Abstract
Cryptococcus neoformans is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization. We applied the protocol to monitor metabolic changes in the pathogen alone and in co-culture with human blood-derived monocytes. We also compared metabolic flux in wild-type C. neoformans, its isogenic 5-PP-IP5/IP7-deficient metabolic mutant kcs1∆, the sister species of C. neoformans, Cryptococcus deuterogattii/VGII, and two other yeasts, Saccharomyces cerevisiae and Candida albicans. Our findings show that in contrast to monocytes and C. albicans, glycolysis and respiration are tightly coupled in C. neoformans and C. deuterogattii, as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that kcs1∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in C. neoformans is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.
Highlights
Cryptococcus neoformans is primarily an AIDS-related opportunistic fungal pathogen responsible for >180,000 deaths per year, mostly in sub-Saharan Africa [1]
We previously demonstrated that utilization of carbon sources other than glucose by C. neoformans depends on intracellular production of inositol pyrophosphate 5-PP-IP5 /IP7 by the IP6 kinase Kcs1 and requires functional mitochondria
The results show that basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) are similar in both strains
Summary
Cryptococcus neoformans is primarily an AIDS-related opportunistic fungal pathogen responsible for >180,000 deaths per year, mostly in sub-Saharan Africa [1]. A lung infection can establish in an immunocompromised host following inhalation of cryptococcal spores or small desiccated yeast cells, or after reactivation of a latent infection [2]. Pathogens 2020, 9, 684 of the lung tissue and blood, with the latter facilitating dissemination to the brain [3,4,5], reviewed in [6]. Growth of C. neoformans is significantly compromised during the hypoxic conditions encountered during infection of the human host lung and the CNS [7,8,9] where infection-induced tissue damage and inflammation create an even more oxygen-restricted local microenvironment [10,11,12]. Functional mitochondria are essential for survival of C. neoformans in the human host, with oxidative phosphorylation being upregulated to cope with hypoxia [8,9]
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