The Role of Extracellular Proteases in CA-MRSA Infections

描述（由申请方提供）：金黄色葡萄球菌是一种高毒力和广泛成功的病原体，推测是人类疾病的最常见原因。目前，它是全世界医院感染的主要病原体，在宿主体内过多的生态位中引起各种疾病。S.金黄色葡萄球菌也是一个主要的公共卫生问题，这是由于持续出现的多药耐药分离株（MRSA），包括那些对最后手段糖肽抗生素（VRSA）具有耐药性的分离株。除此之外，在过去的10年里，严重的侵袭性S。金黄色葡萄球菌感染的健康受试者，缺乏任何诱发因素或连接到医疗机构。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特征的组合。CA-MRSA菌株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调节毒素分泌过多的结果。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菌株中的细胞外蛋白酶过量产生。尽管我们小组的报告证明了胞外蛋白酶对疾病病因的贡献，但它们作为S.金黄色葡萄球菌感染仍不清楚。因此，鉴于CA-MRSA感染的当前临床意义，以及这些菌株超分泌胞外蛋白酶的事实，我们建议明确探索这些酶对S.金黄色葡萄球菌致病性。为了实现这一目标，我们将：1。研究胞外蛋白酶在S.金黄色葡萄球菌生理学和毒力决定簇稳定性。这将通过以下方式进行：i）产生前导CA-MRSA菌株（USA 300）的衍生物，其中4个主要蛋白酶基因座中的每一个都已缺失。然后将该菌株用于：ii）表型表征这些酶对S.金黄色葡萄球菌的生长和生理;和iii）使用蛋白质组学来分析分泌和细胞壁蛋白质组中的改变，探索外切蛋白酶对毒力决定簇稳定性的影响。在体外确定了细胞外蛋白酶的影响后，我们将使用我们的突变株来评估它们对体内毒力的贡献。为了实现这一目标，我们将：2。探讨细胞外蛋白酶对CA-MRSA发病机制的影响。这将通过以下方式进行：i）评估胞外蛋白酶对S.金黄色葡萄球菌皮肤和软组织感染;和ii）使用败血症和传播的鼠模型探索这些酶在严重侵袭性疾病中的作用。 公共卫生相关性：金黄色葡萄球菌是一种高毒性和广泛成功的病原体，被推测是人类感染的最常见原因。随着多重耐药菌株的不断出现，金黄色葡萄球菌（如MRSA），迫切需要了解这种致命病原体引起疾病的机制。该建议旨在了解分泌蛋白酶对S.金黄色葡萄球菌病的病因。

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.