| Summary: | <i>Cryptococcus neoformans</i> 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 <i>C. neoformans,</i> its isogenic 5-PP-IP<sub>5</sub>/IP<sub>7</sub>-deficient metabolic mutant <i>kcs1</i>∆, the sister species of <i>C. neoformans, Cryptococcus deuterogattii/VGII,</i> and two other yeasts, <i>Saccharomyces cerevisiae</i> and <i>Candida albicans.</i> Our findings show that in contrast to monocytes and <i>C. albicans</i>, glycolysis and respiration are tightly coupled in <i>C. neoformans</i> and <i>C. deuterogattii,</i> as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that <i>kcs1</i>∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in <i>C. neoformans</i> is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.
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