Department of Microbiology and Medical Zoology, Medical Sciences Campus, University of Puerto Rico, PO Box 365067, San Juan, PR 00936-5067.
BMC Microbiol. 2011 Jul 11;11:162. doi: 10.1186/1471-2180-11-162.
Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii.
The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM), an inhibitor of HSP90.
Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.
申克孢子丝菌是一种分布广泛的致病双相真菌。它在 25°C 下以透明、有规律分隔的菌丝和梨形分生孢子的腐生形式生长，在 35°C 下以酵母或寄生形式生长。此前，我们对该真菌中的一种钙/钙调蛋白激酶进行了表征。该激酶的抑制剂被观察到抑制申克孢子丝菌的酵母细胞周期。
通过在申克孢子丝菌 DNA 中鉴定出 Dicer-1 同源物，证实了该真菌中存在 RNA 干扰 (RNAi) 机制。使用 RNAi 技术证实了钙/钙调蛋白激酶 I 在申克孢子丝菌双相中发挥作用。酵母细胞用 pSilent-Dual2G(pSD2G) 质粒转化，该质粒带有/不带有钙/钙调蛋白激酶 I(sscmk1)基因的编码区插入物。在 35°C 下使用遗传霉素抗性选择转化体。在将转化体转移到 35°C 的液体培养基中后，RNAi 转化体发育为异常的菌丝团，而不是预期的酵母细胞。在 35°C 下，与空 pSD2G 转化的细胞相比，RNAi 转化体中 sscmk1 基因的表达水平较低。酵母双杂交分析表明，与 SSCMK1 相互作用的蛋白质是热休克蛋白 90 (HSP90) 的同源物。在含有 geldanamycin(GdA，10 μM)的培养基中观察到与 RNAi 转化体相似的真菌生长，GdA 是 HSP90 的抑制剂。
使用 RNAi 技术，我们沉默了该真菌中 sscmk1 基因的表达。RNAi 转化体不能在 35°C 下作为酵母细胞生长，表明对该温度的耐受性降低。酵母双杂交试验观察到 SSCMK1 与 HSP90 的相互作用表明，该激酶通过与 HSP90 的相互作用参与耐热性。SSCMK1 与 HSP90 的 C 末端结构域相互作用，效应蛋白和共伴侣在此与该结构域相互作用。这些结果证实 SSCMK1 是参与申克孢子丝菌双相性的重要酶，是该真菌酵母相发育所必需的。此外，本研究首次报道了申克孢子丝菌的转化和使用 RNAi 技术研究该真菌中基因功能。
