Heterogeneity among Isolates Reveals that Fitness in Low Oxygen Correlates with Aspergillus fumigatus Virulence

Abstract

Previous work has shown that environmental and clinical isolates of Aspergillus fumigatus represent a diverse population that occupies a variety of niches, has extensive genetic diversity, and exhibits virulence heterogeneity in a number of animal models of invasive pulmonary aspergillosis (IPA). However, mechanisms explaining differences in virulence among A. fumigatus isolates remain enigmatic. Here, we report a significant difference in virulence of two common lab strains, CEA10 and AF293, in the murine triamcinolone immunosuppression model of IPA, in which we previously identified severe low oxygen microenvironments surrounding fungal lesions. Therefore, we hypothesize that the ability to thrive within these lesions of low oxygen promotes virulence of A. fumigatus in this model. To test this hypothesis, we performed in vitro fitness and in vivo virulence analyses in the triamcinolone murine model of IPA with 14 environmental and clinical isolates of A. fumigatus Among these isolates, we observed a strong correlation between fitness in low oxygen in vitro and virulence. In further support of our hypothesis, experimental evolution of AF293, a strain that exhibits reduced fitness in low oxygen and reduced virulence in the triamcinolone model of IPA, results in a strain (EVOL20) that has increased hypoxia fitness and a corresponding increase in virulence. Thus, the ability to thrive in low oxygen correlates with virulence of A. fumigatus isolates in the context of steroid-mediated murine immunosuppression.

Importance: Aspergillus fumigatus occupies multiple environmental niches, likely contributing to the genotypic and phenotypic heterogeneity among isolates. Despite reports of virulence heterogeneity, pathogenesis studies often utilize a single strain for the identification and characterization of virulence and immunity factors. Here, we describe significant variation between A. fumigatus isolates in hypoxia fitness and virulence, highlighting the advantage of including multiple strains in future studies. We also illustrate that hypoxia fitness correlates strongly with increased virulence exclusively in the nonleukopenic murine triamcinolone immunosuppression model of IPA. Through an experimental evolution experiment, we observe that chronic hypoxia exposure results in increased virulence of A. fumigatus We describe here the first observation of a model-specific virulence phenotype correlative with in vitro fitness in hypoxia and pave the way for identification of hypoxia-mediated mechanisms of virulence in the fungal pathogen A. fumigatus.

FIG 1

Two common lab strains, CEA10 and AF293, show phenotypic variation and strikingly different virulence in a triamcinolone model of IPA but not a leukopenic model of IPA. (A) Radial growth of CEA10 and AF293 on GMM in normoxia (~21% O₂) and hypoxia (0.2% O₂) at 96 h. (B) Biomass from liquid cultures grown in 1% glucose minimal medium (GMM) in normoxia and hypoxia at 48 h. ***, P = 0.0004; n.s., not significant by unpaired, two-tailed t test. (C) Ratio of biomass in hypoxia to biomass in normoxia, as calculated with values from panel B. *, P = 0.0165 by unpaired, two-tailed t test. (D) Survival analysis of CEA10 (n = 20 from 2 independent experiments) and AF293 (n = 27 from 3 independent experiments) in a triamcinolone model of IPA in CD-1 mice inoculated with 2 × 10⁶ conidia intranasally. **, P = 0.0002 by log rank test. (E) Survival analysis of CEA10 (n = 16) and AF293 (n = 17) in a chemotherapeutic model of IPA in CD-1 mice inoculated with 2 × 10⁶ conidia intranasally. n.s., not significant by log rank test. (F and G) Hematoxylin and eosin (H&E) or Gomori methenamine silver (GMS) stains of lungs 3 days postinoculation from triamcinolone-treated mice (F) and chemotherapy-treated leukopenic mice (G) inoculated with 2 × 10⁶ AF293 or CEA10 conidia. Images are representative of three mice, and all error bars indicate standard errors of the means.

FIG 2

Clinical and environmental isolates show heterogeneity in hypoxic growth. (A) Radial growth of clinical and environmental isolates on GMM under normoxia and hypoxia conditions. (B and C) Four clinical isolates, M35662, F78107, F30186, and W73763 (n = 4) (B), and the remaining environmental isolates (n = 10) (C) reveal heterogeneity in biomass from liquid culture in normoxia (~21% O₂) and hypoxia (0.2% O₂). Data are presented as the means from biological triplicates with error bars representing standard errors of the means.

