Low Glucose Mediated Fluconazole Tolerance in Cryptococcus neoformans

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).

Epidemiological and Clinical Characteristics, Antifungal Susceptibility, and MLST-Based Genetic Analysis of Cryptococcus Isolates in Southern Taiwan in 2013–2020

“…Several factors have been found to affect the susceptibility of Cryptococcus spp. isolates to antifungal drugs, which include the prolonged use of FCZ as a suppressive monotherapy, low bioavailability of FCZ in the infected tissue, fungistatic action of azoles, increased tolerance to FCZ due to replicative aging, low glucose growth condition and limited nutrients, and intrinsic resistance, such as the hetero-resistance of C. neoformans and C. gattii to azoles [ 11 , 13 , 14 , 15 ]. The environmental triazole (tebuconazole) used as an agrochemical pesticide may induce cross-resistance to clinically available azoles in C. neoformans and C. gattii [ 16 ].…”

Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans

“…The observed increased FLC tolerance in low glucose growth conditions, however, is not associated with a change in gene copy number of AFR1 or AFR2. [46]. AFR3 is located on chromosome 3 and the gene copy number was unchanged under low glucose stress (data not shown).…”

“…Stochasticity of life span is still poorly understood in C. neoformans. Exposure to stress, such as glucose deprivation, leads to an increase in FLC tolerance, as the change is only transient and does not become intrinsic to the strain [46]. Low glucose conditions lead to an increase in efflux through increased pump activity dependent on Afr1 [46].…”

“…Exposure to stress, such as glucose deprivation, leads to an increase in FLC tolerance, as the change is only transient and does not become intrinsic to the strain [46]. Low glucose conditions lead to an increase in efflux through increased pump activity dependent on Afr1 [46]. Thus, we explored whether Afr3 contributes to increased FLC tolerance under low glucose growth conditions.…”

“…AFR3 is located on chromosome 3 and the gene copy number was unchanged under low glucose stress (data not shown). It was previously hypothesized that the increase in efflux pump expression is being driven by stress-induced upregulation of transcription factors, as many transcription factors correlated with FLC resistance are upregulated in low glucose [14,46,48]. Afr3, as well as the other characterized ABC transporters (Afr1, Afr2, and Mdr1), are upregulated in 10-generations old cells.…”