Mechanistic Studies on Staphylococcal Quorum Quenching Natural Products

描述(申请人提供)：金黄色葡萄球菌是一种极具问题的病原体。在社区和医疗保健环境中的感染率都在上升，再加上它对抗生素的高度抗药性，这使得金黄色葡萄球菌成为首要的公共卫生问题。事实上，侵袭性耐甲氧西林金黄色葡萄球菌(MRSA)在美国造成的死亡人数超过艾滋病。然而，正在研发的新抗生素的数量正在减少，许多科学家呼吁发现和开发一类新的药物，这种药物可以介导微生物的致病性，而不是生长和生存。葡萄球菌群体感应通路由辅助基因调节系统(AGR)控制，是此类抗病原性药物发现努力的潜在靶点，因为它是葡萄球菌毒力的全球调节因子。在对意大利南部人治疗皮肤和软组织感染的补充和替代医疗(CAM)实践进行广泛研究后，对100多个植物样本进行了鉴定、收集、提取和抗葡萄球菌潜力检测。在这些测试中，包括了对β-溶血素的抑制筛选，？-溶血素是RNAIII的一种翻译蛋白产物，其产生通过agr群体感应途径进行调节。提取物134是从一种常见的树中提取的，含有可食用的果实和药用叶和树皮，发现在亚抑制浓度下对β-溶血素具有很强的剂量依赖性抑制作用。通过对AGR(类型I-IV)的荧光遗传报告的使用，证实了提取物134的剂量依赖的群体猝灭效应。这一活动是重要的，基于先前对agr基因敲除突变体的动物研究，这些突变体表明启动和维持皮肤感染模型的能力减弱。在这项拟议的研究中，我们试图提高我们对提取物134‘S群体猝灭作用的机制基础的理解，并使用体内模型评估这种抗毒力疗法的治疗相关性。这项研究将致力于四个具体目标：1)鉴定和结构鉴定提取物134中的活性成分(S)(或标准化标记化合物)；2)阐明观察到的群体猝灭效应的作用机制；3)测定活性成分(S)的药物代谢和药代动力学参数(Dm/Pk)；以及4)在小鼠模型上评价治疗金黄色葡萄球菌皮肤感染的疗效。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a highly problematic pathogen. Rates of infection in both the community and healthcare setting are on the rise, and coupled with its highly antibiotic-resistant nature, this makes S. aureus a top public health concern. In fact, invasive methicillin-resistant S. aureus (MRSA) is responsible for more deaths in the USA than AIDS. Nevertheless, the number of new antibiotic leads in the pipeline is diminishing, and many scientists have put out a call for the discovery and development of a new class of drugs which could mediate microbial pathogenicity rather than growth and survival. The staphylococcal quorum-sensing pathway, controlled by the accessory gene regulator (agr) system, is a potential target for such anti-pathogenic drug discovery efforts, as it serves as a global regulator of staphylococcal virulence. Following extensive studies on the complementary and alternative medical (CAM) practices of southern Italians in the treatment of skin and soft tissue infection, over 100 plant samples were identified, collected, extracted, and examined for their anti-staphylococcal potential. Among the tests included was a screen for the inhibition of??-hemolysin, a translational protein product of RNAIII, whose production is regulated through the agr quorum-sensing pathway. Extract 134, which is derived from a popular tree with edible fruits and medicinal leaves and bark, was found to exhibit a strong dose-dependent inhibition of ?-hemolysin at sub-inhibitory concentrations for growth. The dose-dependent quorum-quenching effects of Extract 134 were confirmed through the use of fluorescent genetic reporters for agr (types I-IV). This activity is important based upon previous animal studies with agr knockout mutants that show a diminished capacity to initiate and persist in a skin infection model. In the proposed study, we seek to improve our understanding of the mechanistic basis for Extract 134's quorum-quenching effects and evaluate the therapeutic relevance of such an anti-virulence therapy using in vivo models. The study will address four specific aims: 1) identification and structural elucidation of the active constituent(s) (or marker compounds for standardization) in Extract 134; 2) elucidation of the mechanism of action for the quorum-quenching effects observed; 3) determination of drug metabolism and pharmacokinetic parameters (DM/PK) of the bioactive constituent(s); and 4) evaluation of efficacy in treating S. aureus skin infection in a murine model.