Genomics and Virulence of Fonsecaea pugnacius, Agent of Disseminated Chromoblastomycosis

Abstract

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.

Keywords: black fungi; cerebral infection; chromoblastomycosis; dissemination; genome assembly; neurotropism; pathology; virulence.

FIGURE 1

Phylogenomic tree based on the concatenated alignment of genomes of Herpotrichiellaceae family. Species names are given between phylogenomic tree and genome size bars. The genomes size is indicated and the colors are representing the clades of Chaetothyriales summarized in colored boxes.

FIGURE 2

Fonsecaea pugnacius CBS 139214 mtDNA. Rectangles represent annotated genes: in red the rRNAs; in green the tRNAs; in black the other genes and orange inner circle shows reads coverage.

FIGURE 3

The most numerous gene families of Fonsecaea pugnacius based on Gene Ontology annotation separating proteins annotated into three large groups: biological process, cellular components, and molecular functions.

FIGURE 4

Virulence test of Fonsecaea pugnacius using animal models: Tenebrio molitor and Balb/c (Mus musculus). (A) Mortality of the Tenebrio molitor larvaes at intervals of 4, 24, 72, 168, and 240 h post-infection. (B) Fungal burden of Tenebrio molitor larvae tissues at intervals of 4, 24, 72, 168, and 240 h post-infection. (C) Melanization of Tenebrio molitor hemolymph demonstrated by measuring the OD405 nm at 4, 24, 72, 168, and 240 h post-infection. (D) Presence of melanized hyphae in Tenebrio molitor larvae tissue 72 h post-infection. (E) Histopathology of footpad Balb/c tissue with presence of muriform cell after 21 days of intradermal infection. (F) Necrotic lesion in footpad of Balb/c mice after 7 days of intradermal inoculation.

FIGURE 5

Species enzymatous gene profiles and epidemiology of human host pathogenicity are summarized in colored boxes. Cladophialophora bantiana: beige; Exophiala dermatitidis: light green; Fonsecaea pugnacius: medium green; Fonsecaea monophora: light blue; Rhinocladiella mackenziei: medium blue; Verruconis gallopava: dark blue. (A) Carbohydrate-active enzyme (CAZY) annotation separated by classes. (B) MEROPS annotation separated by classes. (C) The species of black fungi related to subcutaneous and cerebral human infection and others affected organs: lungs, lymphatic system, kidneys, spleen, liver, and thyroid gland.