A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

Abstract

Pathogens must compete with hosts to acquire sufficient iron for proliferation during pathogenesis. The pathogenic fungus Cryptococcus neoformans is capable of acquiring iron from heme, the most abundant source in vertebrate hosts, although the mechanisms of heme sensing and acquisition are not entirely understood. In this study, we adopted a chromosomally encoded heme sensor developed for Saccharomyces cerevisiae to examine cytosolic heme levels in C. neoformans using fluorescence microscopy, fluorimetry, and flow cytometry. We validated the responsiveness of the sensor upon treatment with exogenous hemin, during proliferation in macrophages, and in strains defective for endocytosis. We then used the sensor to show that vacuolar and mitochondrial dysregulation and oxidative stress reduced the labile heme pool in the cytosol. Importantly, the sensor provided a tool to further demonstrate that the drugs artemisinin and metformin have heme-related activities and the potential to be repurposed for antifungal therapy. Overall, this study provides insights into heme sensing by C. neoformans and establishes a powerful tool to further investigate mechanisms of heme-iron acquisition in the context of fungal pathogenesis. IMPORTANCE Invasive fungal diseases are increasing in frequency, and new drug targets and antifungal drugs are needed to bolster therapy. The mechanisms by which pathogens obtain critical nutrients such as iron from heme during host colonization represent a promising target for therapy. In this study, we employed a fluorescent heme sensor to investigate heme homeostasis in Cryptococcus neoformans. We demonstrated that endocytosis is a key aspect of heme acquisition and that vacuolar and mitochondrial functions are important in regulating the pool of available heme in cells. Stress generated by oxidative conditions impacts the heme pool, as do the drugs artemisinin and metformin; these drugs have heme-related activities and are in clinical use for malaria and diabetes, respectively. Overall, our study provides insights into mechanisms of fungal heme acquisition and demonstrates the utility of the heme sensor for drug characterization in support of new therapies for fungal diseases.

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

“…We next characterized the phenotypes of mutants lacking SOD1 or SOD2 (constructed in our recent study) [ 13 ]. Growth analysis showed that the mutant lacking SOD1 grew like the wild-type strain in YPD medium (data not shown), but the mutant lacking SOD2 displayed a reduced doubling time ( Figure 2 C) (WT: 84.6 min; sod2 Δ : 93.6 min).…”

“…grubii H99 was used as the wild-type strain. The sod1 and sod2 mutants used in the current study were constructed as previously described [ 13 ]. For fungal cultures, yeast extract-bacto peptone medium supplemented with 2% glucose (YPD) was used.…”

“…By utilizing a cytosolic heme sensor, we demonstrated that deletion of SOD2 caused a significant reduction in intracellular heme levels due to increased ROS in the sod2 mutant, whereas the sod1 mutant displayed similar intracellular heme levels compared with the wild-type strain. In the same study, we also found that inhibition of vacuole function reduced intracellular heme levels, suggesting that the vacuole is an important organelle for maintaining heme homeostasis [ 13 ].…”

Respiring to infect: Emerging links between mitochondria, the electron transport chain, and fungal pathogenesis

“…Mitochondrial transporters and functions for intracellular trafficking of iron and heme are also important for fungal pathogenesis [25][26][27][28]. For example, the use of a genetically encoded heme sensor demonstrated that inhibition of mitochondrial respiration reduced the labile heme pool in the cytosol of C. neoformans [29]. Furthermore, iron acquisition, cell wall integrity, mitochondrial function, and virulence in C. neoformans are dependent on Vps45, a Sec1/ Munc18 (SM) protein that participates in vesicle fusion in the trans-Golgi network-early endosome and the late endosome pathways via interactions with soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins [28].…”

“…For example, the use of a genetically encoded heme sensor demonstrated that inhibition of mitochondrial respiration reduced the labile heme pool in the cytosol of C . neoformans [ 29 ]. Furthermore, iron acquisition, cell wall integrity, mitochondrial function, and virulence in C .…”