Pathogenic Allodiploid Hybrids of Aspergillus Fungi: Current Biology | Current Biology
Jacob L. Steenwyk, Abigail L. Lind, Laure N.A. Ries, Thaila F. dos Reis, Lilian P. Silva, Fausto Almeida, Rafael W. Bastos, Thais Fernanda de Campos Fraga da Silva, Vania L.D. Bonato, André Moreira Pessoni, Fernando Rodrigues, Huzefa A. Raja, Sonja L. Knowles, Nicholas H. Oberlies, Katrien Lagrou, Gustavo H. Goldman, Antonis Rokas
Six Clinical Isolates Previously Characterized as A. nidulans Are Diploid
To gain insights into the genetic diversity of clinical isolates of A. nidulans, we analyzed 7 isolates from patients with different pulmonary diseases and compared them to haploid (A4) and the laboratory-induced diploid (R21/R153) reference strains of A. nidulans (Table 1). Using microscopy-based and/or molecular biology methods, all 7 isolates had previously been identified as A. nidulans, all are similar in appearance when grown in standard laboratory conditions (Figure S1), and two were analyzed as A. nidulans isolates in previous experimental studies [
The fungal exopolysaccharide galactosaminogalactan mediates virulence by enhancing resistance to neutrophil extracellular traps.
]. Examination of DNA content revealed that 6/7 isolates were more similar to the diploid A. nidulans R21/R153 strain than to the haploid A. nidulans A4 strain, suggesting that these 6 isolates are diploid (Figure 1A). The volume of asexual spores (conidia) is frequently proportional to the DNA content of the nucleus [
Deoxyribonucleic acid content of haploid and diploid Aspergillus conidia.
], and examination of their size showed that the same 6 isolates and the diploid A. nidulans strain have significantly larger spores than isolates with haploid genomes (p Figure 1B).
Table 1Isolates Used in This Study
The superscript “T” indicates that the strain is the type strain of the species. See also Figures 1 and S1 and Table S3.
To gain further insight into the genomes of the 6 diploid isolates and 1 haploid isolate, we sequenced them and compared their genome size and gene number with those of representative Aspergillus species known to be haploid (A. clavatus NRRL 1, A. flavus NRRL 3357, A. fumigatus Af293, A. nidulans A4, A. niger CBS 513.88, A. sydowii CBS 593.65, and A. versicolor CBS 583.65) [
Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.
]. We found that the genomes and gene numbers of the diploids were significantly larger (average genome size = 69.09 ± 5.68 Mb; average gene number = 21,321.57 ± 2,342.13) than those of the haploid representative Aspergillus species (average genome size = 32.62 ± 3.05 Mb; average gene number = 11,330.75 ± 1,838.70; Figure 1C; figshare image 1, 10.6084/m9.figshare.8114114; p Table S1). Similarly, examination of gene content completeness revealed a significantly higher number of duplicated near-universally single-copy (BUSCO) genes in the diploids relative to representative Aspergillus species (Figure 1C; p = 0.001; Wilcoxon rank sum test). Thus, we concluded that 6/7 clinical isolates are diploids.
Diploid Clinical Isolates Are Aspergillus latus, a Species of Hybrid Origin
To examine the evolutionary origin of the clinical isolates, we retrieved their calmodulin and β-tubulin sequences and performed molecular phylogenetic analysis in the context of sequences of the two genes from all available taxa in the section Nidulantes phylogeny [
Aspergillus section Nidulantes (formerly Emericella): polyphasic taxonomy, chemistry and biology.
]. We found that the haploid clinical isolate 4060 had nearly identical calmodulin and β-tubulin sequences to other strains of A. spinulosporus and formed a monophyletic group with them, suggesting that it belongs to A. spinulosporus (Figure 2A; figshare: 10.6084/m9.figshare.8114114). Notably, we found that all 6 diploid clinical isolates contained two different copies of the calmodulin and β-tubulin genes; one copy was nearly identical to A. spinulosporus sequences, whereas the other was nearly identical to A. latus ones (Figure 2A; figshare: 10.6084/m9.figshare.8114114), raising the hypothesis that the diploids originated from interspecific hybridization between A. spinulosporus and A. latus.
