Deletion of the Stress Response Gene DDR48 from Histoplasma capsulatum Increases Sensitivity to Oxidative Stress, Increases Susceptibility to Antifungals, and Decreases Fitness in Macrophages.

Logan T Blancett,Mallory B Harmon,Sarah E Scheuermann,Glenmore Shearer,Sydney C Jackson,Kauri A Runge,Jamease C Williams,Lauren A Kennedy,Gabriella M Reyes

doi:10.3390/jof7110981

Abstract

The stress response gene DDR48 has been characterized in Saccharomyces cerevisiae and Candida albicans to be involved in combating various cellular stressors, from oxidative agents to antifungal compounds. Surprisingly, the biological function of DDR48 has yet to be identified, though it is likely an important part of the stress response. To gain insight into its function, we characterized DDR48 in the dimorphic fungal pathogen Histoplasma capsulatum. Transcriptional analyses showed preferential expression of DDR48 in the mycelial phase. Induction of DDR48 in Histoplasma yeasts developed after treatment with various cellular stress compounds. We generated a ddr48∆ deletion mutant to further characterize DDR48 function. Loss of DDR48 alters the transcriptional profile of the oxidative stress response and membrane synthesis pathways. Treatment with ROS or antifungal compounds reduced survival of ddr48∆ yeasts compared to controls, consistent with an aberrant cellular stress response. In addition, we infected RAW 264.7 macrophages with DDR48-expressing and ddr48∆ yeasts and observed a 50% decrease in recovery of ddr48∆ yeasts compared to wild-type yeasts. Loss of DDR48 function results in numerous negative effects in Histoplasma yeasts, highlighting its role as a key player in the global sensing and response to cellular stress by fungi.

Highlights

The H. capsulatum DDR48 gene (HCBG_02772) was originally isolated in our lab from a subtractive cDNA hybridization library enriched to identify transcripts whose expression was up-regulated in Histoplasma mycelia compared to Histoplasma yeasts [44]
We aimed to increase our understanding of the H. capsulatum DDR48 gene, as little is known about DDR48 in general, and there are no prior studies in H
DDR48 was identified as a 315 amino acid protein containing fifteen repeats of the characteristic SYG amino acid sequence that is present in all identified DDR48 amino acid sequences

Summary

Introduction

The stress response gene DDR48 is required to combat oxidative stress, antifungal drugs, and numerous other stressors encountered by fungi. An identifying feature of DDR48 is that it contains multiple repeats of S-Y-G, which seem to be conserved between all fungal species [1,2]. The SYG motif has been characterized as a prion-like domain with low complexity sequence that exhibits RNA binding properties [3,4]. SYG motifcontaining proteins have been identified as components of stress granules, which are unique protein/RNA aggregates involved in RNA quality control, such as maintaining the balance between translating RNAs and mRNA degradation [3,5,6]. DDR48 was identified in stress granules in the pathogenic fungus C. albicans [7], and overexpression of DDR48 in

Objectives

Methods

Results

Conclusion