Histidine Kinase 2-Component Protein of Candida Albicans

白色念珠菌的组氨酸激酶 2 组分蛋白

• 批准号: 7173733
• 负责人: Richard Arthur Calderone
• 金额: $ 33.15万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7173733
• 关键词: Acids; Adherence; Affect; Affinity Chromatography; Anabolism; Animal Model; Bacteria; Biological; Biology; CHES1 gene; Candida albicans; Candidate Disease Gene; Candidiasis; Carbohydrates; Cell Wall; Cells; Collaborations; Condition; Data; Disease; Epitopes; Esophageal Tissue; Event; Facility Construction Funding Category; Farnesol; Genes; Genetic Epistasis; Genetic Transcription; Glucans; Growth; Histidine; Human; Hybrids; Hydrogen Peroxide; In Vitro; Invasive; Knock-out; Laboratories; LacZ Genes; Mannans; Mediating; Mitogen-Activated Protein Kinases; Mutation; Nuclear Translocation; OmpR protein; Oxidants; Oxidative Stress; Pathway interactions; Peroxides; Phenotype; Phosphorylation; Point Mutation; Primary Lesion; Process; Protein C; Proteins; Proteomics; Refractory; Regulation; Reporter; Research; Role; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Stream; Stress; Tetracycline; Tetracyclines; Virulence; design; fungus; in vivo; killings; mutant; neutrophil; promoter; protein protein interaction; protein-histidine kinase; quorum sensing; research study; response; sensor; tool; transcription factor

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.

在过去三年的观察中，我们发现白色念珠菌的 Chklp 二组分组氨酸激酶 [HK] 调节细胞壁生物合成。这些数据是通过分析 chk1 缺失突变体获得的。细胞壁变化的后果是深远的，因为chk1突变体在体外粘附于人食道组织的能力较差，更容易受到人中性粒细胞和过氧化氢的生长抑制和杀伤，并且重要的是，它是无毒的。而且，chk1突变体对白色念珠菌群体感应分子金合欢醇具有抵抗力。除了Chklp之外，白色念珠菌还有另外两种HK蛋白，Slnlp和Nik1/Cos1p，我们的研究表明，每种HK蛋白调节共同功能，但也具有特定功能。例如，所有香港显然都监管 细胞壁酸稳定甘露聚糖表位的甘露糖基化，但只有 Chklp 调节群体感应和氧化应激适应。此外，我们还确定白色念珠菌的 Slnlp 和反应调节蛋白 Ssklp 调节 CHK1 启动子 lacZ 报告基因的表达，表明 HOG1 MAP 激酶途径可能与 Chklp 相互作用。 Chklp 的这些功能属性和调节对于任何双组分信号蛋白的研究都是新的。我们的总体目标是关注细胞壁生物合成和其他过程（例如氧化应激适应）中的 Chklp，以及确定 Chklp 和 Hogtp 调节事件的共性。该提案有三个具体目标。在具体目标1中， 我们将区分原发性病变和与 CHK1 删除相关的细胞壁分解的继发性影响。这一目标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] 来识别对 Chklp（Calderone 实验室）和 Hoglp（Pla 实验室）功能至关重要的蛋白质。目标 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 将重点关注 Chklp 与 Hoglp 的功能反应关系，并包括核易位的研究 Hoglp 在 chkl 突变体的背景下。上位实验旨在检查 Chk1p-Hog1p 相互作用。这项研究的长期目标是加深我们对白色念珠菌（尤其是 Chklp）中二元信号转导的生物学作用和对疾病的贡献的了解。