Karyotype Analysis Of Sporothrix Schenckii And Sir2mRNA Level During Dimorphic Swich

Sporotrichosis clinical manifestations can be divided into fixed, lymphocutaneousand disseminated cutaneous. It is generally known that the virulence of strains and hostimmune status are related with clinical manifestations. Sporothrix schenckii(S. schenckii)grow as mycelia phase at25℃and convert to yeast phase at37℃. It is atemperature-induced phase transition, while the formation of yeast cells is thought to bea requisite for the pathogenicity of S. schenckii. Previous studies show that fungalvirulence and variations of genomic type diversity are related. Chromosomalrearrangements and genome plasticity in fungi are known to be driven through themeiotic process. No sexual cycle has been reported for S. schenckii. So it may be driventhrough variation of karyotype. Silent information regulator (Sir) is proved to beinvolved in phenotypic switching in Saccharomyces cerevisiae(S. cerevisiae) andCandida albicans by organizing chromatin structure. SsSir2like-protein expressionlevel in yeast cells is found to be higher than that in mycelia phase of S. schenckii.Objectivity:(1) Investigate the variation of chromosome karyotype during thetransition from mycelia phase to yeast phase of different clinical manifestations of S.schenckii.(2) Obtain the full-length of the SsSir2gene.(3) Investigate SsSir2mRNAlevel of S. schenckii during the transition from mycelia phase to yeast phase. Methods:(1) Two fixed type clinical isolates and two lymphocutaneous typestandard isolates of S. schenckii was incubates on Sabouraud’s fluid medium at25℃formycelia, and on Brain Heart infusion medium at37℃making sure to switching to yeast.The analysis of chromosomes and molecular karyotype of four strains during myceliaand yeast phase was carried out by the controlled homogenous electric field (CHEF)with Schizosaccharomyces pombe strain972h-and Saccharomyces cerevisiae strainYNN295chromosomes DNA as molecular weight markers.(2) Isolating total RNA tosynthesize cDNA, then the primers was designed based on multiple alignments of thehigh conserved Sir2domains to yielded a fragment homologous to known Sir2. Fivespecific primers3’-RACE and5’-RACE were synthesized based on the cDNA sequenceobtained by the former primers to obtain the full-length cDNA sequence of the SsSir2gene. Finally obtain the full-length sequence of the SsSir2gene.(3) The expressions ofSsSir2transcript in different stages (mycelia, yeast) were measured by real-timeRT-PCR.Results:(1) By the CHEF, total genome size was calculated to be approximately23.6-25.86M. The number of chromosomes varied from six to eight among the differentstrains. The chromosomal sizes, numbers, karyotype between four S. schenckii strainswere slightly different. But the karyotype of the strains between the mycelia and yeastphase is identical.(2) In this report, we isolated total RNA, synthesize cDNA, andcharacterized a Sir homologue gene, designated SsSir2, from yeast-form S. schenckii.The full-length SsSir2cDNA sequence is1753bp in size and contains an open readingframe of1329bp encoding442amino acids. The predicted molecular mass of SsSir2is48.1kDa with an estimated theoretical isoelectric point of4.6.(3) Differentialexpression of the SsSir2in two stages was demonstrated by real-time RT-PCR. Theexpression of SsSir2gene in the yeast cells was1.72-fold higher than that the one inmycelia phase of S. schenckii.Conclusions: The virulence of S. schenckii and variations of genomic type diversitymay be related. But the dimorphic switching process, while the formation of yeast cellswas thought to be a requisite for the pathogenicity of S. schenckii, is not induced by theswitching of karyotype. The SsSir2may be involved in the phenotypic switching andmorphogenesis of yeast phase in S. schenckii.