To test this hypothesis, we analyzed the genome of A. spinulosporus strain NRRL 2395T [
A robust phylogenomic time tree for biotechnologically and medically important fungi in the genera Aspergillus and Penicillium.
] and sequenced the type strain NRRL 200T of A. latus. Examination of the DNA content and asexual spore size of these two species’ genomes showed that A. spinulosporus NRRL 2395T had similar values as clinical isolate 4060 (Figure 1) and was also placed in the same phylogenetic clade (Figure 2A); these findings confirm that clinical isolate 4060 belongs to A. spinulosporus and that A. spinulosporus is one of the parental species involved in the interspecific hybridization event that gave rise to the 6 clinical isolates.
In contrast, the DNA content and spore size of the genome of the type strain of A. latus NRRL 200T were similar to those of the 6 diploid clinical isolates (Figure 1). Furthermore, like the 6 clinical isolates, A. latus NRRL 200T also had two copies of the calmodulin and β-tubulin gene sequences; one copy was nearly identical to A. spinulosporus sequences, and the other copy was closely related to but distinct from A. quadrilineatus sequences (Figure 2A; figshare: 10.6084/m9.figshare.8114114). These results suggest that the 6 diploid clinical isolates belong to A. latus and that A. latus is an allodiploid hybrid species that originated via interspecific hybridization between A. spinulosporus and a species closely related to A. quadrilineatus.
We tested this hypothesis by performing two different sets of analyses. In the first set of analyses, we sequenced, assembled, and annotated the genome of the type strain (NRRL 201T) of A. quadrilineatus. Consistent with our hypothesis that A. latus is an allodiploid hybrid, we found that the A. quadrilineatus genome contains a single copy of the calmodulin and β-tubulin gene sequences, that these sequences form a monophyletic group with their orthologous sequences retrieved from the genome of a different A. quadrilineatus strain (strain CBS 853.96; https://www.ncbi.nlm.nih.gov/sra/SRX5010607), and that the A. quadrilineatus sequences form a sister group with one of the two sets of the A. latus sequences (Figure 2A).
In the second set of analyses, we estimated the sequence divergence of each gene in the genomes of the 7 clinical isolates as well as of A. latus NRRL 200T and A. quadrilineatus NRRL 201T from A. spinulosporus NRRL 2395T. Under this analysis, the genomes of non-hybrids are expected to show a unimodal distribution (e.g., see control non-hybrid, A. fumigatus; Figure 2Bi, left), whereas the genomes of hybrids are expected to show a bimodal distribution whose two modes correspond to the distributions of gene sequence divergence values from each parental genome (e.g., see control hybrid, Zygosaccharomyces parabailii; Figure 2Bi, right). We found that the haploid A. spinulosporus 4060 clinical isolate and A. quadrilineatus NRRL 201T had unimodal distributions reflecting a history devoid of hybridization, and the 6 diploid clinical isolates and A. latus NRRL 200T had bimodal distributions consistent with allodiploidy (Figure 2Bii). Furthermore, all 6 diploid isolates and A. latus NRRL 200T contained nearly equal percentages of A. spinulosporus and A. quadrilineatus-like genes (51.43% ± 0.74% A. spinulosporus: 48.57% ± 0.74% A.-quadrilineatus-like; Figure 2B, pie charts), including nearly the full sets of A. spinulosporus and A.-quadrilineatus-like secondary metabolic gene clusters (Table S2; figshare: 10.6084/m9.figshare.8114114). Putative homeologs exhibited an average nucleotide sequence divergence of 7.15% ± 0.03%, a value very similar to the average divergence of 7.14% observed between the 8,523 orthologs of A. spinulosporus NRRL 2395T and A. quadrilineatus NRRL 201T (Figure S2). These two sets of analyses confirm that the 6 diploid clinical isolates belong to A. latus and that A. latus is an allodiploid hybrid species that originated via interspecific hybridization between A. spinulosporus and a species closely related to A. quadrilineatus.
We next assessed whether the allodiploid hybrid species A. latus stems from a single hybridization event by comparing the genome-scale phylogenies constructed from the A. spinulosporus and the A.-quadrilineatus-like parental genomes of the A. latus isolates (Figure S3). We found that the relationships of the A. latus isolates differed between the two phylogenies (Figure S3). This incongruence may stem from biological reasons (e.g., multiple hybridization events or recombination between the two parental genomes). However, the low level of support for relationships among isolates, especially in the phylogeny from the A. spinulosporus parental genome (Figure S3A), means that we cannot exclude the possibility that the two phylogenies are not statistically significantly different. To test this, we evaluated whether the two topologies were statistically different using the approximately unbiased topology constraint test [
An approximately unbiased test of phylogenetic tree selection.
