Mechanism of azole resistance in Candida glabrata in the presence of immunosuppressant mycophenolic acid

Tome, Miha (author), Raspor, Peter (mentor), Kuchkler, Karl (co-mentor)

Candida glabrata is the second most common cause of Candidemia and other forms of invasive candidiasis in the western world. It has an intrinsic high tolerance and often develops resistance to various antifungals. Understanding the resistance mechanisms is of utmost importance to tackle this problem. The most studied among the yeast pathogens is Candida albicans, however C. glabrata and its pathogenic traits differ from it, especially the proficiency of C. glabrata to adapt to harsh environment and develop resistance. C. glabrata is a close relative to the ale yeast Saccharomyces cerevisiae, they share a high degree of homology, although the regulation and function of some genes can be different. Drug combinations are a valid strategy to combat the resistance, and many combinations are already present in the clinic. Unfortunately, the drug-drug interactions are still mostly only considered for their potentially toxic effect on the host, and their effects on pathogens are usually ignored. On this basis, we tested different combinations of immunosuppressive and antifungal drugs against C. glabrata and S. cerevisiae clinical isolates. We successfully confirmed the synergistic interaction between calcineurin inhibitors (cyclosporine A, Fk506) and antifungals (amphotericin B, itraconazole, and fluconazole). We also discovered clinically relevant antagonism between purine biosynthesis inhibitor mycophenolic acid (MPA) and azole antifungals and explored the mechanism behind it. MPA alleviates the effect of azoles through enhanced activity of efflux pumps, which lowers the bioavailability of azoles thus reducing their effect. The MPA induction of the efflux pumps comes as a cell response to weak lipophilic acid, and via dysfunctional mitochondria regulating the Hog1 osmotic/oxidative stress response and Pkc1 cell wall integrity pathways involving genes HSP12, SSA3, RCK2, ROX1, and YPK1. Ypk1 serine/threonine protein kinase seems to be an integral regulator for drug response and a potential connection between the signaling from the dysfunctional mitochondria, by sensing the sphingolipid homeostasis and triggering the cell wall integrity pathway and drug response. Ypk1 is a promising target for the drug development, since its deletion greatly reduces the tolerance to fluconazole and MPA and diminishes the suppressive antagonistic interaction between the drugs. We also discovered/confirmed 28 gene deletions that significantly change the susceptibility of C. glabrata to fluconazole, 26 for MPA and 17 for the combination of both drugs. All of these genes present potential for further drug development.

• Language: English
• Keywords: Candida glabrata, drug resistance, mechanism of drug resistance, drug combinations, immunosuppressive drugs, antifungals, azoles, functional genomics, fluconazole, mycophenolic acid, Saccharomyces cerevisiae
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Tome]
• Year: 2018
• PID: 20.500.12556/RUL-101911
• UDC: 579.2:582.28:615.2822(043.3)
• COBISS.SI-ID: 926071
• Publication date in RUL: 13.07.2018

Secondary language

• Language: Slovenian
• Title: Mehanizem odpornosti proti azolom v kvasovki Candida glabrata v prisotnosti imunosupresiva mikofenolne kisline

Abstract

Patogena kvasovka Candida glabrata je drugi najpogostejši vzrok kandidemij in drugih oblik invazivnih kandidoz. Ima prirojeno visoko toleranco do antimikotikov in pogosto do njih razvije odpornost. Za spopadanje s tem problemom je razumevanje mehanizmov odpornosti ključnega pomena. Mehanizmi virulence kvasovke C. glabrata se razlikujejo od glavne predstavnice patogenih kvasovk Candida albicans. C. glabrata je namreč bližnji sorodnik s pivsko kvasovko Saccharomyces cerevisiae, z njo si deli mnogo homolognih genov, vendar ti lahko nastopajo z malenkost spremenjeno funkcijo in načinom regulacije. Ena od strategij za borbo proti odpornosti je uporaba kombinirane terapije. Kombinacije zdravil so pogost pojav v kliničnem okolju, vendar se običajno vpliv njihovih interakcije preučuje zgolj za gostitelja. Kakšen učinek imajo te interakcije na patogene, pa je velikokrat spregledano. Na tej podlagi smo testirali učinek različnih kombinacij imunosupresivov in antimikotikov na klinične izolate kvasovk C. glabrata in S. cerevisiae. Potrdili smo sinergijo med kalcinurinskimi inhibitorji (ciklosporin A in Fk506) in antimikotiki (amfotericin B, flukonazol in itrakonazol). Poleg tega smo odkrili nov, klinično relevanten supresivni antagonizem, kjer inhibitor biosinteze purinov mikofenolna kislina (MPA) zmanjša učinkovitost azolov, in raziskali mehanizem, zakaj do tega pride. MPA zmanjša učinek azolov skozi širok stresni odziv s povečanjem aktivnost izlivnih črpalk, kar zmanjša biološko uporabnost azolov. Aktivacija črpalk s strani MPA pride kot odziv celice na šibke lipofilne kisline in z aktivacijo kaskad stresnih odzivov Hog1 (osmotski/oksidativni stres) ter Pkc1 (ohranitev integritete celične stene), verjetno reguliranih skozi disfunkcionalne mitohondrije. Pri tem sodelujejo geni HSP12, SSA3, RCK2, ROX1 in YPK1. Rezultati kažejo v smer, da je serin/treonin protein kinaza Ypk1 integralni regulator za odziv na različna zdravila in morebitna povezava med signalizacijo disfunkcionalnih mitohondrijev in jedra skozi zaznavanje sfingolipidne homeostaze in posledične aktivacije Pkc1 odziva za ohranitev integritete celične stene in odziva na zdravila. Za potrditev tega modela so potrebne še dodatne raziskave. Ypk1 tako predstavlja obetavno tarčo za razvoj novih zdravil/terapij, saj njena delecija močno poveča občutljivost tako na flukonazol kot tudi na MPA in pri tem odstrani supresivno antagonistično interakcijo med zdravili. Odkrili oz. potrdili smo 28 genskih delecij, ki signifikantno spremenijo občutljivost kvasovke C. glabrata na flukonazol, 26 delecij za MPA in 17 za kombinacijo obeh zdravil. Vsi te geni predstavljajo potencial za nadaljnji razvoj zdravil.

• Keywords: Candida glabrata, odpornost na zdravila, mehanizem odpornosti, kombinacije zdravil, imunosupresivi, antimikotiki, azoli, funkcijska genomika, flukonazol, mikofenolna kislina, Saccharomyces cerevisiae