Validation of candidate genes for drug resistance in model organism Saccharomyces cerevisiae

Škofljanc, Neža (author), Petrovič, Uroš (mentor), Mozzachiodi, Simone (co-mentor)

Antibiotic resistance is an increasing threat worldwide due to the high number of microbes developing resistance to the limited range of drugs. Yeasts, as other microbes, can develop antifungal resistance when exposed to these compounds and produce difficult to treat infections. In this work we took advantage of a set of 420 genetic variants that have been mapped as genetic determinants in regulating antifungal resistance in a large cohort of Saccharomyces cerevisiae strains. We looked into those variants and chose the most compelling ones, which map onto four different genes – HSP82, AIM29, PIS1 and FKS1. We applied the CRISPR-Cas9 technology to engineer those variants into genetically different S. cerevisiae strains. Then we tested the constructed strains for their response to two different antifungal drugs, ketoconazole and fluconazole. The tested phenotypes were generation time (i.e., growth speed) and yield (i.e., final population density) in the presence of the drug compared to the drug-free medium. All of the inspected variants, in at least one genetic background, caused a change in drug sensitivity in the engineered strains compared to the parent strains. Furthermore, we measured the growth of the mutants at two different temperatures, 30 °C and 37 °C, to inspect whether the mutations improve or impair the growth of the strains at elevated temperature but saw no such effect. Lastly, we phenotyped the constructed strains for their chronological lifespan and measured a marked increase in the strains with altered variants in AIM29 and FKS1 genes.

• Language: English
• Keywords: Fungal infections, antifungal drug resistance, Saccharomyces cerevisiae, GWAS, CRISPR-Cas9, stress response
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2023
• PID: 20.500.12556/RUL-145350
• COBISS.SI-ID: 149659651
• Publication date in RUL: 19.04.2023

Secondary language

• Language: Slovenian
• Title: Validacija kandidatnih genov za odpornosti proti zdravilom v modelnem organizmu Saccharomyces cerevisiae

Abstract

Odpornost proti antibiotikom predstavlja rastočo grožnjo po vsem svetu zaradi velikega števila mikrobov, ki razvijajo odpornost proti omejenemu naboru zdravil. Tako kot drugi mikrobi so tudi kvasovke, ob njihovi izpostavitvi, sposobne razviti odpornost proti antimikotikom in povzročiti težko obvladljive infekcije. V tem delu smo uporabili nabor 420 genetskih različic, ki so bile kartirane kot genetske determinante za uravnavanje odpornosti proti antimikotikom pri velikem naboru sevov kvasovke Saccharomyces cerevisiae. Po pregledu odkritih različic smo izbrali nekaj najbolj zanimivih, ki se nahajajo v štirih različnih genih – HSP82, AIM29, PIS1 in FKS1. Z uporabo tehnologije CRISPR-Cas9 smo te genetske različice vstavili v genetsko različne seve S. cerevisiae. Nato smo ustvarjene seve fenotipizirali v prisotnosti dveh različnih antimikotikov, ketokonazola in flukonazola. Opazovani lastnosti sta bili generacijski čas (hitrost rasti) in izkoristek (končna gostota populacije) v prisotnosti antimikotika v primerjavi z rastjo v okolju brez antimikotika. Vse proučevane različice so v vsaj enem genetskem ozadju spremenile občutljivost mutiranih sevov na antimikotike v primerjavi z občutljivostjo starševskih sevov. Poleg tega smo primerjali tudi rast ustvarjenih sevov pri dveh različnih temperaturah, 30 °C in 37 °C, da bi preverili, ali predstavljene mutacije izboljšajo ali poslabšajo rast sevov pri povišani temperaturi, vendar razlik nismo opazili. Na koncu smo izmerili še kronološko življenjsko dobo mutant in opazili njeno podaljšanje pri sevih z mutacijami v genih AIM29 in FKS1.

• Keywords: Glivne infekcije, odpornost proti antimikotikom, Saccharomyces cerevisiae, GWAS, CRISPR-Cas9, odziv na stres