Li Xuewei S, Sun Jianing N, Okamoto-Shibayama Kazuko, Edgerton Mira
Department of Oral Biology and Restorative Dentistry, School of Dental Medicine, State University of New York, Buffalo, New York 14214, USA.
J Biol Chem. 2006 Aug 11;281(32):22453-63. doi: 10.1074/jbc.M604064200. Epub 2006 May 23.
Fungicidal activity of Hst 5 is initiated by binding to cell surface proteins on Candida albicans, followed by intracellular transport to cytoplasmic effectors leading to cell death. As we identified heat shock 70 proteins (Ssa1p and/or Ssa2p) from C. albicans lysates that bind Hst 5, direct interactions between purified recombinant Ssa proteins and Hst 5 were tested by pull-down and yeast two-hybrid assays. Pulldown of both native complexes and those stabilized by cross-linking demonstrated higher affinity of Hst 5 for Ssa2p than for Ssa1p, in agreement with higher levels of interactions between Ssa2p and Hst 5 measured by yeast two-hybrid analyses. C. albicans ssa1Delta and ssa2Delta mutants were constructed to examine Hst 5 binding, translocation, and candidacidal activities. Both ssa1Delta and ssa2Delta mutants were indistinguishable from wild-type cells in growth and hyphal formation. However, C. albicans ssa2Delta mutants were highly resistant to the candidacidal activity of Hst 5, although the ssa1Delta mutant did not have any significant reduction in killing by Hst 5. Total cellular binding of 125I-Hst 5 in the ssa2Delta mutant was reduced to one-third that of wild-type cells, in contrast to the ssa1Delta mutant whose total cellular binding of Hst 5 was similar to the wild-type strain. Intracellular transport of Hst 5 was significantly impaired in the ssa2Delta mutant strain, but only mildly so in the ssa1Delta mutant. Thus, C. albicans Ssa2p facilitates fungicidal activity of Hst 5 in binding and intracellular translocation, whereas Ssa1p appears to have a lesser functional role in Hst 5 toxicity.
Hst 5的杀真菌活性是通过与白色念珠菌的细胞表面蛋白结合而启动的，随后通过细胞内转运至细胞质效应器，导致细胞死亡。由于我们从白色念珠菌裂解物中鉴定出了与Hst 5结合的热休克70蛋白（Ssa1p和/或Ssa2p），因此通过下拉实验和酵母双杂交实验检测了纯化的重组Ssa蛋白与Hst 5之间的直接相互作用。对天然复合物以及通过交联稳定的复合物进行下拉实验，结果表明Hst 5对Ssa2p的亲和力高于对Ssa1p的亲和力，这与酵母双杂交分析中测得的Ssa2p与Hst 5之间更高水平的相互作用一致。构建了白色念珠菌ssa1Δ和ssa2Δ突变体，以检测Hst 5的结合、转运和杀念珠菌活性。ssa1Δ和ssa2Δ突变体在生长和菌丝形成方面与野生型细胞没有区别。然而，白色念珠菌ssa2Δ突变体对Hst 5的杀念珠菌活性具有高度抗性，尽管ssa1Δ突变体在Hst 5杀伤方面没有任何显著降低。与ssa1Δ突变体相比，ssa2Δ突变体中125I-Hst 5的总细胞结合量降至野生型细胞的三分之一，而ssa1Δ突变体中Hst 5的总细胞结合量与野生型菌株相似。Hst 5在ssa2Δ突变体菌株中的细胞内转运显著受损，但在ssa1Δ突变体中仅轻微受损。因此，白色念珠菌Ssa2p在结合和细胞内转运方面促进了Hst 5的杀真菌活性，而Ssa1p在Hst 5毒性方面似乎发挥的功能作用较小。
