Abstract
Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).
Highlights
minimum inhibitory concentration (MIC) were performed on four laboratory C. neoformans strains (KN99α, RC2, H99, and JEC21) and four lowpassaged clinical isolates (I55, I114, J9 and J22), all of which were sensitive to FLC at baseline level under 2% glucose conditions (FLC MIC < 2 μg/mL, Figure 1A, Table 1)
The present study demonstrates that low glucose growth conditions greatly enhance
Glucose deprivation leads to enhanced FLC efflux through augmented efflux pump activity, marked changes of the mitochondrial network, and mitochondrial function, including lowered ROS accumulation and higher adenosine tri-phosphate (ATP) production
Summary
Cryptococcal meningitis (CM) is caused predominantly by Cryptococcus neoformans and less frequently by Cryptococcus gattii [1,2]. A global data analysis estimates that this life-threatening fungal infection causes 180,000 deaths annually. CM is primarily diagnosed in patients with advanced HIV/AIDS, rising numbers of CM cases have been reported in immune-competent patients [3,4]. Amphotericin B with or without 5-fluorocytosine (5FC) for two weeks followed by fluconazole (FLC) maintenance therapy [5] is the recommended treatment. FLC monotherapy remains the only option in many countries with limited resources. FLC monotherapy is associated with higher relapse rates. CM mortality can exceed 50% [6,7,8]
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