Functional Characterization of a Novel Oxidative Stress Protection Protein in the Pathogenic Yeast Candida glabrata.

Jane Usher,Hsueh-Lui Ho,Victoria Attah,Yogesh Chaudhari,Ken Haynes

doi:10.3389/fgene.2020.530915

Abstract

Candida species are important pathogens of humans and the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of invasive candidosis, the incidence of Candida glabrata infections has rapidly grown. The reason for this increase is not fully understood, but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerant to stresses especially oxidative stress. Taking the approach that using the model organism, Saccharomyces cerevisiae, with its intrinsic sensitivity to oxidative stress, we hypothesized that by expressing mediators of stress resistance from C. glabrata in S. cerevisiae, it would result in induced resistance. To test this we transformed, en-masse, the C. glabrata ORFeome library into S. cerevisiae. This resulted in 1,500 stress resistant colonies and the recovered plasmids of 118 ORFs. Sequencing of these plasmids revealed a total of 16 different C. glabrata ORFs. The recovery of genes encoding known stress protectant proteins such as GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified a C. glabrata unique ORF that confers oxidative stress resistance. We set to characterise this gene herein, examining expression in oxidative stress sensitive strains, comet assays to measure DNA damage and synthetic genetic array analysis to identify genetic interaction maps in the presence and absence of oxidative stress.

Highlights

Biological systems function in constantly changing complex environments, where they are subject to wide ranging perturbations
Previous studies have demonstrated that the C. glabrata response to in vitro exerted oxidative stress is very similar to that observed upon phagocyte engulfment, both at the level of gene expression (Kaur et al, 2007; Fukuda et al, 2013), where the up-regulation of genes encoding functions related to stress adaptation and nutrient recycling overlap substantially, and in growth kinetics (Kaloriti et al, 2012) where in both environments, approximately 20% of C. glabrata cells survive initial contact with a substantial delay occurring prior to growth re-commencing
We hypothesized that expression of effectors of C. glabrata stress resistance in S. cerevisiae would lead to increased oxidative stress resistance

Summary

Introduction

Biological systems function in constantly changing complex environments, where they are subject to wide ranging perturbations. Previous studies have demonstrated that the C. glabrata response to in vitro exerted oxidative stress is very similar to that observed upon phagocyte engulfment, both at the level of gene expression (Kaur et al, 2007; Fukuda et al, 2013), where the up-regulation of genes encoding functions related to stress adaptation and nutrient recycling overlap substantially, and in growth kinetics (Kaloriti et al, 2012) where in both environments, approximately 20% of C. glabrata cells survive initial contact with a substantial delay occurring prior to growth re-commencing These data sets demonstrate that in vitro oxidative stress is a realistic model of host-induced stress and the ability to survive oxidative stress is an important virulence determinant for pathogens. Understanding this network, and the role that selected components play in stress resistance, is essential to the long-term development of small molecule inhibitors

Methods

Results

Conclusion