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Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis

Nature Microbiology volume 9, pages 346–358 (2024)Cite this article

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Abstract

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite–drug–fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.

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Fig. 1: Glucose strongly induces cryptococcal tolerance to AmB.
Fig. 2: Glucose induces nuclear localization of Mig1, enabling AmB tolerance.
Fig. 3: Mig1-mediated AmB tolerance leads to decreased therapeutic efficacy in mice with cryptococcal meningitis.
Fig. 4: Mig1 mediates AmB tolerance by inhibiting ergosterol production.
Fig. 5: IPC competes with AmB for ergosterol, limiting AmB efficacy in vitro and in vivo.

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Data availability

All sequencing data are archived on GEO (accession: GSE188965). All other data needed to evaluate the conclusions are available within the Article or its Supplementary Information. Source data are provided with this paper. Any additional data are available from the corresponding author.

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Acknowledgements

We thank members of the Wang lab, and Y. Zhao and E. Yang for sharing constructs and protocols and scientific discussions; Y. Wang for providing the Rhizopus arrhizus strains; T. Liu for providing C. neoformans H99 strain harbouring PH3-GFP-NOP1; G. Liu for quantification of glucose in CSF; B. Shi for lipidomic analysis; L. Su for the time-lapse experiment; and T. Zhao for intracellular ROS measurement. This work was supported by the National Key Research and Development Program (2022YFC2303000 and 2021YFC230000, L.W.; 2021YFA0911300, P.H.; and 2021YFC2100600, X.T.; http://www.most.gov.cn/); the National Natural Science Foundation of China (32100153, P.H.; 31970077, G.-j.H.; 82172291, M.C.; and 82073789, C.L.; http://www.nsfc.gov.cn/); and the CAS Interdisciplinary Innovation Team (L.W.; http://www.cas.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Lei Chen, Xiuyun Tian, Lanyue Zhang.

Authors and Affiliations

  1. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Lei Chen, Xiuyun Tian, Lanyue Zhang, Wenzhao Wang, Pengjie Hu, Shibin Li, Zhenghao Shen, Weixin Ke, Guang-jun He & Linqi Wang

  2. University of Chinese Academy of Sciences, Beijing, China

    Lanyue Zhang, Zhenghao Shen & Linqi Wang

  3. Medical Research Institute, College of Pharmaceutical Sciences, Southwest University, Chongqing, China

    Zhongyi Ma, Leixin Ye, Guojian Liao & Chong Li

  4. Department of Microbiology, University of Georgia, Athens, GA, USA

    Yeqi Li & Xiaorong Lin

  5. Department of Infectious Diseases and Clinical Microbiology, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China

    Xin Fan

  6. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Yao Wu

  7. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Guanghou Shui

  8. Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China

    Meng Xiao

  9. Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijing, China

    Meng Xiao

  10. Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Disease, Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing, China

    Ying Yang

  11. Institute of Biological Science and Technology, Guangxi Academy of Sciences, Nanning, Guangxi, China

    Wenxia Fang

  12. Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China

    Fan Bai

  13. Department of Dermatology, Shanghai Key Laboratory of Medical Mycology, Changzheng Hospital, Naval Medical University, Shanghai, China

    Min Chen

Authors
  1. Lei Chen
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Contributions

All authors contributed to the data analysis. L.C. and L.W outlined the study; L.C., X.T., L.Z., W.W., P.H., Z.M., Y.L., S.L, Z.S., X.F., L.Y., W.K., Y.W., G.S., M.X., G.-j.H., Y.Y., F.B., G.L., M.C., W.F., X.L., C.L. and L.W. designed the experiments. L.C., X.T. and L.Z. conducted most of the studies; L.C. conducted the bioinformatic analysis; L.Z., W.W. and Y.W. analysed the data; M.X., Y.Y., G.S. and M.C. contributed reagents/materials/analysis tools; and L.C., X.T., X.L. and L.W. wrote the manuscript with contributions from other authors.

