Histidine Kinase 2-Component Protein of Candida Albicans

在我们过去三年的观察中，我们发现白色念珠菌的ChKLP双组分组氨酸激酶[HK]调节细胞壁的生物合成。这些数据是通过对chk1缺失突变体的分析获得的。细胞壁变化的后果是深远的，因为chkl突变体在体外较不能附着于人类食道组织，更容易受到人类中性粒细胞和过氧化氢的生长抑制和杀伤，而且重要的是，是无毒的。此外，chk1突变体对白念珠菌群体感应分子法尼醇不敏感。除了ChKLP，白念珠菌还有另外两种HK蛋白，SLnlp和Nik1/Cos1p，我们的研究表明，每一种HKs都调节共同的功能，但也有特定的功能。例如，所有香港交易所显然都在监管 细胞壁酸稳定甘露聚糖表位的甘露糖化，但只有CHKLP调节群体感应和氧化应激适应。此外，我们已经确定白念珠菌的SLNLP和反应调节蛋白Ssklp调节CHK1-启动子-LacZ报告基因的表达，这表明HOG1MAP途径可能与ChkLp相互作用。ChKLP的这些功能属性和调控对于任何双组分信号蛋白的研究都是新的。我们的总体目标是重点研究ChkLp在细胞壁生物合成和其他过程中的作用，如氧化应激适应，以及确定ChkLp和Hogtp调控的事件的共性。这项提议有三个具体目标。在具体目标1中， 我们将区分与CHK1缺失相关的细胞壁分解中的原发损害和继发影响。这一目的将通过使用四环素可调节启动子来实现。同样在目标1中，将引入CHK1的点突变来区分几个关键结构域的功能。第二个具体目标将利用蛋白质组学、微阵列和串联亲和层析[TAP]来鉴定对ChkLp(Calderone实验室)和Hoglp(Pla实验室)功能至关重要的蛋白质。AIM 2包括与J.Brown(TAP)和J.Kerwin(蛋白质组学)实验室的重要合作。具体目标3将侧重于CHKLP和Hoglp的功能反应关系，并包括对核转位的研究 在chkl突变体的上下文中的Hoglp。上位性实验旨在研究Chk1p-Hog1p的相互作用。这项研究的长期目标是增加我们对两组分信号转导在白念珠菌中的生物学作用和在疾病中的贡献的理解，特别是CHKLP。

Among our observations during the past three years, we have shown that the Chklp two-component histidine kinase [HK] of Candida albicans regulates cell wall biosynthesis. These data were achieved through an analysis of the chkl deletion mutant. The consequences of the cell wall changes are profound as the chkl mutant is less able to adhere to human esophageal tissue in vitro, is more susceptible to growth inhibition and killing by human neutrophils and hydrogen peroxide, and, importantly, is avirulent. Also, the chkl mutant is refractory to the C. albicans quorum sensing molecule, farnesol. In addition to the Chklp, C. albicans has two other HK proteins, Slnlp and Nik1/Cos1p, and our studies have shown that each of the HKs regulate common functions but also have specific functions. For example, all HKs apparently regulate mannosylation of the cell wall acid-stable mannan epitopes, but only Chklp regulates quorum sensing and oxidative stress adaptation. In addition, we have determined that the Slnlp and response regulator protein of C. albicans, Ssklp, regulate expression of a CHK1-promoter-lacZ reporter suggesting that the HOG1 MAP kinase pathway may interact with Chklp. These functional attributes and regulation of Chklp are new to studies of any two-component signal protein. Our overall objective is to focus upon Chklp in cell wall biosynthesis and other processes such as oxidant stress adaptation, as well as to identify the commonality of events regulated by Chklp and Hogtp. There are three specific aims in this proposal. In specific aim 1, we will differentiate the primary lesion from secondary affects in the cell wall disassembly that are associated with the deletion of CHK1. This aim will be achieved by using the tetracycline regulatable promoter. Also in aim 1, point mutations in CHK1 will be introduced to distinguish functions of several key domains. The second specific aim will utilize proteomics, microarrays, and tandem affinity chromatography [TAP] to identify proteins critical to the functions of Chklp (Calderone lab) as well as Hoglp (Pla lab). Aim 2 includes important collaborations with the labs of J. Brown (TAP) and J. Kerwin (Proteomics). Specific aim 3 will focus upon the functional response relationships of Chklp with Hoglp and include studies on nuclear translocation of Hoglp in the context of the chkl mutant. Epistasis experiments are designed to examine Chk1p-Hog1p interactions. The long-term objective of this research is to increase our understanding of the biological roles and contribution to disease of 2-component signal transduction in C. albicans, especially Chklp.