Analysis of the pathogenic fungus Trichophyton rubrum gene expression during ex vivo human skin infection and the effect of the antifungal drug ambruticin

PERES NT; GRAS DE; SANCHES PR; FALCÃO JP; ROCHA LB; ROSSI MA; MAZUCATO M; ROSSI A; PRADE RA; MARTINEZ-ROSSI NM

Ribeirão Preto

Encuentro; XXXIII Meeting of the Brazilian Society for Immunology; 2008

Brazilian Society for Immunology

Introduction and Objectives: Trichophyton rubrum is the most prevalent  dermatophyte isolated from skin and nail infection in humans, and although therapy  is efficient, resistant strains to the most used antifungal drugs had been reported.  Furthermore, the available antifungal drugs act upon a few cellular targets, making  the discovery of new targets and development of new drugs extremely necessary. Ambruticin is a compound with antifungal properties, probably interfering with osmotic regulation. Due to the high prevalence of infections caused by T. rubrum, and the  lack of knowledge about the infection process, the aim of this work is to establish and  validate the efficacy of an ex vivo skin infection system to analyze gene expression  during skin interaction. The effect of Ambruticin was also evaluated and T. rubrum up  and down-regulated genes in response to this drug during skin infection were  identified. Methods and Results: An ex vivo human skin infection was used followed by  suppression subtractive hybridization (SSH) to identify T. rubrum expressed genes  during fungal-host interaction, and also in response to Ambruticin. After 96 hours of ex vivo infection, conidia germinated and hyphal penetration was observed with scan  electron microscopy. SSH and differential screening generated 116 unique  Expressed Sequence Tags (ESTs) during ex vivo skin infection. In response to Ambruticin, 93 up-regulated and 40 down-regulated genes were isolated and annotated. Moreover, 82 novel T. rubrum genes were isolated, which expression may be regulated during host interaction or in response to Ambruticin, being somehow involved in T. rubrum-skin interaction/adaptation. Conclusion: The ex vivo skin infection system established is suitable for molecular studies about host-pathogen interaction and evaluation of the effect of antifungal drugs. The identification of T. rubrum genes expressed during infection could reveal the molecular aspects required for its survival in the host milieu, providing new insights into metabolic adaptations performed by T. rubrum during host infection. Furthermore, Ambruticin presents antifungal properties against T. rubrum in vitro, leading to a swollen of the hyphal-tips and regulating the expression of genes  involved in different cellular processes in this medically important fungus.