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Correspondence to Linqi Wang.

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Extended data

Extended Data Fig. 1 Glucose at a concentration similar to that in brain tissue is sufficient to induce AmB tolerance.

a, Cells were cultured in glucose-free RPMI medium supplemented with glucose before challenge with 1 hr 10 × MIC AmB treatment. The percent survival was normalized to survival without AmB. Shaded color represents SD of three biological replicates. b, The stimulatory effect of glucose on AmB tolerance is independent of its growth- promoting activity. The influences of different metabolites on the growth of a wildtype C. neoformans strain were evaluated relative to glucose (Down). Yellow dots represent metabolites with similar fungal growth-promoting effect to glucose (0.8–1.2 times CFU to glucose). Histogram indicating the influence of metabolites with similar fungal growth-promoting effect to glucose on the AmB tolerance (Up). Dots are representative of means of two independent experiments. c, AmB MIC levels of the wildtype strain cultured at different glucose concentrations were measured with a method described by CLSI. Yellow indicates fungal cell survival under corresponding drug concentration. d, A minimum duration for killing 99% of cells (MDK99) test of AmB using a wildtype C. neoformans strain treated at 10 × MIC. Cells were cultured on glucose-free RPMI medium supplemented with 0, 0.25, or 4 mM glucose for 6 hrs before exposure to AmB. Data represent mean ± SD (n = 3). Two-tailed unpaired Student’s t test in (d).

Source data

Extended Data Fig. 2 Evaluation of the effects of glucose oxidase, MnO2 and gluconic acid on stimulation of AmB tolerance.

a, H2O2 concentrations following GOX-catalyzed glucose conversion and MnO2 treatment were monitored. Data represent the mean ± SD (n = 3). ND, not detected. b, C. neoformans cells were treated at 1 hr 10 × MIC AmB treatment and cultured in YP broth supplemented with 2% glucose or 2% galactose and the noted factors. Data represent the mean ± SD (n = 3).

Source data

Extended Data Fig. 3 Glucose, but not its metabolic intermediates, induces AmB tolerance in C. neoformans.

Results from metabolite-AmB screening showed that neither the glucose metabolites generated during glycolysis (left) nor those during the tricarboxylic cycle (right) showed detectable impact on AmB tolerance in wildtype C. neoformans.

Source data

Extended Data Fig. 4 Glucose repression (GR) mediates AmB tolerance in C. neoformans.

a, Wildtype C. neoformans cells were cultured in media containing 1% glucose and 1% galactose (left), or in media containing only 2% galactose (right). Sugar metabolism was evaluated by HPLC. Cell growth was determined by measuring the absorbance at 600 nm (A600). Data are shown as mean ± SD (n = 3). b, 2-DG, a non-metabolizable analog of glucose, inhibits the growth of C. neoformans when added with an alternative carbon source (galactose) but has no effect on growth when added with a preferred carbon source. c, Percent survival of wildtype cells cultured in media containing 2% galactose or 2% galactose with 0.02% 2-DG upon 10 × MIC AmB exposure for 1 hr. Data are shown as mean ± SD (n = 3). Two-tailed unpaired Student’s t test in (c). Sugar con., sugar concentration; 2-DG, 2-deoxyglucose.

Source data

Extended Data Fig. 5 Mig1 plays a critical role in glucose repression and glucose-induced AmB tolerance in C. neoformans.

a, Wildtype and mig1Δ mutant cells were spotted onto the noted YP plates, which were photographed after 3 days. Images are representative of more than three trials. b, Wildtype and mig1Δ mutant cells were cultured in media containing 2% glucose. Cell growth was determined by measuring the absorbance at 600 nm (A600). Data are shown as mean ± SD (n = 3). c, Survival curves of wildtype and mig1Δ cells cultured in the presence of 2% glucose at noted concentration of AmB. Data represent the mean ± SD (n = 3).

