Preliminary Studies On The Expression And Molecular Mechanisms Of The M35 Family In Trichophyton Rubrum And T.mentagrophytes

Trichophyton rubrum and T.mentagrophytes are the most common causative agents of dermatophytosis,a disease that affects millions of people worldwide.Secretory proteases are important virulence factors for dermatophytes to invade the hosts.Dermatophytes possess multiple copies of genes encoding secretory proteases,and these molecules are differentially expressed by different conditions(e.g.,substrate,pH,etc.).Objective: The most representative strains of T.rubrum and T.mentagrophytes were selected as experimental strains to investigate the growth characteristics of the two species of dermatophytes,to study the differences in the expression of the M35 family in the two species of dermatophytes,and to construct two molecular knockout models of Np IIc and Np IId,which are constantly highly expressed,and to initially explore their role in the process of dermatophytes invasion of the host.Experimental methods: The six main components are as follows,briefly described:1.Fungal culture and identificationIdentification of T.rubrum and T.mentagrophytes by macro-phenotypic,micro-structural and molecular biological techniques,and comparative analysis of the colony morphology and microscopic structure of the two fungi.2.Effect of different nutrients on the growth characteristics of T.rubrum and T.mentagrophytesT.rubrum and T.mentagrophytes were inoculated on media with different nutrients(nitrogen source,glucose,keratin powder)and the changes in growth rate,colony morphology and colour of the colonies were dynamically observed and photographed and recorded.3.Screening of culture systems for the stimulation of protease secretion by T.rubrum and T.mentagrophytesTr1(Trichophyton Agar Nr.1)medium was used as the basic control and other nutrients(nitrogen source,glucose,keratin powder)were added or subtracted to the experimental group and keratin-azure was added to the medium.The protease secretion of the culture system was dynamically examined and photographed.4.Changes in gene expression of the M35 family of T.rubrum and T.mentagrophytes under different pH conditionsThe culture system was established to stimulate protease secretion and the pH was adjusted to 4,6 and 8.Mycelium was harvested for total RNA extraction and reverse transcription to obtain c DNA,and RT-PCR was used to detect the expression of each gene in the M35 family at different pH values.5.Construction of T.rubrum NpⅡc and NpⅡd mutant strains by CRISPR/Cas9Primers were designed for the gene-specific fragments;sgRNA was obtained by in vitro transcription,and sgRNA was transformed into the protoplasts of strain STRB18(ΔURA3)together with Cas9 protein and Ura3 fragment,and as the target DNA was positioned and cut to form a double-strand break,the cells repaired the break by the exogenous Ura3 fragment to cause the mutation,and the mutation was detected by bi-directional nutrient.The target transformants were selected by screening and their extracted DNA was verified by PCR amplification and sequencing.6.Dynamic measurement of keratinase secretion in wild-type and mutant types(SCNB19 and SCNB20)The keratinase secretion of T.rubrum wild type,mutant SCNB19(ΔNpⅡc)and SCNB20(ΔNpⅡd)was measured by the addition of keratin-azure combined with spectrophotometry.The results of the experiment: a brief description in six parts as follows1.Fungal culture and identificationT.rubrum typically grew as fluffy to wooly,pinkish colonies with a moderate growth rate,whereas T.mentagrophytes grew as powdery to cottony,yellowish colonies with a higher growth rate.Microscopically,T.rubrum sparsely developed pear-shaped microconidia,whereas T.mentagrophytes frequently sporulated with rounded to broadly clavated microconidia.2.Effect of different nutrients on the growth characteristics of T.rubrum and T.mentagrophytesBoth T.rubrum and T.mentagrophytes grew radiatively in the medium,with T.rubrum growing most vigorously on unmodified Tr1 medium and SC medium.Growth levels were slightly worse on media containing only amino acids.Colony density was sparsest in keratin powder medium.T.rubrum produce pigment in glucose medium,but not when it is not added.T.mentagrophytes grows in a similar state to T.rubrum,except that in SC basal medium,T.mentagrophytes grows at a significantly slower rate than other media,but produces the most pigment.3.Screening of culture systems for stimulating protease secretion by T.rubrum and T.mentagrophytesAt week 2,a blue colour was clearly observed in the keratin powder medium,suggesting the secretion of proteases.In contrast,no colour change was observed in any of the other media.4.Changes in gene expression in the M35 family of T.rubrum and T.mentagrophytes at different pH conditionsIn T.rubrum and T.mentagrophytes,the expression of NpⅡa,NpⅡb and NpⅡe in the M35 family was relatively stable and low at different pH values,NpⅡc was expressed at high levels in T.rubrum and NpⅡd was expressed in higher amounts in T.mentagrophytes than in the other M35 family proteases.In addition,M35 family genes were found to be more actively expressed in acidic and neutral conditions than in alkaline conditions.5.Construction of T.rubrum NpⅡc and NpⅡd mutant strains by CRISPR/Cas9technologyThe target transformants were selected by bi-directional nutritional screening,and their extracted DNA was verified by PCR amplification and sequenced for comparison.The results suggested that the URA3 gene fragment was successfully inserted into the NpⅡc and NpⅡd genes,and the ΔNpⅡc mutant strain SCNB19 andΔNpⅡd mutant strain SCNB20 were successfully obtained.6.Dynamic measurements of keratinase secretion in wild-type and mutant strains(SCNB19 and SCNB20)At all time points tested,the protease activity of the wild type was higher than that of the mutant strains.A peak in protease activity was reached at day 5 for the wild-type strain and SCNB20,and at day 7 for SCNB19.At day 15,the enzyme production of wild-type and mutant strains was almost identical and then gradually increased and reached a peak again at day 20.Conclusions1.Through experiments on the effect of different nutrients on the growth characteristics of T.rubrum and T.mentagrophytes it was found that 1)dermatophytes could not grow in the presence of a carbon source(glucose)alone and without a nitrogen source;2)if there was no carbon source(glucose),a nitrogen source could be used as both a carbon source and a nitrogen source;3)the type of nitrogen source(amino acids or keratin powder)varied with the growth pattern of dermatophytes,which grows better under amino acid nutrition;4)glucose may play an important role in the production of dermatophyte pigments.2.Both the acidic environment and keratin powder contribute to the induction of M35 family protease expression.3.NpⅡc and NpⅡd are important for keratinolytic and activation of dermatophytes pathogenesis,but presumably their biological functions go beyond keratinolytic and their more molecular functions need to be further explored.4.The enrichment of protease genes in dermatophytes allows for complex regulation of protease secretion in response to the changing external environment.