Genomic and Phenotypic Heterogeneity of Clinical Isolates of the Human Pathogens Aspergillus fumigatus, Aspergillus lentulus, and Aspergillus fumigatiaffinis

Abstract

Fungal pathogens are a global threat to human health. For example, fungi from the genus Aspergillus cause a spectrum of diseases collectively known as aspergillosis. Most of the >200,000 life-threatening aspergillosis infections per year worldwide are caused by Aspergillus fumigatus. Recently, molecular typing techniques have revealed that aspergillosis can also be caused by organisms that are phenotypically similar to A. fumigatus but genetically distinct, such as Aspergillus lentulus and Aspergillus fumigatiaffinis. Importantly, some of these so-called cryptic species are thought to exhibit different virulence and drug susceptibility profiles than A. fumigatus, however, our understanding of their biology and pathogenic potential has been stymied by the lack of genome sequences and phenotypic profiling of multiple clinical strains. To fill this gap, we phenotypically characterized the virulence and drug susceptibility of 15 clinical strains of A. fumigatus, A. lentulus, and A. fumigatiaffinis from Spain and sequenced their genomes. We found heterogeneity in drug susceptibility across species and strains. We further found heterogeneity in virulence within each species but no significant differences in the virulence profiles between the three species. Genes known to influence drug susceptibility (cyp51A and fks1) vary in paralog number and sequence among these species and strains and correlate with differences in drug susceptibility. Similarly, genes known to be important for virulence in A. fumigatus showed variability in number of paralogs across strains and across species. Characterization of the genomic similarities and differences of clinical strains of A. lentulus, A. fumigatiaffinis, and A. fumigatus that vary in disease-relevant traits will advance our understanding of the variance in pathogenicity between Aspergillus species and strains that are collectively responsible for the vast majority of aspergillosis infections in humans.

Keywords: Aspergillus; antifungal drug susceptibility; cryptic species; drug resistance; genetic determinants of virulence; genomics; strain heterogeneity; virulence.

FIGURE 1

High heterogeneity in drug susceptibility profiles among Spanish strains of three closely related Aspergillus pathogens. (A) Principal component analysis (PCA) carried out for antifungal susceptibility testing. PC1 (Dim1) explains most of the variation (37.2% of the variation) and is able to separate A. fumigatus from other two species, whereas an overlap is observed in cryptic species (A. lentulus and A. fumigatiaffinis). (B) Antifungal susceptibility testing was carried out using the EUCAST reference microdilution method. The minimum inhibitory concentration (MIC) was obtained for AMB, VCZ, PCZ, and ICZ and the minimum effective concentration (MEC) was obtained for TRB, CPF, MCF, and AND. A lower scale is shown for echinocandins (bottom panel). Antifungal classes are A: polyenes; B: azoles; C: allylamines; D: echinocandins. AMB, amphotericin B; ICZ, itraconazole; VCZ, voriconazole; PCZ, posaconazole; CPF, caspofungin; MCF, micafungin; AND, anidulafungin; TRB, terbinafine.

FIGURE 2

High heterogeneity of virulence levels among Spanish strains of three closely related Aspergillus pathogens. We found significant heterogeneity in the survival curves between strains within A. fumigatiaffinis (A), A. lentulus (B), and A. fumigatus (C) (Benjamini and Hochberg adjusted p-values using the log-rank test are shown). We also tested differences between species (considering each strain as a biological replicate), and found that the virulence profiles of Spanish strains of all three species are not significantly different (D) (p-value using the log-rank test is shown).

FIGURE 3

Genomics of the three closely related Aspergillus pathogens. (A) Genome-scale phylogeny of the section Fumigati species used in this study and additional species with sequenced genomes. The A. viridinutans clade is presented as a sister clade. Spanish strains sequenced in this work are colored in red (A. fumigatus), blue (A. lentulus) and black (A. fumigatiaffinis). The newly sequenced A. fumigatiaffinis strains form a separated group that is closely related to A. novofumigatus. All A. lentulus strains in this work group together and share an ancestor with A. lentulus IFM54703, the only sequenced strain in this species to date. The A. fumigatus strains sequenced in this work form different internal groups in the clade with other strains in the species (e.g., strains CNM-CM8714 and CNM-CM8812 group together and strains CNM-CM8686 and CNM-CM8689 form another group). (B) A. fumigatiaffinis and A. lentulus shares the highest number of common orthogroups and A. fumigatiaffinis displays the highest number of species-specific orthogroups. We considered species-specific orthologs those that were present in at least one strain of a given species, with no representative from another species. (C) Orthogroups shared by all and “all but one” strains are the most frequent in three closely related Aspergillus pathogens. A. lentulus, A. fumigatus, and A. fumigatiaffinis have 9,008, 8,321, and 9,423 orthologous genes present in all strains, respectively. The five largest combinations of orthogroups are shown. As expected, the most frequent combination of orthogroups are those in all strains but one.

FIGURE 4

Changes in important genes related to antifungal susceptibility in the three Aspergillus pathogens. (A) Products of genes related to antifungal resistance, Cyp51A (azoles) and Fks1 (echinocandins), display species- and strain-specific polymorphisms. Only the positions with changes in at least one strain are shown (substitutions or insertions/deletions). Blue triangles highlight important amino acid changes in positions 98, 121, and 289 in Cyp51A and in hot spot 2 (HS2) of Fks1. Red triangles indicate insertions/deletions. (B) Promoter region of the cyp51A gene displays strain-specific mutations among Spanish strains of three closely related Aspergillus pathogens. Well-known tandem repeat regions in antifungal-resistant strains of A. fumigatus are shown between positions 70–140 in the alignment (i.e., TR34 and TR46, observed in CNM-CM8714 and CNM-CM8057, respectively, delimited by two blue arrows in upper part). Polymorphisms in cryptic species were also identified, for instance, the short deletions exclusively found in the cryptic species (either in A. fumigatiaffinis or A. lentulus) around positions 230–250. Red arrow and red font indicate the start codon. (C) Phylogeny of Cyp51 gene family (protein sequences) reveals three different members (Cyp51A, Cyp51B, and the putative Cyp51C) in A. fumigatiaffinis. Ultrafast Boostrap Approximation and SH-aLRT support values are shown.

FIGURE 5

Orthogroups for virulence determinants reveals variable number of paralogs among the three closely related Aspergillus pathogens. We searched for 215 known genetic determinants of virulence in A. fumigatus Af293 in the species of interest and found they were grouped into 203 orthogroups. 146/203 were found in single copy across all strains and are not shown here. The cladogram above the species reflects similarities between strain presence/absence patterns. A. fumigatus Af293 shows a different pattern compared to other strains of A. fumigatus, grouping with one of the A. lentulus strains (CNM-CM8927). This may reflect the phylogenetic divergence of A. fumigatus strain Af293 from other species members. Conidial pigment polyketide synthase alb1 (Afu2g17600) is one of the genetic determinants of virulence with highest number of copies in cryptic species (n = 7) when compared to A. fumigatus strains (n = 4). Gene identifiers in A. fumigatus Af293 are highlighted in bold. Color scale indicates the number of genes found within the orthogroup.