Source data

Extended Data Fig. 6 The mechanism by which Mig1 affects glucose-induced AmB tolerance revealed by time-series transcriptome analyses.

a, Percent survival of C. neoformans wildtype and mig1Δ cells incubated with AmB at 10 × MIC in YP broth with 2% glucose (Glu) or 2% galactose (Gal). Wildtype and mig1Δ cells with or without AmB treatment for 10, 20, or 30 minutes were collected for further transcriptome analyses. Data represent the mean ± SD (n = 3). b, Principal component analysis (PCA) of dynamic transcriptome data of C. neoformans wildtype and mig1Δ cells treated with AmB (10 × MIC). Each plot represents the mean from three biological repeats. c, Plotting the 7 dimensions determined to be statistically significant by permutation analysis. d, Pearson correlations of metabolite alteration, Mig1 function and AmB treatment to dimensions 1–7. e, Biological functional enrichment analysis of genes specifically regulated by Mig1 in response to glucose. f, Fold change of genes related to ergosterol synthesis under different conditions. Data shown in the figure are from the AmB-untreated samples. Two-sided Wald test in DESeq2 in (f).

Source data

Extended Data Fig. 7 Beanplot illustrating relative lipid composition levels of wildtype and mig1Δ cells in the presence of 2% glucose.

PC: phosphatidylcholines; LPC: lyso-PC; IPC: inositolphosphorylceramide; MIPC: mannosyl inositolphosphorylceramide; PhytoCer: phytoceramides; SPH: sphingosine. Two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 8 AmB tolerance is induced by inhibiting ergosterol production and is associated with reduced AmB accumulation.

a, AmB MIC levels of the wildtype cells grown under AmB sensitive conditions (2% galactose) pre-treated with 1 μg/mL terbinafine. b, Relative AmB levels in wildtype and mig1Δ cells cultured on YP medium supplemented with 2% glucose or 2% galactose or 2% galactose with 8 μg/mL terbinafine. Data represent the mean ± SD (n = 3). Two-tailed unpaired Student’s t test in (b).

Source data

Extended Data Fig. 9 Pretreatment with AbA does not improve fungicidal efficacy of AmB in the erg6Δ mutant strain.

a, Relative IPC levels of wildtype cells cultured in the presence of 2% glucose with or without AbA treatment (1 μg/mL). Boxplots display the 25th, 50th (median) and 75th quantiles as well as the minimum and maximum values. b, Percent survival of erg6Δ cells cultured in the presence of 2% glucose after 1 hr 10 × MIC AmB treatment with or without AbA (1 μg/mL). Data represent the mean ± SD (n = 3). c, Relative ergosterol levels of wildtype and erg6Δ cells in the presence of 2% glucose. Data represent the mean ± SD (n = 3). ND, not detected.

Source data

Extended Data Fig. 10 The potential AmB-potentiating effect of AbA in a mouse model of cryptococcosis.

a, Experimental design of brain infections and treatments of antifungals as noted. Mice were infected intravenously with C. neoformans at day 0. b, Percent survival of wildtype in mouse brains at 7 days after infection, with AmB or AmB-AbA treatment (without liposomal encapsulation). Bars represent the mean ± SD (n = 3). c, Comparison of AbA content in the brain of mice treated with liposome-encapsulated AbA and without liposome-encapsulated AbA as determined by LC-MS/MS. Data represent the mean ± SD (n = 3). d, Relative brain fungal burdens of wildtype at 7 days after infection, with or without treatment with liposome-encapsulated AbA. Data represent the mean ± SD (n = 10). NS, not significant (two-tailed unpaired Student’s t test in (d)). Lipid: liposome encapsulation.

Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1–5.

Source data

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Chen, L., Tian, X., Zhang, L. et al. Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis. Nat Microbiol 9, 346–358 (2024). https://doi.org/10.1038/s41564-023-01561-1

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