Genome Sequencing and Comparative Genomics Analysis Revealed Pathogenic Potential in Penicillium capsulatum as a Novel Fungal Pathogen Belonging to Eurotiales

Abstract

Penicillium capsulatum is a rare Penicillium species used in paper manufacturing, but recently it has been reported to cause invasive infection. To research the pathogenicity of the clinical Penicillium strain, we sequenced the genomes and transcriptomes of the clinical and environmental strains of P. capsulatum. Comparative analyses of these two P. capsulatum strains and close related strains belonging to Eurotiales were performed. The assembled genome sizes of P. capsulatum are approximately 34.4 Mbp in length and encode 11,080 predicted genes. The different isolates of P. capsulatum are highly similar, with the exception of several unique genes, INDELs or SNPs in the genes coding for glycosyl hydrolases, amino acid transporters and circumsporozoite protein. A phylogenomic analysis was performed based on the whole genome data of 38 strains belonging to Eurotiales. By comparing the whole genome sequences and the virulence-related genes from 20 important related species, including fungal pathogens and non-human pathogens belonging to Eurotiales, we found meaningful pathogenicity characteristics between P. capsulatum and its closely related species. Our research indicated that P. capsulatum may be a neglected opportunistic pathogen. This study is beneficial for mycologists, geneticists and epidemiologists to achieve a deeper understanding of the genetic basis of the role of P. capsulatum as a newly reported fungal pathogen.

Keywords: Penicillium capsulatum; comparative genomics; genome sequencing; novel fungal pathogen.

FIGURE 1

Phylogenetic analysis of Eurotiales based on all of the single-copy orthologues among 40 whole genome sequences. Maximum Likelihood tree was inferred by RAxML based on single-copy orthologs with best model JTT+I+G+F identified using ProtTest. The tree was rooted with Mucor circinelloides and Rhizopus oryzae. Bootstrap percentages of the maximum likelihood analysis were presented at the nodes.

FIGURE 2

Phylogenetic analysis based on 43 virulence-related genes shared among 22 related species. The heat map on the right represented the number of virulence-related genes in each strain.

FIGURE 3

Venn diagram showing shared orthologous groups among genomes of Talaromyces marneffei. P. capsulatum. Aspergillus fumigatus. P. paxilli. The numbers marked behind “Groups” in each region represent for orthologous groups, and the numbers following C1∼C4 means the counts of genes in the strains.

FIGURE 4

Genome comparison of strains CBS 134186 and ATCC 48735. (A) Collinearity plot. The forward matches were displayed in red, while the reverse matches were displayed in blue. (B) Difference between two strains. The left part of the circle indicates CBS 134186 and the right part indicates ATCC 48735. Rings from the outermost to the center: (1) Scale marks of the genome. (2) SNPs. Bases of A, T, G, and C were represented in red, blue, green, and yellow. (3) GC content. The ratio of GC sites per 10 kb were plotted in red (> = 50%) and green (<50%). (4) Special genes. Blastp similarity ranged from 100% (blue) to 0% (red). Genes with lower sequence similarity tended to hotter orange and red tones. (5) INDELs in blue links. (C) Mauve plot of two strains. Collinear contigs were sorted in proper order.

FIGURE 5

Comparison of the differences of morphological characteristics between two P. capsulatum strains CBS 134186 and ATCC 48735, which were cultured in 25°C on PDA medium. (magnification ×63 for (A,D), magnification ×100 for (B,C,E,F), scale bars = 10 μm).