Regulation of hemolysin genes in Bacillus

描述(申请人提供)：炭疽芽孢杆菌是炭疽病的病原体，对其毒力因子的研究主要集中在质粒编码的胶囊以及致死性和水肿性毒素。关于形成孔洞的溶血素的研究报道很少。有限研究的一个主要原因是炭疽杆菌被普遍认为是一种非溶血性病原体，尽管溶血素基因存在于基因组中。然而，最近的研究表明，炭疽杆菌在一定条件下可以产生溶血素。另一种非溶血性芽胞形成菌枯草芽孢杆菌也携带可能的溶血素基因。该研究计划的长期目标是调查溶血素是否在炭疽杆菌的致病性中发挥作用，例如成功地从巨噬细胞逃逸到细胞外环境。拟议的研究将旨在了解溶血素等毒力因子是如何随着环境变化和调控基因的单一突变而诱导的。“潜在”毒力基因的激活可能会对各种细菌感染产生强烈影响。本研究的具体目的是：1)通过基因组图谱和DNA序列分析，筛选能激活枯草杆菌溶血素基因(S)的调节性突变(S)。2)通过与溶血素基因启动子的转录LacZ融合，研究β-溶血素基因的调控突变和氧限制的诱导机制。3)研究硝酸盐对枯草杆菌β-溶血素基因厌氧表达和炭疽杆菌需氧/厌氧表达的刺激作用。硝酸盐依赖诱导需要硝酸还原酶活性的假设将通过使用枯草杆菌硝酸还原酶突变体和测量炭疽杆菌需氧培养的硝酸还原酶活性来检验。将编码C反应蛋白家族转录调节因子(S)的炭疽杆菌基因(S)导入枯草杆菌，以确定该调控因子是否能够在有氧条件下激活呼吸硝酸还原酶操纵子的表达，从而导致枯草杆菌中β-溶血素的有氧诱导。

DESCRIPTION (provided by applicant): Studies of virulence factors of Bacillus anthracis, the causative agent of anthrax, have mainly focused on plasmid-encoded capsule as well as lethal and edema toxins. Very few studies have been reported on pore-forming hemolysins. A primary reason for the limited studies is that B. anthracis is generally accepted as a nonhemolytic pathogen, although hemolysin genes are present in the genome. However, recent studies have shown that B. anthracis can produce hemolysin under certain conditions. Another nonhemolytic sporeformer Bacillus subtilis also carries putative hemolysin genes. The long-term objectives of the research plan are to investigate whether hemolysin plays a role in pathogenicity of B. anthracis such as successful escape from macrophage into extracellular milieu. The proposed studies will aim at understanding how virulence factors such as hemolysins are induced in response to environmental changes and as a result of a single mutation in regulatory genes. The activation of 'latent' virulence genes could have a strong impact on various bacterial infections. The specific aims of the proposal are: 1) To identify regulatory mutation(s) that activate ?-hemolysin gene(s) in B. subtilis by genome mapping and DNA sequence analysis. 2) To characterize the Induction mechanism of the ?-hemolysin gene by the regulatory mutation and by oxygen limitation using a transcriptional lacZ fusion to the promoter of the hemolysin gene. 3) To investigate the stimulatory effect of nitrate on anaerobic expression of ?-hemolysin gene in B. subtilis and on aerobic/anaerobic expression in B. anthracis. The hypothesis that nitrate-dependent induction requires nitrate reductase activity will be examined by using a B. subtilis nitrate reductase mutant and measuring nitrate reductase activity of aerobic B. anthracis cultures. B. anthracis gene(s) encoding putative transcriptional regulator(s) of the CRP-family will be introduced into B. subtilis to determine whether the putative regulator is able to activate expression of the respiratory nitrate reductase operon under aerobic conditions, and thus leading to the aerobic induction of ?-hemolysin in B. subtilis.