Characterization of C. diptheria virulence determinants

摘要：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)表征参与白喉隐翅虫中血红素运输的因素；3)开发白喉隐翅虫的遗传工具，以更准确地定义各种遗传系统的功能。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。一种ABC(三磷酸腺苷结合盒)运输系统参与了利用血红素和血红蛋白作为白喉弯曲菌及其相关病原菌的铁源。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蛋白是一种脂蛋白，被认为是血红素的细胞表面受体。建立了白喉弧菌和溃烂弧菌明确突变的构建系统。编码血红素受体hmuT的基因在这两种棒状杆菌中都存在突变。溃烂梭菌中hmuT基因的破坏导致不能利用血红素作为铁源，而白喉隐翅虫中hmuT的失活对血红素的运输没有影响，这表明该物种具有额外的血红素运输机制。溃烂弧菌的血红素转运基因也被克隆和测序，与白喉弯曲菌的血红素转运基因有大约70-80个同源性。

Summary: 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) the characterization of factors involved the transport of heme in C. diphtheriae and 3) the development of genetic tools in C. diphtheriae to more accurately define the function of various genetic systems. 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. An ABC (ATP-binding cassette) type transport system is involved in the utilization of heme and hemoglobin as iron sources in C. diphtheriae and the related pathogen C. ulcerans. The three proteins identified in this system are homologous to proteins involved in heme transport from gram-negative bacteria. The HmuT protein, which is a lipoprotein, is proposed to be the cell surface receptor for heme. A system for constructing defined mutation was developed for both C. diphtheriae and C. ulcerans. Mutations in the gene encoding the heme receptor, hmuT, were constructed in both Corynebacterium species. Disruption of the hmuT gene in C. ulcerans resulted in an inability to use heme as an iron source, while inactivation of hmuT in C. diphtheriae had no effect on heme transport, suggesting this species has an additional mechanism for transporting heme. The heme transport locus from C. ulcerans was also cloned and sequenced and was shown to be approximately 70-80 homologous to the heme transport locus from C. diphtheriae.