IDENTIFICATION AND CHARACTERIZATION OF VIRULENCE DETERMI

该项目的目的是确定和表征白喉棒状杆菌的毒力决定因素，以便了解这种重要病原体如何引起疾病。白喉白喉毒素是白喉白喉的主要毒力决定因素，其表达受铁的调控，很可能还有其他毒力因子与白喉白喉毒素协同调控。在感染过程中获取铁的能力对许多细菌病原体致病至关重要。该实验室早期的研究表明，在白喉链球菌中，hmuO基因是利用血红素和血红蛋白中的铁所必需的。最近的研究集中在；1)了解hmuO基因调控的分子机制，2)hmuO蛋白的纯化和活性表征，3)白喉白喉血红素转运相关因素的表征。hmuO启动子的转录已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，并受到白喉毒素抑制蛋白（DtxR）和铁的负调控。本实验室的研究表明，hmuO启动子的血红素依赖性激活是通过一个双组分信号转导系统发生的，该系统需要chrA和chrS基因，它们分别编码应答调节因子和传感器激酶。ChrS蛋白可以检测细胞外环境中的血红素，并通过磷酸化机制将信号传递给ChrA蛋白，然后ChrA蛋白激活hmuO基因的转录。ABC （atp结合盒）型运输系统被证明是白喉白喉利用血红素和血红蛋白作为铁源所必需的。在该系统中鉴定的三种蛋白质与参与革兰氏阴性细菌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，在体外被证明与血红素琼脂糖结合，并且被证明是一种脂蛋白。这些发现表明HmuT具有结合血红素的能力，并进一步表明这种假定的受体通过其n端的脂质片段锚定在细胞表面。

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.