Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis

Abstract

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Figure 1. Genome organization of P. brasiliensis.

Alignment of Pb18 supercontigs to the five chromosomes from the optical map are shown. Tracks (left to right) show gene frequency (blue), transposon frequency (red), synteny with P. lutzii (yellow), and synteny with Histoplasma (purple).

Figure 2. Phylogeny of the dimorphic fungi, shared gene content, and clade-specific carbohydrate and protein metabolism.

The phylogeny was inferred by maximum likelihood using RAxML from a concatenated alignment of 309 single copy orthologs shared across all taxa. All nodes on the phylogeny were supported with bootstrap values of 100%. Colored bars indicate orthologs shared across all taxa (green), orthologs shared across some taxa (yellow), Paracoccidioides-specific (blue) and species-specific genes (red). Numbers of genes for each taxon involved in carbohydrate metabolism and peptidase activity are shown. Categories with asterisks at the bottom of the column are significantly enriched in the non-dimorphic fungi relative to the dimorphic fungi. Carbohydrate active enzyme (CAZY) categories include glycoside hydrolases (GH), glycosyltransferases (GL), carbohydrate esterases (CE), and pectate lysases (PL). Numbers of carbohydrate transporters (CT) are also listed. Peptidase (MEROPS) categories include aspartic (A), cysteine (C), glutamic (G), metallo (M), serine (S) and threonine (T). The ratio of MEROPS genes to CAZY genes for each genome is shown on the right.

Figure 3. Growth by Uncinocarpus reesii on carbohydrates, amino acids, and peptides as the sole carbon source after a 7-day period.

The experiment was done in triplicate; circles represent the normalized median absorbance at 590 nm and bars represent standard error. The dotted line indicates 0 normalized absorbance. Numerical growth values for all compounds can be found in Table S9.

Figure 4. Phylogenetic relationships of FunK1 kinases estimated by maximum likelihood with RAxML .

Lineage-specific expansions can be seen in Paracoccidioides (red) and Coccidioides (blue).

Figure 5. Conservation of FunK1 functional domains.

The active site (domain VIB) and substrate specificity region (domain VIII) are shown for Pezizomycotina, including the dimorphic fungi (FunK1 Pezi), the mushroom Coprinopsis cinerea (Ccin), and ‘typical’ eukaryotic protein kinases (ePKs). The domain numbering follows standard protein kinase nomenclature . FunK1 proteins share a unique serine at the active site position 8 (green triangle) compared to a lysine found in eukaryotic protein kinases. Additionally, FunK1 proteins have glutamate residues at positions VIB 9 and VIII 10 (red triangles), suggesting the recognition of arginine-containing substrates.