Giant transposons promote strain heterogeneity in a major fungal pathogen

Abstract

Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity which confounds diagnostic predictability. Yet the genetic mechanisms driving strain-to-strain variation remain poorly understood. Here, we determined the extent to which Starships-giant transposons capable of mobilizing numerous fungal genes-generate genetic and phenotypic variability in the opportunistic human pathogen Aspergillus fumigatus. We analyzed 519 diverse strains, including 11 newly sequenced with long-read technology and multiple isolates of the same reference strain, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales relevant for experimental reproducibility. Starship-mobilized genes encode diverse functions, including known biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in genome structure, gene content and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.

Importance: No "one size fits all" option exists for treating fungal infections in large part due to genetic and phenotypic variability among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress towards achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the human fungal pathogen Aspergillus fumigatus: giant transposons called Starships that span dozens of kilobases and mobilize fungal genes as cargo. By conducting a systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome and transcriptome levels. The Starship compendium we develop will not only help predict variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining how Starships impact clinically-relevant phenotypes, such as antifungal resistance and pathogenicity.

Keywords: Aspergillosis; Aspergillus fumigatus; secondary metabolism; strain heterogeneity; transposable element; transposon.

Figure 1:

At least 20 distinct Starships carrying hundreds of protein-coding genes vary in their presence/absence across Aspergillus fumigatus strains. A) Top: a SNP-based maximum likelihood tree of 220 A. fumigatus strains from Lofgren et al. 2022 (visualized as a cladogram for clarity), color-coded according to phylogenetic clade as defined by Lofgren et al. 2022 (10). Bottom: A heatmap depicting the presence (gray) and absence (white) of 20 high-confidence Starships in each isolate. B) Schematics and sequence alignments of the type elements from 20 high-confidence Starships, where links between schematics represent alignable regions ≥500bp and ≥80% nucleotide sequence identity and arrows represent predicted coding sequences. C) A donut chart summarizing the number of distinct types of Starships by the quality of their prediction. D) A Venn diagram indicating the number of shared and unique high-confidence Starships in the reference strains Af293 and CEA10 (Table S7). E) Pairwise genome alignments between the CEA10 isolate from BioSample SAMN28487501 and the CEA10-derived A1163 isolate sequenced in this study demonstrating Starship movement. Nebuchadnezzar h1 is present on Chromosome 6 in CEA10 (CP097568.1: 3645813–3698867) and absent at the corresponding locus in A1163 (NODE10: 188873–188877). Conversely, Nebuchadnezzar h1 is present on Chromosome 4 in A1163 (NODE6: 77737–130777) but absent from the corresponding locus in CEA10 (CP097566.1:1344957–1344961).

Figure 2:

Starships are enriched in accessory genes whose presence/absence and genomic location vary across Aspergillus fumigatus strains. All panels visualize data derived from 13 reference-quality A. fumigatus assemblies and the 20 high-confidence Starships (n = 459 elements total). A) Box-and-whisker plots summarizing the total percentage of nucleotide sequence and predicted genes carried by Starships per genome. B) Donut charts summarizing the percentages of gene orthogroups in the core, accessory, singleton, and Starship-associated compartments of the A. fumigatus pangenome (Table S10). C) Iceberg plots summarizing the genomic locations of the single best BLASTp hits (≥90% identity, ≥33% query coverage) to the cargo genes from the type elements of all 20 high-confidence Starships (left) and two individual Starships (right; Table S12). Each column in the iceberg plot represents a cargo gene and is color-coded according to the genomic location of hits (full dataset in Fig. S5).

