TheHistoplasma capsulatum DDR48Gene Is Required For Survival Within Macrophages, Response To Oxidative Stress, And Resistance to Antifungal Drugs

Abstract

AbstractHistoplasma capsulatum (Hc)is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually.Hcgrows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. This manuscript is focused on characterizing the DNA damage-responsive geneHcDDR48.HcDDR48was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase ofHcgrowth. Upon further analysis we found thatHcDDR48is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. We found thatHcDDR48is involved in oxidative stress response, antifungal drug resistance, and survival within resting and activated macrophages. Growth ofddr48Δyeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found thatddr48Δyeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To testHcDDR48’s involvementin vivo, we infected resting and activated RAW 264.7 murine macrophages withHcyeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in theddr48Δstrain compared to wildtypeHclevels. Herein, we demonstrate the importance of maintaining a functional copy ofHcDDR48in order forHcyeasts to sense and respond to numerous environmental and host-associated stressors.ImportanceHistoplasma capsulatumis an intracellular pathogen of phagocytes, where it subverts immune recognition and avoids killing by the innate immune system. Macrophages provide a permissive environment forHcreplication and killing only occurs upon the onset of the T-cell driven adaptive immune response.Hchas evolved numerous virulence factors that aid in its survival against host-derived ROS and RNSin vivo. While these virulence factors have been described in past years, only a few reports describing the regulation of these genes and how this intricate system leads to fungal survival. In this study, we characterized the stress response geneDDR48and determined it to be indispensable forHcsurvival within macrophages.HcDDR48regulates transcript levels of superoxide dismutases and catalases responsible for detoxification of ROS and contributes to antifungal drug resistance. Our studies highlightDDR48as a potential target to controlHcinfection and decrease the severity of the disease process.