Agrobacterium tumefaciens-Mediated Transformation of Fonsecaea monophora and Fonsecaea erecta for Host-Environment Interaction Studies.

Cristina Isabel Ferrer Villena,Larissa Fernandes,Edvaldo Da Silva Trindade,Renata Rodrigues Gomes,Camille Silva Florencio,Maria Eduarda Grisolia,Sybren De Hoog,Amanda Bombassaro,Vania Aparecida Vicente

doi:10.3390/jof6040325

Abstract

The fungal genus Fonsecaea contains etiological agents of human chromoblastomycosis, a (sub)tropical, (sub)cutaneous implantation disease caused by plant contact. The invasive potential differs significantly between species. Infections by Fonsecaea monophora are believed to originate from the environment and the species has been reported as one of the main causative agents of the disease, but also of cases of primary brain infection. The epidemiology of the disease has not been fully elucidated and questions related to its infection route and virulence are still to be clarified. The environmental species Fonsecaea erecta was isolated from organic material and living plants in endemic areas for chromoblastomycosis in Brazil. The present paper describes Agrobacterium tumefaciens-mediated transformation (AMT) of the environmental species F. erecta and the pathogenic species F. monophora. We propose the use of Agrobacterium transformation for future gene function studies related to Fonsecaea virulence and pathogenicity. We evaluated the co-cultivation ratios 1:1, 10:1 and 100:1 (Agrobacterium:conidia) at 28 °C during 72 h. pAD1625 and pCAMDsRed plasmids were inserted into both species. Confirmation of transformation was realized by hph gene amplification and Southern blot determined the amount of foreign DNA integrated into the genome. In order to evaluate a potential link between environmental and clinical strains, we obtained red fluorescent transformants after pCAMDsRed insertion. We observed by confocal fluorescence microscopy that both F. monophora and F. erecta were able to colonize the palm Bactris gasipaes, penetrating the epidermis. These results contribute to understanding the ability of Fonsecaea species to adapt to different environmental and host conditions.

Highlights

Chromoblastomycosis is a neglected occupational disease [1] caused by several dematiaceous fungi
We describe Agrobacterium tumefaciens-mediated transformation (AMT) of F. erecta, an environmental species isolated from a living plant, and the pathogenic species F. monophora, transformed with pCAMDsRed and pAD1625
The MIC concentration was determined as 25 μg/mL of hygromycin, but 100 μg/mL was the concentration which was used for the selection of transformants mediated by A. tumefaciens

Summary

Introduction

Chromoblastomycosis is a neglected occupational disease [1] caused by several dematiaceous fungi. Chromoblastomycosis may be acquired by inoculation of pathogenic agents following penetrating trauma [9]; the infection process and the origin of the disease are still not entirely clear [7,16,17,18]. Fonsecaea species and their relatives are asexual Ascomycetes belonging to the family Herpotrichiellaceae (order Chaetothyriales) containing numerous agents of human infection and with a significant predisposition to grow in human tissue [16,19]. Fonsecaea comprises cryptic species (Fonsecaea pedrosoi, Fonsecaea pugnacius, Fonsecaea monophora and Fonsecaea nubica) potentially causing disease [12,15], but the invasive potential differs significantly between species [20,21,22]

Methods

Results

Conclusion