Figure 3:

Starships and their insertion sites are distributed across all major chromosomes in the Aspergillus fumigatus genome. A) A Circos plot summarizing all inserted Starships (in red) and all genomic regions containing either an empty insertion site or a fragmented Starship (in black, along perimeter and labeled with the r prefix) in the 8 chromosomes of the A. fumigatus reference strain Af293 (Table S14). All genomic regions contain a Starship insertion in some other individual from the 519 strain population. B) Barcharts summarizing the genotypes of segregating genomic regions associated with the four most active high-confidence elements in the 519 strain population (Table S16; full dataset in Fig. S7). If an isolate did not have any Starships within a given region, it was assigned either an “empty” or “fragmented” genotype (Methods). C) A scatterplot summarizing the relationship between the number of genomic regions containing a given Starship and the total number of copies of that Starship in the 519 strain population, where each point represents one of the 20 high-confidence Starships. A line derived from a linear regression model is superimposed, with shaded 95% confidence intervals drawn in gray. D) Alignments of Tardis h1 copies +/− 50kb of flanking sequence across 14 genomic regions. Links between schematics represent alignable regions ≥1000bp and ≥90% nucleotide sequence identity.

Figure 4:

Starships mobilize adaptive traits and generate allelic diversity among Aspergillus fumigatus strains. Schematics and alignments of: A) Starship Lamia h2 and h4, which carry the biosynthetic gene cluster (BGC) encoding the polyketide secondary metabolite fumigermin (45), shown inserted at 3 independent sites. B) Starship Gnosis h2 carrying the BGC encoding the terpene secondary metabolite fumihopaside A (38), shown inserted at 2 independent sites. C) a large region on Chromosome 3 previously identified as an idiomorphic BGC (36) containing multiple segregating Starship insertions and various combinations of putative BGCs, including a a non-reducing polyketide synthase (NR-PKS) BGC carried by Starship Osiris h4. Starships from the Osiris navis specifically insert in 5S rDNA sequence and are predicted to fragment it. D) Eight Starships in the reference strains Af293 and CEA10 that all carry homologs of biofilm architecture factor A (bafA)(33). Starship Nebuchadnezzar h1 (Neb. h1) carries bafA as part of the H_AAC (hrmA-associated gene cluster), while Starship navis10-var35 carries bafB as part of H_BAC and Starship Osiris h3 carries bafC as part of H_CAC (33). Only a portion of Starship Lamia h4 is visualized for figure legibility. All data for A-D was collected from the 519 A. fumigatus strain population (Table S5). Links between schematics represent alignable regions ≥5000bp and ≥95% nucleotide sequence identity and arrows represent predicted coding sequences. Abbreviations: polyketide synthase (PKS); non-ribosomal peptide synthetase (NRPS); highly reducing (HR).

Figure 5:

A) A heatmap of transcript abundances (log₁₀TPM) of Starship captain tyrosine recombinase genes (top) and genes within the hrmA-associated cluster (HAC; bottom), collapsed across treatment replicates, for 11 RNAseq studies from A. fumigatus Af293. B) Results from differential expression tests for select Starships, treatment categories, and strain combinations. Differentially expressed genes (DEGs) based on a single study are shown as log₂ fold-change (log₂FC) in black with standard error bars, whereas DEGs identified across multiple studies are represented with summarized log₂FC values from a random effects model (REM) and coloured by “sign-consistency”, the number of studies that reported DEGs in the positive (+1) or negative (−1) direction, centered around 0. Labels in bold font represent DEGs that are significantly differentially expressed in more than one study. C) The results of a weighted gene co-expression network analysis (WGCNA) constructed from 14 “antifungal”, “infection”, and “nutrient” RNAseq studies represented using UMAP clustering (89) based on the co-expression eigengene values for all genes in the A. fumigatus Af293 genome. Non-Starship genes present within modules of interest are shown in blue, while all Starship genes are shown in red, with those genes within modules of interest having a black outline. D) Genes within modules that were significantly associated with samples from specific treatment categories or studies were used to construct sub-networks which contained the top 10 edges in the network that were made between any pair of genes or any gene and a Starship captain. The connections between genes (edges) are based on the topological overlap matrix (TOM) for each module, and have been 0–1 scaled. E) Boxplots of eigengene values from WGCNA, akin to a weighted average expression profile, indicate the extent of co-expression (correlation) of the genes present within each module. Pairwise comparisons of module eigengene values determined if a module was significantly associated with samples from specific treatment categories or studies (Fig. S16).

Figure 6:

Contributions of Starships to strain heterogeneity in Aspergillus fumigatus.