]. Using the A. spinulosporus data matrix, we found that we could not reject the topology inferred based on the A.-quadrilineatus-like data matrix as statistically inferior; similarly, we could not reject the A. spinulosporus topology when we using the A.-quadrilineatus-like data matrix (p = 0.50 for both tests). These results are consistent with the hypothesis that the two parental genomes of A. latus share the same evolutionary history.
To provide more insight on whether the two parental genomes A. latus hybrids undergo recombination, we first examined whether A. latus hybrids undergo the sexual cycle to produce sexual spores (ascospores). We found that all A. latus hybrids produce sexual spores and that the viability of these spores is similar to that of the sexual spores of their parental species (figshare image 2, 10.6084/m9.figshare.8114114). We next examined whether any contigs in the genomes of A. latus isolates had evidence of recombination events. Examination of long (≥100 kb) contigs revealed that most genes in most contigs contained genes from one or the other parent and that very few contigs contained substantial percentages of genes from both parents (Figure S4). For example, only an average of 2.67% ± 0.71% contigs per A. latus hybrid genome contained substantial percentages of genes from both parental species (Figure S4). However, interpretation of these data is challenging for two reasons. First, the high sequence similarity of the two parental genomes means that identification of parent of origin for highly conserved genes is difficult and likely explains the sporadic presence of one or a handful of genes from one parent in contigs comprised mostly of genes from the other parent. Second, alignment of several of the contigs that contain large numbers of genes from both parents to the A. nidulans A4 reference genome suggests that they are often patchworks of A. nidulans contigs; for example, a long stretch of an A. latus contig that matches one parent is homologous to A. nidulans chromosome 5 and the rest of the contig, which matches the other parent, is homologous to A. nidulans chromosome 7. The absence of A. latus contigs that contain genes from both parental species and map to a single A. nidulans chromosome suggests that A. latus contigs that contain genes from both parental species may stem from assembly artifacts. These results suggest that A. latus hybrids likely undergo little to no recombination between the two parental genomes.
The Genomes of the A. latus Allodiploid Hybrid Isolates Are Stable
To assess the genome stability of the A. latus isolates, we began by examining the gene content completeness of each parental genome. We found that each parental genome contained nearly all of the 1,315 BUSCO genes from the fungal phylum Ascomycota (93.50% ± 1.88% A. spinulosporus and 94.30% ± 0.40% A. quadrilineatus like; figshare image 3, 10.6084/m9.figshare.8114114). Considering that gene content completeness from each parent is only slightly below that from haploid representative species (average = 96.33% ± 0.78%; min = 95.70%, A. spinulosporus; max = 97.3%, A. nidulans A4), these results suggest little loss of each parental genome by either aneuploidy or loss of heterozygosity events.
To further test this observation genome-wide, we examined the fraction of orthologous genes shared between the A. spinulosporus NRRL 2395T strain and the parental genomes of A. latus isolates that stem from A. spinulosporus. We found that the A. spinulosporus parental genomes from A. latus hybrids shared a minimum of 9,227/9,611 orthologous genes with A. spinulosporus NRRL 2395T; the sole exception was A. latus NRRL 200T, which shared 8,749 orthologs (figshare: 10.6084/m9.figshare.8114114). Interestingly, the A. spinulosporus parental genome of A. latus NRRL 200T shows by far the highest evolutionary rate in our phylogenomic analyses (Figure S3), suggesting that the A. spinulosporus parental genome of this strain may be more genetically unstable than those of the clinical isolates.
