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Pan-drug resistance and hypervirulence in a human fungal pathogen are enabled by mutagenesis induced by mammalian body temperature

Nature Microbiology volume 9, pages 1686–1699 (2024)Cite this article

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Abstract

The continuing emergence of invasive fungal pathogens poses an increasing threat to public health. Here, through the China Hospital Invasive Fungal Surveillance Net programme, we identified two independent cases of human infection with a previously undescribed invasive fungal pathogen, Rhodosporidiobolus fluvialis, from a genus in which many species are highly resistant to fluconazole and caspofungin. We demonstrate that R. fluvialis can undergo yeast-to-pseudohyphal transition and that pseudohyphal growth enhances its virulence, revealed by the development of a mouse model. Furthermore, we show that mouse infection or mammalian body temperature induces its mutagenesis, allowing the emergence of hypervirulent mutants favouring pseudohyphal growth. Temperature-induced mutagenesis can also elicit the development of pan-resistance to three of the most commonly used first-line antifungals (fluconazole, caspofungin and amphotericin B) in different Rhodosporidiobolus species. Furthermore, polymyxin B was found to exhibit potent activity against the pan-resistant Rhodosporidiobolus mutants. Collectively, by identifying and characterizing a fungal pathogen in the drug-resistant genus Rhodosporidiobolus, we provide evidence that temperature-dependent mutagenesis can enable the development of pan-drug resistance and hypervirulence in fungi, and support the idea that global warming can promote the evolution of new fungal pathogens.

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Fig. 1: Identification and phenotypic characterization of two independent cases of human infection with a previously undescribed fungal pathogen, R. fluvialis, based on the CHIF-NET surveillance programme.
Fig. 2: Identification of parallel resistance mechanisms in the Rhodosporidiobolus pathogen.
Fig. 3: Evaluation of the in vivo adaptation of different Rhodosporidiobolus species in an immunocompromised mouse model.
Fig. 4: Pseudohyphal growth promotes the virulence of R. fluvialis and mouse infection induces the emergence of pseudohyphal mutants.
Fig. 5: Heat stress induces the accumulation of intracellular ROS, which is an important factor promoting mutagenesis of R. fluvialis.
Fig. 6: Heat stress induces the emergence of pan-resistance variants in different Rhodosporidiobolus species.

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Data availability

All data needed to evaluate the conclusions in the paper are present in the paper or in Supplementary Information. The sequencing data have been deposited at NCBI under the BioProject PRJNA990320. Source data are provided with this paper.

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Acknowledgements

We would like to thank S. Gao and S. Hu for important advice on R. fluvialis NJ103 and TZ579 genome assembly. We are grateful to all participants in the CHIF-NET programme. This study was financially supported by the National Key Research and Development Program of China (2022YFC2303000, L.W.); the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-038, Y. Xu); the National High Level Hospital Clinical Research Funding (2022-PUMCH-C-052, M.X.); the CAS Interdisciplinary Innovation Team (L.W.), and the Scientific Research Program of the Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University (YCT202302 and CG202305, J.H.).

Author information

Author notes

  1. These authors contributed equally: Jingjing Huang, Pengjie Hu, Leixin Ye.

Authors and Affiliations

  1. Department of Clinical Laboratory, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, China

    Jingjing Huang

  2. Department of Laboratory Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Jingjing Huang, Xinfei Chen, Jinhan Yu, Meng Xiao, Yingxing Li, Ge Zhang & Yingchun Xu

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

    Pengjie Hu, Leixin Ye, Zhenghao Shen, Fang Liu, Yuyan Xie, Lei Cai, Feng-yan Bai & Linqi Wang

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

    Leixin Ye, Zhenghao Shen, Yuyan Xie & Linqi Wang

  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. National Centre for Infectious Diseases, Tan Tock Seng Hospital, Novena, Singapore

