IDENTIFICATION AND CHARACTERIZATION OF VIRULENCE DETERMI

毒力测定的鉴定和表征

• 批准号: 6101142
• 负责人: MICHAEL P SCHMITT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6101142
• 关键词: Corynebacterium; Corynebacterium diphtheriae; bacterial cytopathogenic effect; bacterial genetics; diphtheria toxin; genetic library; genetic mapping; genetic promoter element; heme; heme oxygenase; hemoglobin; host organism interaction; iron; iron metabolism; microorganism metabolism; nucleic acid sequence; transferrin; virulence

The purpose of this project is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. It was shown in earlier studies from this laboratory that the hmuO gene is required for the utilization of iron from heme and hemoglobin in C. diphtheriae. Recent studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) purification and characterization of the activity of the HmuO protein and 3) the characterization of factors involved the transport of heme in C. diphtheriae. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. The ChrS protein is proposed to detect heme in the extracellular environment and transmit this signal via a phosphotransfer mechanism to the ChrA protein, which then activates transcription of the hmuO gene. An ABC (ATP-binding cassette) type transport system was shown to be required for the utilization of heme and hemoglobin as iron sources in C. diphtheriae. The three proteins identified in this system are homologous to proteins involved in heme transport from gram-negative bacteria. The HmuT protein, which is proposed to be the cell surface receptor for heme, was shown in vitro to bind to heme agarose, and it was shown to be a lipoprotein. These findings suggest that HmuT has the capacity to bind heme and further suggests that this putative receptor is anchored to the cell surface by a lipid moiety at its N-terminus.

本项目的目的是鉴定和表征白喉棒状杆菌的毒力决定因素，以了解这种重要的病原体是如何致病的。白喉毒素是白喉隐翅虫的主要毒力决定因素，其表达受铁的调控，很可能是额外的毒力因子与白喉毒素的毒力因子协同调节。在感染期间获得铁的能力是许多细菌病原体致病的关键。本实验室的早期研究表明，hmuO基因是白喉隐翅虫利用血红素和血红蛋白中的铁所必需的。最近的研究主要集中在：1)了解hmuO基因调控的分子机制，2)HmuO蛋白的纯化和活性鉴定，3)白喉隐翅虫中参与血红素运输的因素的鉴定。HmuO启动子的转录被证明受到双重调控，其中表达受到血红素来源(如血红素或血红蛋白)的正调控，而受白喉毒素抑制蛋白(DtxR)和铁的负调控。本实验室的研究表明，依赖于血红素的hmuO启动子的激活是通过一个两组分的信号转导系统进行的，该系统需要chra和chrS基因，这两个基因分别编码一个反应调节因子和一个传感器激酶。CHRS蛋白被认为是为了检测细胞外环境中的血红素，并通过磷酸转移机制将这一信号传递给Chra蛋白，然后Chra蛋白激活hmuO基因的转录。在白喉隐翅虫中，利用血红素和血红蛋白作为铁源需要一种ABC(三磷酸腺苷结合盒)型转运系统。该系统中鉴定的三种蛋白质与来自革兰氏阴性细菌的参与血红素运输的蛋白质同源。HmuT蛋白被认为是血红素的细胞表面受体，体外实验表明它与血红素琼脂糖凝胶结合，是一种脂蛋白。这些发现表明，HmuT具有与血红素结合的能力，并进一步表明，这种假定的受体通过其N端的脂类部分锚定在细胞表面。