Examination of loss of heterozygosity and aneuploidy events in A. latus genomes revealed relatively little evidence for either. Two isolates contained loss of heterozygosity regions. The A. latus NRRL 200T strain contained an ~1.2-Mb region homologous to the end of A. nidulans chromosome VIII that contained two copies of the A.-quadrilineatus-like parental genome and lacked a copy of the A. spinulosporus genome. This region contains several BUSCO genes, which explains why this strain has a higher proportion of missing BUSCO genes from the A. spinulosporus parental genome compared to the 6 clinical isolates (figshare image 3, 10.6084/m9.figshare.8114114). The clinical isolate MO46149 contained an ~1-Mb region homologous to the beginning of A. nidulans chromosome V with two copies of the A. spinulosporus genome and lacked a copy of the A.-quadrilineatus-like genome (Data S1). We did not find evidence for chromosome-scale aneuploidies (Data S1).
Lastly, by comparing the gene lengths of homeolog pairs as a signature of pseudogenization, we found evidence of pseudogenization in at least one gene among an average of 11.67% ± 0.004% homeologs (Figure S2). These results suggest that the genomes of the A. latus allodiploid hybrids are generally stable, that loss of heterozygosity is rare, that major aneuploidies have not occurred, and that both genes in ~88% of homeolog pairs are intact.
Hybrids Exhibit Wide Variation for Infection-Relevant Traits
To examine variation in infection-relevant traits between the hybrid isolates, one of their known parental species (A. spinulosporus), the closest known relative of their other parental species (A. quadrilineatus), and the species they were originally identified as (A. nidulans), we tested the virulence of all isolates in an invertebrate disease model and phenotypically characterized them across a wide variety of infection-relevant conditions, including interactions with host immune cells, drug susceptibility, oxidative stress, iron starvation, and temperature stress (Figures 3 and S5). Principal-component analysis (PCA) and examination of the traits with the greatest contributions to the observed variance among isolates revealed two major findings. First, the 7 A. latus hybrids exhibit substantial heterogeneity in their phenotypic profiles (Figure 3A). Second, the A. latus hybrids are phenotypically distinct from A. nidulans and their parental species but are more similar to A. spinulosporus than to A. quadrilineatus (Figure 3A). Among the traits tested, those with the largest contributions to the observed variation among isolates and species were interactions with host immune cells, antifungal drug susceptibility, and oxidative stress resistance (figshare image 3A and 4, 10.6084/m9.figshare.8114114). Here, we discuss exemplary phenotypic traits that highlight these two major findings (see Figure S5 for other phenotypes).
Phenotypic variation or strain heterogeneity among A. latus hybrids was observed for nearly every trait measured (Figures 3 and S5). For example, examination of virulence in the invertebrate greater wax moth (Galleria mellonella) model revealed substantial variation among isolates (p Figure 3B). Specifically, we observed that A. latus isolate ASFU1710 was the most virulent and A. latus isolate MO46149 was the least virulent. Similarly, we found substantial strain heterogeneity in how much lytic and non-lytic NETosis (a process where neutrophils release neutrophil extracellular traps, or NETs, to kill microbes) [
Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens.
] was stimulated by A. latus hybrid isolates (Figure 3C). For example, A. latus NIH did not substantially stimulate NETosis although A. latus ASFU1710 did. Strain heterogeneity was less pronounced for other traits, such as hyphal viability, drug susceptibility, and oxidative stress, yet all exhibited variation across isolates (Figures 3D–3F). One A. latus isolate that was consistently different from the rest is MO46149; for example, this isolate was twice as susceptible to hyphal killing by neutrophils compared to the other A. latus isolates and it was the isolate most sensitive to the antifungal caspofungin, as well as the isolate most tolerant to the oxidative stressor paraquat.
Phenotypic variation was also pronounced when we compared infection-relevant traits between A. latus, its two parental species, and A. nidulans (Figure 3A). For example, we found that A. latus hybrids (and A. quadrilineatus) were less susceptible to killing by neutrophils compared to A. spinulosporus (Figure 3D). In contrast, we found A. latus isolates (and A. spinulosporus) differed in their susceptibility to low doses of caspofungin (Figure 3E) or to high doses of oxidative stress (Figure 3F) from A. quadrilineatus and A. nidulans.
In summary, we found substantial heterogeneity among A. latus hybrid isolates as well as between A. latus and closely related or parental species for diverse infection-relevant traits. Generally, A. latus hybrids are more similar to their known parent, A. spinulosporus, compared to the closest known relative of their other parent, A. quadrilineatus. Importantly, A. latus hybrids are also phenotypically distinct from A. nidulans, the species they were originally misdiagnosed as.