Chromosome-Level Genome Assembly of a Human Fungal Pathogen Reveals Synteny among Geographically Distinct Species

Abstract

Histoplasma capsulatum, a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent hosts. Histoplasma is endemic in the Ohio and Mississippi River Valleys in the United States and is also distributed worldwide. Previous studies have revealed at least eight clades, each specific to a geographic location: North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B), Eurasian, Netherlands, Australian and African, and an additional distinct lineage (H81) comprised of Panamanian isolates. Previously assembled Histoplasma genomes are highly fragmented, with the highly repetitive G217B (NAm 2) strain, which has been used for most whole-genome-scale transcriptome studies, assembled into over 250 contigs. In this study, we set out to fully assemble the repeat regions and characterize the large-scale genome architecture of Histoplasma species. We resequenced five Histoplasma strains (WU24 [NAm 1], G217B [NAm 2], H88 [African], G186AR [Panama], and G184AR [Panama]) using Oxford Nanopore Technologies long-read sequencing technology. Here, we report chromosomal-level assemblies for all five strains, which exhibit extensive synteny among the geographically distant Histoplasma isolates. The new assemblies revealed that RYP2, a major regulator of morphology and virulence, is duplicated in G186AR. In addition, we mapped previously generated transcriptome data sets onto the newly assembled chromosomes. Our analyses revealed that the expression of transposons and transposon-embedded genes are upregulated in yeast phase compared to mycelial phase in the G217B and H88 strains. This study provides an important resource for fungal researchers and further highlights the importance of chromosomal-level assemblies in analyzing high-throughput data sets. IMPORTANCE Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe. Phylogenetically distinct species of Histoplasma have been isolated and sequenced. However, for the commonly used strains, genome assemblies have been fragmented, leading to underutilization of genome-scale data. This study provides chromosome-level assemblies of the commonly used Histoplasma strains using long-read sequencing technology. Comparative analysis of these genomes shows largely conserved gene order within the chromosomes. Mapping existing transcriptome data on these new assemblies reveals clustering of transcriptionally coregulated genes. The results of this study highlight the importance of obtaining chromosome-level assemblies in understanding the biology of human fungal pathogens.

Keywords: Histoplasma capsulatum; genome assembly; long-read sequencing.

FIG 1

Histoplasma genomes are assembled at the chromosomal level. The newly assembled chromosomes of Histoplasma genomes are shown as black horizontal lines. Chromosomes or contigs are plotted largest to smallest. Telomeres are shown with black vertical lines at the end of each chromosome when present. Contigs from the previous assemblies are overlaid using alternating colors. Repeat regions (LTR transposons) are indicated with purple blocks below each chromosome. Genes of interest are displayed above each chromosome.

FIG 2

The Histoplasma genomes are highly syntenic. Chromosomes are sorted largest to smallest, and regions of synteny are colored according to the WU24 chromosomes in other strains. Purple bars below the chromosomes indicate the repetitive (transposon) regions.

FIG 3

G186AR strain contains two duplicated regions. (A) Coverage for mapping G186AR Illumina reads onto the current G184AR assembly. (B, C) BLASTN-based dotplots for the duplicated regions. (D, E) Coverage for mapping G184AR and G186AR ONT reads onto the current G184AR assembly.

FIG 4

Transposon-embedded genes show yeast-phase enriched expression patterns. (A) Schematic of transcript classification by proximity to LTR transposon; viz., “LTR” (orange), transcript overlaps transposon annotation; “in” (blue), transcript is between two transposon annotations within 50 kb of each other; “near” (green), transcript is within 50 kb of a transposon annotation; “other” (yellow), transcript does not fall into any of the previous categories. (B) Violin plots of yeast/hyphae expression ratios from (18) or (35); *, taken from the Kallisto analysis of (12), colored according to the same scheme as panel A. Significant differences by Wilcoxon rank sum test are indicated: **, P < 2.2e-16, *, P = 4.7e-15.