The role of glucose in inducing tolerance to amphotericin B for fungal meningitis treatment

By Dr. Priyom Bose, Ph.D.Reviewed by Benedette Cuffari, M.Sc.Jan 18 2024

A recent Nature Microbiology investigates the mechanism responsible for amphotericin B (AmB) tolerance of Cryptococcus neoformans. 

Study: Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis. Image Credit: Kateryna Kon / Shutterstock.com

Background

C. neoformans is a fungal pathogen that causes meningitis, which causes approximately 180,000 deaths annually worldwide. Currently, AmB is the only fungicidal drug available to treat fungal meningitis.

Genetic resistance to AmB is not a common phenomenon for meningitis-causing fungi, including C. neoformans. However, the clinical outcomes of cryptococcal meningitis are significantly dependent on AmB resistance.

Fungistatic tolerance describes the ability of a fungi sub-population to grow at drug concentrations above the minimum inhibitory concentration (MIC). It is imperative to understand whether AmB tolerance can be triggered in vivo by host factors.

About the study

The impact of host metabolites on the efficacy of AmB to eliminate C. neoformans was assessed based on a metabolite-drug screening strategy. To this end, a BIOLOG phenotype microarray containing 340 metabolites was used for the screening.

Time-kill curve-based approaches, such as assessing the minimum duration for killing 99% of cells (MDK99), were used to quantify fungal tolerance to AmB. 

Study findings

A higher average cell survival percentage was observed in cells grown in culture media containing phosphorus-, nitrogen-, and sulfur-based metabolites. In contrast, this type of tolerance was not observed in cells grown in media containing carbon-based metabolites. Since BIOLOG-nitrogen, -phosphorus, and -sulfur plates contained a high level of glucose as a carbon source, it is possible that glucose induced AmB tolerance.

Based on experimental results, a positive correlation between glucose concentration and AmB tolerance was established. However, when glucose was replaced with galactose, this type of fungal tolerance to AmB was not observed. Importantly, glucose-induced AmB tolerance was independent of macronutrients.

MDK99 assessment revealed that brain glucose between two and five millimolar (mM) could strongly induce AmB tolerance. Human cerebrospinal fluid (CSF) was used for further evaluation, as it is the main site of C. neoformans infection during meningitis.

In the control CSF sample, glucose was removed using a glucose oxidase (GOX)-based strategy. Here, AmB tolerance was observed in C. neoformans-infected human CSF samples. Interestingly, no AmB tolerance was observed in C. neoformans cells that were cultured in glucose-free enzymatically treated CSF samples.

The fungal regulator Mig1 was found to be a key determining factor that leads to glucose-induced AmB tolerance during life-threatening cryptococcal meningitis. The mouse model of cryptococcal meningitis revealed that most C. neoformans-infected cells in mouse brain tissue exhibited nuclear localization of Mig1. Thus, brain glucose may induce Mig1-mediated GR and exert AmB tolerance.

Further animal-based experiments confirmed the role of Mig1 in cryptococcal virulence. Importantly, Mig1 does not influence AmB resistance; however, it is a gene that is specific for antifungal tolerance.

The underlying mechanism by which Mig1 mediates AmB tolerance is through inhibiting the synthesis of ergosterol, which is the primary target of AmB. Mig1 also promotes the production of inositolphosphorylceramide (IPC), which competes with AmB for ergosterol. 

Lipidomic analyses indicated that the disruption of Mig1 leads to changes in several membrane lipid component levels, which suggests that Mig1 could play an important role in linking AmB tolerance and membrane integrity. Taken together, the identification of Mig1 as a tolerance-specific gene has many potential medical implications, as it could be used as a therapeutic target to improve clinical outcomes.

The current study also elucidated the role of AbA in exacerbating AmB activity against tolerant C. neoformans cells. Although AbA alone exhibited limited efficacy in treating cryptococcal brain infections, a better therapeutic effect was observed in a mouse model of cryptococcal meningitis when combined with AmB. Notably, the therapeutic efficacy of AbA-AmB superseded the clinically recommended AmB-flucytosine combination.

Conclusions

The current study revealed that glucose, which is abundantly present in the brain, induces fungal AmB tolerance through the glucose repression regulator Mig1 during meningitis. Future research is needed to better understand whether host factors other than glucose influence drug tolerance. Furthermore, additional studies are needed to determine whether high blood glucose levels influence fungicidal tolerance during fungemia.