| Among more than100,000different species of fungi, there are six dimorphicfungi: Blastomyces dermatitidis, Histoplasma capsula, Coccidioides immitis,Paracoccidioides immitis， Penecillium marnefiii, Sporothrixschenckii. These fungichange morphology once spores are inhaled into a mammalian host from hyphalmolds in the environment to pathogenic yeast forms. Compared with the other fivekinds of two-phase fungi, sporotrichosis is on high incidence, can cause skin,subcutaneous tissue, as well as nearby lymphatic chronic infection, leading tosuppuration, ulceration and exudation, even involving the muscles, bones, centralnervous system and other organs.The sporotrichosis pathogens is Sporothrixschenckii,it has biphasic:25℃, hyphal, was gray to brown mold-like colony growth, atmicroscopic, is slender septate hyphae, the sporulation cell undifferentiated hyphaeformation is small, clustered distribution of conidia. Conidia have not hyphal buddingform of yeast cells, non-pathogenic;37℃, yeast, cream or tan, yeast-like colonies atmicroscopic, was round or oval yeast-like cells, narrow base may issue an elongatedcigar-like buds on histopathology in cell morphology, has pathogenicity. The processof Sporothrix mycelial phase transformed into yeast phase is considered the process ofSporothrix pathogenic formation, in-depth study of the conversion helps to reveal thepathogenesis of sporotrichosis. Biphasic fungi transformation is a multi-factorinvolved extremely complex process, and fungi with its signaling system feel andcascadly transmite the environment signal to the cell, causing the expression of specific genes and exercise the function. Previous studies by our group have reported24proteins by comparing protein expression of hyphal and yeast phase, including thetwo-component signal system histidine kinase DRK1gene, we conducted this study inorder to study the role of DRK1in Sporothrix pathogenicity.Objectives:1, clone Sporothrix histidine kinase DRK1gene.2, Build a RNAinterference vector by Agrobacterium tumefaciens-mediated suitably for a variety offilamentous fungi.3, Study the impact of DRK1gene of Sporothrix mycelial andyeast cells on growth, the transformation from mycelium to yeast phase, cell wallcomposition, the downstream gene expression and Sporothrix pathogenic.4, Makesure the potential efficacy of astragalus on the host immune status and adjuvanttreatment of sporotrichosis.Methods:1, By RACE technology to get the histidine kinase DRK1cDNAfull-length, clone DRK1the genome and find intron. Identificate functional domains2,Build an intermediate vector PCB301, PCB309, PUC-PUT PUC-PDUDT, importfungi common promoter PtrpC, terminator TtrpC, the hairpin structure gus gene,hygromycin resistance gene Hypr, positive and negative sequences of interferenceDRK1, finally build interference carrier PCB309-PDUDT. Optimizate the reactionsystem of the Agrobacterium transformed, to determine the type and concentration ofthe inducing agent in the induction medium, the best time of the induced culture andthe concentration of hygromycin.3, Compared the wild-type strain, the idling strain(vector PCB309) and DRK1interference strain to observe colony growth,morphology, optical microscopy, electron microscopy structure, Congo Red andZymolyase sensitivity test, the downstream gene Ste20, cAMP expression levels,differences of mice infection model of local lesions inflammatory infiltration to clearDRK1gene function.4, Compared the infection model the local lesions inflammatoryinfiltration between the Astragalus administration group and the non-administeredgroup sporotrichosis to clear the potential efficacy of astragalus to adjust the hostimmune status and sporotrichosis adjuvant therapy.Results:1, Complete SsDRK1cDNA total length is4743bp, the open readingframe is4071bp, encoding1356amino acid. Molecular weight is147.3kDa andisoelectric point is5.46. And similarity of dermatitis Blastomycosis DRK1gene is65%. The Sporothrix DRK1is a soluble histidine protein kinase, transmembraneregion is not found, the basic structure of the signal includes feelings district,connected areas and functional areas.2, Build the8825bp interference vectors PCB309-PDUDT. Optimize Agrobacterium-mediated transformation system: makesure the concentration of acetosyringone is200μmol/L, at25°C48h in the dark, theconcentration of hygromycin is100mg/L. successfully implementated the Sporothrixthe histidine kinase DRK1gene interference, after interference gene expression levelsreduced89%.3, Down DRK1gene expression, colonies grow delayed in diameterfrom1.1CM0.6CM; melanin production reduced, interference strain suspensionbecomes faint; cultivated96H, bacteria dry weight decreased from2.9689g to0.9274g; spore formation reduced, cultivated1W, spectrophotometric values are from1.15to0.55; hyphae and spores phenotypic changes: cell wall rough, loose change;components of the cell wall changed. sensitivity of the Congo red and Zymolaseincreased; downstream pathway genes the Ste20and cAMP expression levels weresignificantly lower, reduced by83%and73%respectively; virulence of the strainsreduced, the reactions caused by inflammation of the skin lesions were mild, thenumber of local lesions in mice between the wild-type strain and interference straininfection neutrophil infiltration were97.5±10,29.4±5; mononuclear cells were90.2±8,30.0±4.4, Astragalus can adjust the host immune function, reduce spore in miceinfected with the inflammatory response, the number of neutrophil infiltration of locallesions before and after treatment were97.5±10,75.7±2. The mononuclear cellswere90.2±8,72.0±2.Conclusion:1, Determine Ss DRK1is4743bp,1356aa, a kind of membranesoluble histidine protein kinase.2, Build a RNA interference suitable filamentousfungal gene mediated by Agrobacterium tumefaciens.3, Ss DRK1gene in growth,morphogenesis, cell wall composition, in the formation of pathogenic have importantrole, located in upstream of the signal transduction pathway MAPK and cAMP-PKA.4, Astragalus may adjust the the mouse host immune function, may play a potentialrole in support of the treatment of sporotrichosis. |
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