Study On The Drug Resistance Of Clinical Candida Albicans And The Discovery Of Selective Inhibitors Of Azole-resistant Strains

In recent years,due to the extensive use of broad-spectrum antimicrobial agents and the operation of organ transplant,antineoplastic chemotherapy and hematodialysis,fungal infection has become one of the most common clinical infection,and Candida albicans is a major pathogenic fungus.Azoles are widely used in clinic to treat life-threatening invasive fungal infections due to the high efficacy,excellent bioavailability,low toxicity and easy access.However,drug resistance appeared as azole antifungals broadly and chronically used.Therefore,understanding of the molecular mechanisms of azole resistance in C.albicans will benefit the development of new drugs and guidelines for the clinical treatment.Through the determination of minimum inhibitory concentration(MIC),we have screened 61 clinical C.albicans obtained from two hospitals in Jinan,and 22 isolates were found to be resistant to more than 2 kinds of azole antifungals.Of these,18 strains exhibited azole resistance at 30? but became susceptible to azole drugs when the temperature was raised to 35? or even higher.The remaining four strains(28A,28D,281,and 28J)possessed a stable high-level resistance to five types of azole drugs at either temperature.The underlying mechanisms of azole resistance were explored by flow cytometry,real-time qPCR,gene sequencing,and heterologous expression in yeast.Among these azole-resistant strains,eight isolates showed elevated efflux pumps activity,which was consistent with the high-level expressions of the efflux pump-encoding genes CDR1,CDR2 or MDR1.Elevated expression of ERG11 was also observed in seven resistant isolates,and some mutations in its regulator UPC2 were further found.Several new mutations were identified in the ERG3,ERG11,and zinc-finger transcription factor genes(CAP1 and TAC1);the mutations were predominantly harbored by the stably azole-resistant isolates.A combined mutations inERG3 resulting in the S265Y,N324S,and N322D substitutions in Erg3 at least partially increases fluconazole resistance in C.albicans.In addition,we found that azole resistance of all these strains relied on the existence of Hsp90,and could be reversed by the use of Hsp90 or calcineurin inhibitors.Our study deepens the understanding of the local determinants of azole resistance,and may lead to further insights that will aid the development of novel targeted treatments for C.albicans infections.In C.albicans infections,azole-susceptible isolates often co-colonize with azole-resistant ones in the host or a subpopulation of susceptible isolates evolved into resistant ones under the stress of antifungal agents.The formed resistant organisms dominate the whole population as the prolonged use of antifungal agents.Natural products,especially from the medicinal plant,are still major sources of antifungal drugs.In our previously studies,five natural molecules including riccardin D(RD),retigeric acid B(RAB),pyridoxatin(PYR),diorcinol D(DD),biatriosporin D(BD)were reported to show potential antifungal activity.To simulate clinical C.albicans infection status,we developed a co-culture screen assay with GPF-labeled susceptible isolate and resistant isolates with different resistance mechanisms,and reevaluate those five natural products to fing compounds that could selectively inhibit the azole-resistant strains.All the tested compounds showed antifungal activity against both susceptible and resistant strains.BD exhibited selective inhibitory activity against both YEM13 and YEM15,but showed no inhibitory difference between other strains.While PYR and DD tend to inhibit the susceptible strain.RAB and RD displayed selective inhibitory activity against MDR1-activated strains resulting from either Mdrl promoter activation or gain-of-function of MRR1 mutations.MDR1 upregulation is one of the most common mechanisms of clinical azole resistance.Thus,uncovering the mechanisms of selectively antifungal mechanism of RAB and RD would benefit the treatment of C.albicans infections.We utilized HPLC to measure the intracellular contents of RAB and RD in susceptible strain and three MDP1-activated azole-resistant strains.The intracellular contents of tested agents in azole-resistant strains were greater than those in susceptible strain.We then analyzed the membrane sterols and lipids composition by using GC-MS.All the MDR1-activated azole-resistant strains have a certain distinction in the sterol and lipid contents.The investigations revealed that the alteration of cell membrane components,which lead to cell membrane fluidity and permeability change,might confer the differential permeability of RAB and RD into MDR1-activated strains.The expressions of ERG genes were down-regulated in MDR1-activated strains,indicating that the upregulation of MDR1 may result in impaired ergosterol biosynthesis.RAB and RD were reported that they could modulate the ergosterol pathway and reduce ergosterol synthesis to retard C.albicans growth.Therefore,RAB or RD treatment aggregated the disorder of ergosterol biosynthesis in MDR1-activated strains,resulting in a decrease of ergosterol and a slight increase of sterol intermediates.In this thesis,we found 22 azole-resistant isolates of 61 clinical Candida albicans strains,including four stably-resistant strains.Through the analysis,several resistance mechanisms were identified,including some.new mutations,which enrich the resistance mechanisms of C.albicans,and deepen the understanding of clinical azole resistance.Furthermore,we developed a co-culture assay to reevaluate five natural products,and clarified the mechanism that RAB and RD can selectively inhibit MDR1-activated strains.Overall,our findings pave the way for developing alternative strategies against resistant pathogenic fungi.