| BackgroundTinea capitis is a relatively common superficial fungal infection.It was primarily caused by the fungal-infected animals and patients through direct physical contact or indirect transmission through fungal-contaminated materials.Tinea capitis was once under control.Recently,as the people lifestyle was changed and more and more people in cities liked to keep pets,moreover the antibiotics were abused and corticosteroid and immuosupressor was used increasly,the incidence of M.canis infections,and in particular tinea capitis,has been increasing in China year by year.It was reported that M canis is one of the most common dermatophytes to cause human tinea capitis at home and abroad.Tinea capitis is more common in prepubescent children,and there was asymptomatic at the initial stage of infection.As M.canis is a type of zoophilic fungi,the clinical manifestations of tinea capitis infected by M.canis are intense inflammatory reactions,including severe itching of the scalp,red scaly papules around hair shafts,and breaking-off of hairs severe inflammation leading to subcutaneous pustular formation and hair loss.In serious cases the infection may cause permanent hair loss and leave cosmetic scars.As a result,M.canis infections affect patients physically and psychologically and may be a significant burden for society.However,the detail mechanism of caused by tinea capitis remains unclear.Therefore,it is essential necessary to understand the underlying mechanism of pathogenesis and thus,prevent it.It was reported that proteases secreted by M.canis and host immune reactions were associated with the pathogenic mechanisms of tinea capitis.The reported proteases contained keratinase,elastase,collagenase,esterase,ceramidase and phosphatase,etc.Keratinase represented by two families of subtilases and metalloproteases was primarily pathogenic factor.Other proteases,such as cysteine dioxygenase,fungalysin and dipeptidyl peptidases were also demonstrated to serve important roles in fungal infection.Since tinea capitis occurs almost exclusively in children,in our previous study two forward and reverse suppression subtractive libraries of M.canis incubated on mineral medium with child scalp tissue vs adult scalp tissue and child scalp tissue vs child glabrous skin using the suppression subtractive hybridization(SSH)technique were constructed,the complementary deoxyribonucleic acid(cDNA)genes expressed differentially of each library screened were regared as pathogenic factors.FSH1 gene was one of the screened potential pathogenic factor.The expression levels of the FSH1 gene were 44.6-fold in child scalp tissue induced group than in child scalp tissue induced group.Moreover,the mRNA expression of FSH1 was higher in M canis strains isolated from tinea capitis lesions than in those from tinea corporis lesions.It was also higher in M canis strains induced by scalp or foreskin tissue than before inducing.These results suggested FSH1 was important for the pathogenicity of M.canis.To verify this assumption,the gene function of FSH1 in the pathogenicity of M.canis was investigated in the present study.Objective1.Cloning and sequence analysis of the full-length cDNA of microsporum canis serine hydrolases(FSH1)gene.2.Construction of FSH1 interference mutants.3.Compared the phenotype and pathogenicity of the pathogenic dermatophyte M.canis before and after FSH1 sclienced.Then presumption the function of FSH1 whether it it the virulence factor of M.canis in tinea capitis.4.Etablishment of the green fluorescent protein expression system and analysis of subcellular localization of FSH1 gene in microsporum canis.Methods1.Total RNA was extracted from the microsporum canis strain.Primers was designed according to the previous screened FSH1 gene fragment.Rapid cDNA end amplification(RACE)was used to clone the full-length cDNA sequence of FSH1 gene.And the obtained sequence was analyzed and the protein encoded was presumed by bioinformatics methods.2.pCB309-PFUFT-FSH1 knockdown vector was constructed.FSH1 was knocked down using double-stranded RNA interference mediated by Agrobacterium tumefaciens.Construction the Reverse transcription-quantitative PCR analysis was used to confirm gene knockdown.3.The wild-type and FSH1-i strains were grown on SDA and Rice medium.The phenotype between them was compared,containing the colony growth rate and morphology,mycelial and macroconidia morphology under light microscopy,scanning and transmission electron microscopy.The wild-type and FSH1-i strains were infected the geniea pigs.The pathogenicity between them was also compared by observed the clinical symptom of the infected skin,the number of mycelial and spore in the the infected hair,and the pathological change stained with hematoxylin and eosin solution and Alcian blue periodic acid-Schiff(PAS).4.The FSH1 and EGFP gene were amplified by PCR technique