Exploring the Influence of an Intracellular Aminopeptidase on S. aureus Virulence

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，它是世界范围内社区和医院获得性感染的主要病原体，在宿主体内过多的生态位中引起各种疾病。随着抗生素耐药性的出现，以及对最后手段抗生素耐药的临床分离株的出现，迫切需要对这种微生物的致病机制进行深入的探索。我们的小组先前发表了许多报告，记录了分泌蛋白酶作为金黄色葡萄球菌感染的毒力因子的作用。然而，到目前为止，这种危险细菌的细胞内蛋白水解酶尚未被探索为毒力影响实体。在我们实验室对这些酶的影响进行筛选时，我们发现了细胞内亮氨酸氨基肽酶（LAP, pepZ）的突变体，该突变体强烈影响金黄色葡萄球菌的致病潜力。具体来说，我们通过实验室和临床菌株、局部和全身性感染以及人类和小鼠疾病模型证明了pepZ突变体的显著衰减。这些发现不应被低估：很少有细胞内氨基肽酶被证明与细菌毒力有关；使得金黄色葡萄球菌LAP非常独特。重要的是，应该注意到，观察到的衰减(i)不是由简单的生长缺陷引起的；并且（ii）不是金黄色葡萄球菌中其他氨基肽酶的共同特征。在酶学上，氨基肽酶从蛋白质底物上切割n端氨基酸。因此，我们的中心假设是pepZ突变细胞不能处理关键的细胞靶标，导致适应度下降和发病机制。初步数据显示，在pepZ破坏后细胞内和细胞外蛋白质组的改变支持了这一点。因此，我们将通过1.探讨LAP对金黄色葡萄球菌致病的贡献。鉴定金黄色葡萄球菌细胞内的LAP靶点：在使用蛋白质组学工具鉴定病理生理底物之前，这将通过绘制LAP的扩展底物特异性来实现。还将探讨LAP与鉴定底物相互作用的亲和性和反应动力学。然后我们会。确定LAP和PrsA对毒力决定因子分泌的影响：初步数据表明，pepZ突变体的分泌组受到显著影响，这可能是由LAP靶向PrsA折叠酶介导的。由于已知PrsA在多种革兰氏阳性病原体中介导蛋白质分泌和毒力，我们将探索LAP对PrsA加工和运输的影响，然后评估这两种蛋白质在毒力决定因素分泌中的作用。我们认为，这一分析将提供对一种新型酶的独特见解，使我们能够更好地了解金黄色葡萄球菌的致病特性，并可能有助于合理开发新的治疗方法。

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 a leading agent of both community and hospital- acquired infections worldwide, causing a variety of ailments in a plethora of ecological niches within the host. With the advent of antibiotic resistance, and the emergence of clinical isolates resistant to last resort antibiotis, a thorough exploration of the pathogenic mechanisms employed by this organism is urgently required. Our group has previously published a number of reports documenting the role of secreted proteases as virulence factors in S. aureus infections. However, until now, the intracellular proteolytic enzymes of this dangerous bacterium have not been explored as virulence affecting entities. During a screen in our lab on the impact of such enzymes, we identified a mutant in an intracellular leucine aminopeptidase (LAP, pepZ) that strongly influences the pathogenic potential of S. aureus. Specifically we demonstrated significant attenuation of pepZ mutants using laboratory and clinical strains, localized and systemic infections, and human and murine models of disease. These findings should not be undersold: few intracellular aminopeptidases have ever been shown to contribute to bacterial virulence; making the S. aureus LAP highly unique. Critically, it should be noted that the observed attenuation (i) does not result from a simple growth defect; and (ii) is not a common feature for other aminopeptidase in S. aureus. Enzymatically, aminopeptidases cleave N-terminal amino acids from protein substrates. Thus, our central hypothesis is that pepZ mutant cells fail to process key cellular targets, leading to decreased fitness, and pathogenesis. This is supported by preliminary data that reveals alterations in intracellular and extracellular proteomes upon pepZ disruption. Accordingly, we will explore the contribution of LAP to S. aureus disease causation by 1. Identifying LAP Targets within S. aureus Cells: This will be achieved by mapping the extended substrate specificity of LAP, before identifying pathophysiological substrates using proteomic tools. The affinity and reaction kinetics of LAP interaction with identified substrates will also be explored. We will then 2. Determine the influence of LAP and PrsA on virulence determinant secretion: Preliminary data suggests that secretomes are markedly affected in pepZ mutants, and that this might be mediated by LAP targeting the PrsA foldase. As PrsA is known to mediate protein secretion and virulence in a variety of Gram-positive pathogens, we will explore the influence of LAP on PrsA processing and trafficking, before assessing the role of both proteins in virulence determinant secretion. We contend that this analysis will provide a unique insight into a novel enzyme, enable us to better understand the pathogenic properties of S. aureus, and may aid in the rational development of new therapeutic treatments.