Characterization of epidermal infection by the dermatophyte Trichophyton benhamiae and involvement of fungal subtilisin proteases

Abstract

Background: Dermatophytosis are superficial infections of keratinized structures of the host. Because of high and increasing prevalence, and because they are hard to treat, these infections represent important topics to investigate.

Aim: The main objective of this project is to characterize epidermal infection by the zoophilic T.benhamiae species through the development of an in vitro model of infection on reconstructed human epidermis (RHE). The resulting model is compared with the model previously developed for studies of epidermal infection by the anthropophilic Trichophyton rubrum species. To bring further information over the role played by subtilisin-type proteases in these infectious processes, the model of dermatophytosis is also explored using strains of T. benhamiae deleted for the subtilisin-6 (SUB6) gene.

Methods: RHE were infected by topical application of arthroconidia produced from T. benhamiae cultures, and containing adequate numbers of CFU to create invasion of RHE similar to what is observed in vivo (i.e. restricted to cornified layer) during dermatophytosis. The development of infection was then characterized through morphological observation, absolute quantification of infecting agents via qPCR, and assessment of the epidermal barrier integrity by measurement of transepithelial electrical resistance and Lucifer Yellow permeability. Simultaneously, keratinocyte responses to the infection were analysed by monitoring expression and release of cytokines and antimicrobial peptides (AMP), respectively via RT-qPCR and ELISA.

Results: The model established for T. benhamiae infection exhibits characteristics very similar to in vivo lesions where dermatophytes invade the cornified layer, leading to alterations of the epidermal barrier integrity and to the expression and release of cytokines and AMP by keratinocytes. A superior capacity of infection is suggested for T. benhamiae in comparison with T. rubrum, providing potential explanations to the more severe lesions induced in vivo by this zoophilic species. Besides, the analysis of strains of T. benhamiae belonging to the two different phenotypes described for this dermatophyte species (i.e. yellow and white) is highlighting differences in spore production and in the respectivevirulence of the strains. Finally, the strain deleted for SUB6 gene appears more aggressive on RHE than non-deleted control strains, suggesting eventual compensatory mechanisms.

Conclusion: A model of infection by T. benhamiae was developed to characterize infectious processes, as well as of keratinocytes responses. Through infection by different strains, including deletant, this model reveals differences between both yellow and white phenotypes of T. benhamiae, and suggests that compensatory mechanisms might be responsible to promote infection when SUB6 expression is deleted.

Date of Award	18 Jan 2021
Original language	English
Awarding Institution	University of Namur
Supervisor	Yves Poumay (Supervisor)