FIG 3

Clinical and environmental isolates present a spectrum of virulence in the triamcinolone model of IPA but not in a chemotherapeutic model. (A and B) Data are separated into two distinct graphs for easy viewing. Triamcinolone-treated CD-1 mice inoculated with 2 × 10⁶ conidia of 02-10 (n = 12), 47-9 (n = 11), 02-30 (n = 12), 47-10 (n = 16), 47-4 (n = 16), 47-60 (n = 12), 47-7 (n = 12), 02-46 (n = 13), W72310 (n = 16), or 47-57 (n = 16) reveal a range of virulence levels across the isolates. (C) Clinical isolates (n = 16 per strain) inoculated in triamcinolone-treated CD-1 mice at 2 × 10⁶ conidia reveal reduced heterogeneity in virulence (curves not significantly different by log rank test; P = 0.2296). (D) Survival of mice treated with both cyclophosphamide and triamcinolone (chemotherapeutic model) were inoculated with 2 × 10⁶ conidia of 47-4, 02-10, or EVOL20 (n = 12 per strain). In this model, all three strains produce curves that are indistinguishable from one another (log rank, P = 0.1851 for 47-4 and 02-10).

FIG 4

Higher ratio of hypoxia-to-normoxia biomass correlates with increased virulence in the triamcinolone model of IPA. (A) Ratio of liquid culture biomass in hypoxia (0.2% O₂) to normoxia (~21% O₂) for environmental and clinical isolates. (B to D) Spearman rank order correlations calculated from H/N ratio and median survival (n = 16; r = −0.7867; P = 0.0003) for all strains (B) (clinical isolates [diamonds], lab-utilized “WT” strains [squares], and environmental isolates [circles]) and separated by environmental (n = 10; r = −0.7031; P = 0.0268) (C) and clinical (n = 6; r = −0.9429; P = 0.0390) (D) isolates. (E and F) Spearman rank order correlations and median survival for all 16 strains of hypoxia-only biomass (r = −0.6788; P = 0.0038) (E) and normoxia-only biomass (r = 0.5199; P = 0.0390) (F).

FIG 5

Serial passaging of a strain with low hypoxia fitness increases growth and virulence in triamcinolone-treated mice. (A and B) Radial growth (A) and liquid biomass (B) on GMM in normoxia (~21% O₂) and hypoxia (0.2% O₂). ***, P = 0.0001; *, P = 0.0448, by unpaired, two-tailed t test. (C) Ratio of hypoxia biomass to normoxia biomass for EVOL20 compared to the parental AF293 strain. *, P = 0.0111 by unpaired, two-tailed t test. (D) Survival analysis with a low-dose inoculum (1 × 10⁵ conidia) in the triamcinolone-treated CD-1 mice (*, P = 0.0296 by log rank test; n = 10 per strain). (E) Hematoxylin and eosin (H&E) or Gomori methenamine silver (GMS) stains of lungs 3 days postinoculation from triamcinolone-treated CD-1 mice inoculated with 1 × 10⁵ conidia. Images are representative of 3 mice.

FIG 6

Hypoxia influences a myriad of biological processes in A. fumigatus and the host. Hypoxia has been shown to impact several key factors, many of which have identified roles in virulence. Transcriptomics reveals increased expression of glycolytic enzymes as well as enzymes of the γ-aminobutyrate (GABA) shunt (62). Hypoxia has been shown to cause thickening of the cell wall with increased exposure of β-glucan (39). Ergosterol biosynthesis and iron homeostasis are also transcriptionally influenced by hypoxia (64), and increased iron content in the fungi has been demonstrated under hypoxic conditions (63). Last, the cellular response to reactive oxygen species and endoplasmic reticulum stress is altered in hypoxia as shown by increased alternative oxidase activity (AOX) (61) and a hypoxic growth defect in ΔireA (74). UPR, unfolded protein response. Hypoxia-mediated host changes include increased neutrophil recruitment, survival, and effector functions through activation of HIF-1α.