The Role of Extracellular Proteases in CA-MRSA Infections

描述（由申请人提供）：金黄色葡萄球菌是一种高毒力和广泛成功的病原体，据推测是人类疾病的最常见原因。目前，它是世界范围内医院感染的主要病原体，在宿主体内的大量生态位中引起各种疾病。由于不断出现多重耐药分离株（MRSA），包括对最后手段糖肽抗生素（VRSA）耐药的分离株，金黄色葡萄球菌也是一个主要的公共卫生问题。除此之外，在过去10年中，健康受试者中严重侵袭性金黄色葡萄球菌感染的病例出现了令人不安的迅速增加，缺乏任何易感因素或与医疗保健机构的联系。显然，MRSA的高毒力菌株已经在社区中进化（CA-MRSA），具有独特的毒力因子和抗性性状的组合。CA-MRSA菌株的毒力和传播增强的原因尚不清楚；然而，最近的研究表明，这可能是agr调节毒素过度分泌的结果。其中包括细胞外蛋白酶，我们和其他人已经证明，与HA-MRSA分离株相比，CA-MRSA菌株中细胞外蛋白酶过量产生。尽管我们小组的报告证明了细胞外蛋白酶对疾病病因的贡献，但它们在金黄色葡萄球菌感染中作为关键毒力决定因素的作用仍不清楚。因此，考虑到CA-MRSA感染目前的临床意义，以及这些菌株高分泌细胞外蛋白酶的事实，我们建议明确探索这些酶在金黄色葡萄球菌发病机制中的作用。为此，我们将：1。探讨细胞外蛋白酶在金黄色葡萄球菌生理和毒力决定稳定性中的作用。这将通过：i)产生一个CA-MRSA先导菌株（USA300）的衍生物，其中4个主要蛋白酶位点都被删除。该菌株将用于：ii)表型表征这些酶对金黄色葡萄球菌生长和生理的体外贡献；iii)利用蛋白质组学分析分泌和细胞壁蛋白质组的变化，探讨外蛋白酶对毒力决定稳定性的影响。在体外确定细胞外蛋白酶的影响后，我们将使用我们的突变菌株来评估它们对体内毒力的贡献。为实现这一目标，我们将：探讨细胞外蛋白酶对CA-MRSA发病机制的影响。这将通过：i)评估细胞外蛋白酶对金黄色葡萄球菌皮肤和软组织感染的作用；ii)利用小鼠脓毒症和传播模型探索这些酶在严重侵袭性疾病中的作用。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a highly virulent and widely successful pathogen, which is speculated to be the most common cause of human disease. Currently, it is the leading agent of nosocomial infections worldwide, causing a variety of ailments in a plethora of ecological niches within the host. S. aureus is also a major public health concern due to the continued emergence of multi-drug resistant isolates (MRSA), including those resistant to last resort glycopeptide antibiotics (VRSA). In addition to this, over the last 10 years, there has been a disturbing and meteoric increase in cases of severe invasive S. aureus infections in healthy subjects, lacking any predisposing factors or connections to healthcare settings. Apparently, hypervirulent strains of MRSA have evolved in the community (CA-MRSA), possessing unique combinations of virulence factors and resistance traits. The reason for the enhanced virulence and transmission of CA-MRSA strains is unclear; however recent work suggests that it may be the result of hypersecretion of agr regulated toxins. Included amongst these are extracellular proteases, which ourselves and others have shown to be overproduced in CA-MRSA strains compared to HA-MRSA isolates. Despite reports by our group demonstrating the contribution of extracellular proteases to disease causation, their role as key virulence determinants in S. aureus infections is still unclear. Therefore, given the current clinical significance of CA-MRSA infections, and the fact that these strains hyper secrete extracellular proteases, we propose to definitively explore the contribution of these enzymes to S. aureus pathogenesis. To achieve this we will: 1. Investigate the role of extracellular proteases in S. aureus physiology and virulence determinant stability. This will be carried out by: i) Generating a derivative of the leading CA-MRSA strain (USA300) in which each of the 4 major protease loci has been deleted. This strain will then be use to: ii) Phenotypically characterize the in vitro contribution of these enzymes to S. aureus growth and physiology; and iii) Explore the effect of exoproteases on virulence determinant stability using proteomics to analyze alterations in secreted and cell wall proteomes. Having determined the impact of the extracellular proteases in vitro we will then use our mutant strain to assess their contribution to virulence in vivo. To achieve this we will: 2. Explore the impact of extracellular proteases on CA-MRSA pathogenesis. This will be carried out by: i) Assessing the contribution of extracellular proteases to S. aureus skin and soft tissue infections; and ii) Exploring the role of these enzymes in severe invasive disease using a murine model of sepsis and dissemination. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human infection. With the continued emergence of multi-drug resistant isolates of S. aureus (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal seeks to understand the contribution of secreted proteases to S. aureus disease causation.