N DE COI¹, T YAMADA², K SALAMIN¹, M MONOD<sup>1

1</sup>Department of Dermatology, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland., ²Teikyo University Institute of Medical Micology, Hachioji, Tokyo, Japan

Background: We obtained a complete gene expression profile of the genes coding for proteins secreted by A. benhamiae (dermatophyte) during infection using RNA-seq analysis. The expression profile of genes encoding secreted proteins was found to be very different from those during in vitro growth using various substrates. In particular, the most upregulated genes encoding secreted proteases during infection were those encoding subtilisins (SUB6, SUB7, SUB8 and sUB10), which have never been detected in all in vitro conditions tested. SUB6 was known to be a major allergen in the related dermatophyte Trichophyton rubrum.
Objective: The objective of this work was to characterize SUB6 and SUB7 as recombinant proteins. Recombinant SUB6 and SUB7 could not be obtained using either Escherichia coli, or Pichia Pastoris or Aspergillus expression systems. Therefore, we developed an Arthroderma benhamiae system to produce dermatophyte secreted proteins for further characterization and antigen tests.
Methods: A plasmid pNDC1 was designed for the production of dermatophyte secreted proteins under the control of the Translation Elongation Factor 1 (TEF1) promoter, and Agrobacterium mediated transformation was used.
Results: Transformants grown in minimal liquid medium (MM) were tested for proteolytic activity using N-Suc-Ala-Ala-Pro-Phe-pNA as a substrate. Activity varied in a 1 to 10 ratio from one clone to another. The transformants with maximum SUB6 or SUB7 activity were retained for further investigations. Western blot analyses revealed that SUB6 or SUB7 were indeed secreted by the selected transformants as strong signals were obtained with antibodies. A yield of 10µg/ml culture supernatant was obtained for both Sub6 and SUB7. Characterization of individual recombinant SUBs is underway.
Conclusion: The developed Arthroderma benhamiae expression system revealed to be efficient to obtain dermatophyte secreted proteins which could not be produced using heterologous expression systems.