    Clement K. M. Tsui

  7. Faculty of Medicine, University of British Columbia, Vancouver, Canada

    Clement K. M. Tsui

  8. Department of Clinical Laboratory, Jinling Hospital, Medical School of Nanjing University, Nanjing, China

    Weiping Wang

  9. Faculty of Health Sciences, University of Macau, Macau SAR, China

    Koon Ho Wong

  10. Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China

    Koon Ho Wong

  11. MoE Frontiers Science Center for Precision Oncology, University of Macau, Macau SAR, China

    Koon Ho Wong

Authors
  1. Jingjing Huang
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  2. Pengjie Hu
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Contributions

All authors contributed to the data analysis. J.H., P.H., L.Y., Z.S., X.C., F.L., Y. Xie, J.Y., X.F., M.X., C.K.M.T., L.C., G.Z., F.B., Y. Xu and L.W. designed the experiments. J.H., P.H., L.Y. and Z.S. conducted most of the studies. J.H. and P.H. constructed most of the strains and conducted the phenotypic assays. Z.S. conducted most of the bioinformatics assays. J.H. and P.H. performed the SEM experiments. J.H., P.H. and L.Y. performed the animal experiments. L.Y. conducted the macrophage phagocytosis assays. L.W., Y. Xu, F.B., L.C., K.H.W., W.W. and Y.L. contributed reagents, materials or analysis tools. L.W., K.H.W. and P.H. wrote the paper with contributions from all other authors.

Corresponding authors

Correspondence to Yingchun Xu or Linqi Wang.

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Nature Microbiology thanks David Denning and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic and phenotypic analysis of Rhodosporidiobolus species NJ103 and TZ579.

a. Comparison of the ITS and D1/D2 sequences of NJ103, TZ579 and their closest relatives. b. Phylogenetic analysis of NJ103, TZ59 and R. fluvialis strains based on ITS and D1/D2 sequences. The ITS and D1/D2 sequences of nine isolates of R. fluvialis species were obtained from the GenBank database. c. Colony and cellular morphology of strains from eight Rhodosporidiobolus species. Colonies and cells were photographed after 10 days of growth on YPD plate. Scale bars, 200 μm (upper panel), 10 μm (bottom panel). Images are representative of three independent experiments conducted with similar results.

Extended Data Fig. 2 Phenotypic assays of nine strains from Rhodosporidiobolus species.

Strains of Rhodosporidiobolus species were cultured for 12 h in YPD liquid medium at 25 °C. Cells were washed with distilled water and 5-fold serially diluted and then spotted (3 μl of each dilution) onto YPD containing the indicated stress inducing reagents. Sorbitol to induce osmotic stress; NaCl, KCl, CaCl2, CdSO4 or Cr2(SO4)3 to induce salt stress or heavy-metal stress; hydrogen peroxide, tert-butyl hydroperoxide, paraquat or diamide to induce oxidative stress; CoCl2 to induce hypoxic stress; SDS, DMSO, calcofluor white or Congo red to induce cell membrane/cell wall stress; dithiothreitol to induce endoplasmic reticulum stress; methyl methanesulfonate, hydroxyurea and cisplatin to induce genotoxic stress; cycloheximide or brefeldin A to inhibit protein biosynthesis or transport; or antifungal agents (amphotericin B, fluconazole, caspofungin, nystatin, monesin, cyclosporine A or rapamycin). The abbreviations used are the same as those in the phenome heat map in Fig. 1d. Images are representative of three independent experiments conducted with similar results.

Extended Data Fig. 3 R. fluvialis could rapidly generate 5-fluorocytosine-resistant mutants.

a. 5-flucytosine resistance rate for R. fluvialis NJ103. Approximately 5 × 105 cells of R. fluvialis NJ103 or the control strain C. neoformans H99 were plated on RPMI medium containing 500 μg/mL 5-flucytosine and incubated at 25 °C for 1 week. Data are presented as the mean ± SD of three independent experiments, two-tailed, unpaired Student’s t-test. b. The R. fluvialis NJ103 MIC values for 5-flucytosine with test results read at different time points.

Source data

Extended Data Fig. 4 Genome-wide comparison of high-quality genome assemblies of two R. fluvialis strains.

