Genomic and Phenotypic Trait Variation of the Opportunistic Human Pathogen Aspergillus flavus and Its Close Relatives

Abstract

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus can cause both aspergillosis and fungal keratitis infections, but closely related species are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related species, Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified >3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three nonpathogens. We characterized secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species, whereas salinity did not affect production of any species. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate fungal disease model Galleria mellonella, we found virulence of strains of the same species varied widely; A. flavus strains were not more virulent than strains of the nonpathogens. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 h postinfection, but not at 72 h. Our work identifies variations in key phenotypic, chemical, and genomic attributes between A. flavus and its nonpathogenic relatives and reveals extensive strain heterogeneity in virulence that does not correspond to the currently established clinical relevance of these species. IMPORTANCE Aspergillus flavus is a filamentous fungus that causes opportunistic human infections, such as aspergillosis and fungal keratitis, but its close relatives are considered nonpathogenic. To begin understanding how this difference in pathogenicity evolved, we characterized variation in infection-relevant genomic, chemical, and phenotypic traits between strains of A. flavus and its relatives. We found extensive variation (or strain heterogeneity) within the pathogenic A. flavus as well as within its close relatives, suggesting that strain-level differences may play a major role in the ability of these fungi to cause disease. Surprisingly, we also found that the virulence of strains from species not considered to be pathogens was similar to that of A. flavus in both invertebrate and murine models of disease. These results contrast with previous studies on Aspergillus fumigatus, another major pathogen in the genus, for which significant differences in infection-relevant chemical and phenotypic traits are observed between closely related pathogenic and nonpathogenic species.

Keywords: Aspergillus flavus; aspergillosis; biosynthetic gene cluster; comparative genomics; evolution; fungal keratitis; genomes; genomics; pathogenicity; phenotypic variation; secondary metabolism; secondary metabolites.

FIG 1

The taxonomic identity of newly sequenced strains was consistent with Aspergillus section Flavi phylogeny. A maximum likelihood phylogeny was constructed using 2,422 single-copy orthologs from 30 strains of 20 Aspergillus species, with A. niger as the outgroup. Additional information about strains used can be found in Table 5. Numbers near branches are bootstrap values calculated from 1,000 replicates. Strains sequenced as part of this study are highlighted, with A. nomiae NRRL 6108 in orange, A. flavus in pink (NRRL 501 in light pink, NRRL 1957 in dark pink), A. arachidicola in blue (IC26645 in dark blue, IC26646 in light blue), and A. parasiticus in purple (NRRL 502 in dark purple, NRRL 2999 in light purple).

FIG 2

Strains of the same species did not differ substantially in number of predicted protein families present in their genomes. Upset plot shows the number of shared protein families for orthogroups with at least 20 proteins. Linked black circles under the bar plot indicate strains sharing orthologous protein families. Numbers above bars indicate exact numbers of shared families.

FIG 3

Strains of the same species did not differ substantially in their predicted biosynthetic gene clusters (BGCs). (A) Stacked bar plot of predicted BGCs. Each bar adds up to the total number of predicted BGCs, with the type of BGC indicated by color. (B) Synteny plot comparison of the aspergillic acid BGC from all seven strains. Arrows represent genes, and vertically shaded areas between arrows indicate sequence similarity. (C) Diagram of unique (singletons) and shared BGC families, calculated by BiGSCAPE.

FIG 4

Genetic determinants of virulence were identified in all strains; data shown here summarize presence or absence of 50 Aspergillus flavus genetic determinants of virulence. All genes were identified in all strains except aflR, norA, and hexA, which were absent in A. flavus NRRL 501. Colors represent the A. flavus experimental virulence assay for each gene (from published literature). Genes which were studied using an animal model are in shown in light or dark blue. Additional information is provided in Table S2 of our supplementary information on the FigShare website.

FIG 5

The metabolic profiles of Aspergillus flavus strains were more similar at 37°C than room temperature. The metabolomic profiles of A. flavus NRRL 501 and NRRL 1957 were almost identical at 37°C, showing very similar metabolites in the UPLC-MS analysis; most of the metabolites identified were fatty acids and ergosterol derivatives. In contrast, the profiles were significantly different at room temperature. (A) Principal-component analysis for all strains at 37°C. Circles represent both presence of metabolites and relative abundance. (B) Principal-component analysis for all strains at room temperature. Circles represent both presence of metabolites and relative abundance. (C) Hierarchical clustering of strains based on metabolite profiles at 37°C. (D) Hierarchical clustering of strains based on metabolite profiles at room temperature.

FIG 6

Iron starvation and cell wall stress impacted growth of A. flavus strains differently, but hypoxic conditions impacted A. flavus strains similarly. For each of the seven Aspergillus strains, radial growth is expressed as a ratio of colony radial diameter (in centimeters) of growth under the stress condition divided by the colony radial diameter in the control (solid minimal medium). Not all significant comparisons are shown. (A) Hypoxic stress was induced by incubating plates in 1% O₂ and 5% CO₂. Statistical significance of growth differences among species was primarily driven by growth of A. parasiticus NRRL 2999, which was significantly less impacted by hypoxia than other strains. (B) Iron starvation was induced through growth on iron-depleted substrate in the presence of gallium. All species with multiple strains exhibited strain heterogeneity, and A. parasiticus grew significantly better under iron starvation conditions than other species. Other species comparisons between species were nonsignificant. (C) Cell wall perturbation was induced by adding Congo red to the medium. A. flavus NRRL 501 was most impacted by Congo red at both concentrations, and A. parasiticus NRRL 502 was impacted the least. At both concentrations, strains of A. flavus had significantly different responses to cell wall stress (P < 0.0001). Cell wall stress also impacted A. parasiticus strains differently (P < 0.0001). Strains of A. arachidicola did not have significant growth differences. ns, not significant; *, P ≤ 0.05; **, P < 0.005; ***, P < 0.0005 (ANOVA).

FIG 7

Aspergillus flavus was not significantly more virulent than related nonpathogenic species in an invertebrate model of fungal disease. Cumulative survival of Galleria mellonella larvae inoculated with 1 × 10⁴ asexual spores (conidia) of an Aspergillus strain or a PBS control. (A) Survival for larvae inoculated with either A. flavus NRRL 1957 or NRRL 501. All pairwise comparisons between the two strains and the control group were statistically significant. (B) Survival for larvae inoculated with either A. parasiticus NRRL 2999 or NRRL 502. A. parasiticus NRRL 2999 was not statistically different from the control group, but NRRL 502 was statistically different from both the control and NRRL 2999. (C) Survival for larvae inoculated with either A. arachidicola IC26645 or IC26646. All pairwise comparisons between the two strains and the control group were statistically significant. (D) Survival for larvae inoculated with A. nomiae NRRL 6108. Survival of NRRL 6108 was statistically different from the control survival.

FIG 8

Section Flavi species infected eyes as well as A. fumigatus did in a murine model. (A) Slit-lamp images of a representative animal for each infection group. (B) Clinical score analysis of all slit-lamp images revealed reduced disease severity in the A. arachidicola group at 48 h postinfection (n = 5/group). (C) Corneal thickness measured by optical coherence tomography similarly revealed reduced structural alteration in the A. arachidicola group at 48 h (n = 5). (D) CFU analysis on resected corneas revealed indistinguishable fungal burden at 72 h postinfection between infection groups. A.arach, A. arachidicola IC26646; A.flav, A. flavus NRRL 1957; A.fum, A. fumigatus Af293; A.para, A. parasiticus NRRL 2999.