatethanol inhibited filamentation in Spider+glucose medium,but 2 ~ 4% ethanol in YPD+ serumstimulated the filamentation of SC5314 and sdh2?/?.Malonate did inhibit aerobic respiration and caused intracellular ethanol accumulation,but the corresponding solid hyphae formation of C.albicans had no defects.In addition,we found that intracellular ethanol content of adh1?/? was significantly lower than that wild type,but solid hyphae-forming ability was not stronger than the wild-type and the sensitivity to ethanol was not lower than the wild-type.In General,the intracellular ethanol content has nopositive correlation with filamentation ability of C.albicans.As SDH2 worked in both TCA cycle and ETC,we further investigate the effect of both processes on filamentation of C.albicans.Malonate and carboxin are the special inhibitor of SDH,respectively inhibiting the function of succinate dehydrogenase in TCA cycle and function of passing electronics to ubiquinone in ETC.The wild-type C.albicans treated with 1.5 mM carboxin showed significantly reducing virulence and exihibited the same phenomenon asSDH2 deletion strain.In contrast,the same concentration of malonateand FUM12 deletion mutantcouldn't affect thevirulence and filamentation ofC.albicans.Above results suggested that mitochondrial ETCdisfunction was the main reasonofC.albicansfilamentation defection.We further investigated the mechanism ofhow ETC function affecting filamentation ofC.albicans.We found sdh2?/? had normal ATP production and mitochondrial potential,but an increasing ROS(reactive oxygen species)production compared to wild type strain.Additon of reducing substances(Vitamins E,Proanthocyanidins)to reduce ROS production could recover the solid hyphae forming ability of sdh2?/?.Moreover,ROS inducing substance could indeed inhibit C.albicas hyphae formation.In addition,lower oxygen content in the environment(hypoxia,20% CO2),to reduceintracellular ROS production,could also restorefilamentation of sdh2?/? in solid mediium.Above resluts implied that the accumulation of ROS cause by deletion of SDH2 in C.albicans is the main reason of filamentation defect.Collectively,SDH2 deletion affected ETC function,resulting in an increasingintercellular ROS,which defectedC.albicans in hyphae formation and pathogenicity.This work indicates the possibility of developing mitochondrial electrontransport chain related genes tothe new anti-fungal target.