Circos maps of the whole genomes of R. fluvialis NJ103 (a) and TZ579 (b). The outer ring represents the karyotype. A = GC content, B = gene density, C = repeat density. All statistics are calculated for windows of 100-Kb. Inter-scaffold interactions are displayed in the inner-most ring. c. Gene collinearity between the R. fluvialis TZ579 subgenomes. Each line connects one pair of homologous genes. d. Gene collinearity between the R. fluvialis NJ103 and TZ579 genomes. Each line connects one pair of homologous genes. e. K-mer distribution and contig frequency analyses. Seventeen k-mer depth distribution of whole-genome Illumina reads. f. R. fluvialis NJ103 and TZ579 cells were cultured on YPD plate for 1 day and FACS analysis was performed using cells stained with propidium iodide. Histogram was plotted representing 10,000 cell events.

Source data

Extended Data Fig. 5 The two subgenomes of R. fluvialis TZ579 share a high degree of sequence identity.

Distribution of amino acid sequence identity level between homologous genes from the two subgenomes of R. fluvialis TZ579.

Source data

Extended Data Fig. 6 Synteny maps of the MAT loci in R. fluvialis NJ103 and TZ579.

The mating pheromone (RHA) and receptor (STE3) genes are shown in green and orange, respectively. Additional genes that are present in the MAT locus are shown in blue.

Source data

Extended Data Fig. 7 Duplication of ERG11 gene is not the cause of the high fluconazole resistance observed in the R. fluvialis.

a. Gene deletion experiments on the ERG11-1 gene in R. fluvialis NJ103. The wild type showed a 492 bp band when amplified with internal primers (PCR1) and a 6,088 bp band that could not be digested by EcoR V when amplified with external primers (PCR2). erg11-1Δ showed no bands when amplified with internal primers, and showed a 5,854 bp band that could be digested by EcoR V to produce two bands of 4,178 bp and 1,786 bp when amplified with external primers. b. Gene deletion experiments on the ERG11-2 gene in R. fluvialis NJ103. The wild type showed a 434 bp band when amplified with internal primers (PCR1) and a 5,973bp band that could not be digested by EcoR V when amplified with external primers (PCR2). erg11-2Δ showed no bands when amplified with internal primers, and showed a 5,971 bp band that could be digested by EcoR V to produce two bands of 3,996 bp and 1,975 bp when amplified with external primers. c. MIC (left) and spotting susceptibility (right) assays of WT, erg11-1Δ, and erg11-2Δ stains. MIC values were tested as described in Materials and Methods. For spotting susceptibility assays, cells of different strains were spotted onto RPMI agar containing 100 μg/mL fluconazole (FLC). C. neoformans H99 was used as a control. Images are representative of three independent experiments conducted with similar results.

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Extended Data Fig. 8 Phenotypic assays of R. fluvialis NJ103 WT and crtYBΔ mutant strains.

Cells of different strains were cultured for 12 h in YPD liquid medium at 25 °C. Cells were washed with distilled water and 5-fold serially diluted and then spotted (3 μl of each dilution) onto YPD containing the indicated stress inducing reagents. Images are representative of three independent experiments conducted with similar results.

Extended Data Fig. 9 Incubation at 37 °C is a key factor in inducing the formation of pseudohyphal variants.

R. fluvialis cells were pre-cultured in RPMI medium at 25 °C or 37 °C in the presence or absence of CO2 for 2 days and the cells were then plated on YPD agar at 25 °C until colonies were formed. Data are presented as the mean ± SD of six independent experiments, two-tailed, unpaired Student’s t-test.

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Extended Data Table 1 Antifungal susceptibility profiles of nine strains from Rhodosporidiobolus species
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Huang, J., Hu, P., Ye, L. et al. Pan-drug resistance and hypervirulence in a human fungal pathogen are enabled by mutagenesis induced by mammalian body temperature. Nat Microbiol 9, 1686–1699 (2024). https://doi.org/10.1038/s41564-024-01720-y

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