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

Abstract

Histoplasma capsulatum (Hc) is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows 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 gene HcDDR48. HcDDR48 was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hc growth. Upon further analysis we found that HcDDR48 is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. We found that HcDDR48 is involved in oxidative stress response, antifungal drug resistance, and survival within resting and activated macrophages. Growth of ddr48Δ yeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found that ddr48Δ yeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To test HcDDR48s involvement in vivo, we infected resting and activated RAW 264.7 murine macrophages with Hc yeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in the ddr48Δ strain compared to wildtype Hc levels. Herein, we demonstrate the importance of maintaining a functional copy of HcDDR48 in order for Hc yeasts to sense and respond to numerous environmental and host-associated stressors.

Cprp—An Unusual, Repetitive Protein Which Impacts Pleuromutilin Biosynthesis in the Basidiomycete Clitopilus passeckerianus

“…It has been shown to respond to a wide range of environmental stresses, including exposure to DNA damaging chemicals, heat-shock, osmotic stress, oxidative stress, DNA replication stress and exposure to antifungals (McClanahan and McEntee, 1986;Treger and McEntee, 1990;Miralles and Serrano, 1995;Karababa et al, 2004;Hromatka et al, 2005;Setiadi et al, 2006;Hao et al, 2009;Tkach et al, 2012;Mostafa and Awad, 2015). A recent study found that in H. capsulatum DDR48 confers some resistance to the antifungal azoles via the regulation of sterol biosynthesis (Reyes, 2019;Blancett et al, 2020), which is consistent with an increase in ddr48 expression in azole-resistant isolates of C. albicans (Mostafa and Awad, 2015). DDR48 is also known to be required for H. capsulatum pathogenicity and survival in macrophages, likely due a role regulating the enzymes needed to detoxify reactive oxygen species (ROS) (Blancett, 2019;Blancett et al, 2020).…”

“…A recent study found that in H. capsulatum DDR48 confers some resistance to the antifungal azoles via the regulation of sterol biosynthesis (Reyes, 2019;Blancett et al, 2020), which is consistent with an increase in ddr48 expression in azole-resistant isolates of C. albicans (Mostafa and Awad, 2015). DDR48 is also known to be required for H. capsulatum pathogenicity and survival in macrophages, likely due a role regulating the enzymes needed to detoxify reactive oxygen species (ROS) (Blancett, 2019;Blancett et al, 2020). Regulating complex pathways in response to environmental factors is perfectly consistent with a potential role in the regulation of secondary metabolites such as pleuromutulin, which are known to be produced under specific environmental conditions.…”

Utilization of aHistoplasma capsulatumzinc reporter reveals the complexities of fungal sensing of metal deprivation

“…H. capsulatum RNA was isolated by mechanical disruption with 0.5 mm glass beads, extraction with TRIzol™ (Invitrogen), and precipitated with ethanol, as previously described (44,45). Genomic DNA was removed via DNase digestion and column purification using the PureLink™ RNA Mini Kit (Invitrogen).…”