from the template of the plasmid containing FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP,respectively.The vector DNA was obtained by double restriction of recombinant plasmids pCAMBIA-LRP-EGFP as template using Sna B I/Kpn I.Then the EGFP expression plasmid and the fusion FSH1-EGHP plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into vector DNA plasmids respectively,and identified with PCR and sequencing.The two binary vectors were transformed into m.canis by Agrobacterium tumefanciens-mediated method.The gene EGFP and fusion gene FSH1-EGFP were expressed under the regulation by the fungul universal promoter Ptrpc and terminator Ttrpc.The cellular localization of the fusion protein was observed using confocal laser scanning microscopy.Results1.The full-length cDNA of FSH1 gene was cloned succefully.It was 945bp and included a 44bp 5’untranslated region(URT),a 70bp 3’URT and a 831bp of open reading frame(ORF)that encoded protein of 276 amino acid residues.BLAST analysis showed the cloned cDNA sequence was 100%identity with that of encoding the microsporum canis domain-containing protein.2.The knockdown vector pCB309-PFUFT was constructed succefully.Many FSH1-I mutants were screened.qPCR analysis demonstrated that the degree of knockdown was stable and efficient,and the expression of the FSH1 gene in FSH1-i mutant was～70%lower compared with the wild-type strain.3.The phenotype and pathogenicity of the pathogenic dermatophyte M.canis regulated by FSH1 gene was analysed successfully.The colony growth rate and morphology were not different between the wild-type strain and FSH1-i mutants.The color of the colonies of the wild-type strain appeared more orange compared with the FSH1-i mutant when cultured on both the SDA solid medium and rice medium,and the colonies were thinner in the wild-type compared with the FSH1-i mutant when cultured on rice medium.The shape of the wild-type strain and FSH1-i mutants were spindle-like,but the macroconidia septa of the FSH1-i mutant exhibited a reduced number or complete loss.The size and shape of the growing hyphae did not appear different between the wild-type strain and the FSH1-i mutant.SEM micrographs revealed that the hyphae of the FSH1-i strain did not exhibit any visible changes when compared with the wild-type strain.The macroconidia of the FSH1-i strain were typical,with a warty and spindle appearance.The macroconidia of the wild-type strain had a reproductive organelle with a well-defined surface.In the FSH1-i strain,there were a reduced number of papillary nodules of the macroconidia on the surface.SEM images revealed that the nuclei and mitochondria appeared normal in shape in the wild-type strain,and the plasma membrane folding was regular and uniform.In the mutant strain,significant morphological changes were observed.The majority of the cell membrane appeared irregular and thinner,and there were empty spaces in the cytoplasm.Additionally,cellular structures were partly degraded,and organelles exhibited marked degradation.The diameter of the skin erythema lesions were smaller in the animals infected with the FSH1-i strain compared with the animals infected with the wild-type strain.Spores and hyphae were observed in the hair from the skin lesions infected by wild-type and FSH1-i mutant strains.The severity of the inflammation in histopathological changes was higher in the wild-type group compared with the FSH1-i mutant group on day 14 PI.Considerably more fungal endothrix spores and hyphae were observed in the skin lesions of animals infected with the M.canis wild-type strains compared with the FSH1-i mutants4.The green fluorescent protein expression system in microsporum canis was firstly constructed successfully.The fusion gene FSH1-EGFP was firstly expressed and the green fluorescent signals were observed on cytoplasm and nucleolus of the transformed microsporum canis in a granular or cluster formConclusions1.The full-length cDNA of FSH1 gene was 945bp and included a 44bp 5’untranslated region(URT),a 70bp 3’URT and a 831 bp of open reading frame(ORF)that encoded protein of 276 amino acid residues.The conserved domain region was serine hydrolase.2.The knockdown vector of FSH1 gene in M.canis was also suitablely used for other gene function.3.FSH1 gene was one of the pathogenical factors in M.canis.It was relative with the form of the sepa of macrocondia.It was also important influence on membrane,cytoplasm and organelles of M.canis.4.The green fluorescent protein expression system in microsporum canis can be also used for other protein subcellular localization.5.The fusion gene FSH1-EGFP were locazition in cytoplasm and nucleolus of the transformed microsporum canis.It was foundation for studying the pathogenicity of FSH1 in M.canis and developing of antifungal drug in tinea capitis. |
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