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

探索细胞内氨肽酶对金黄色葡萄球菌毒力的影响

• 批准号: 8827670
• 负责人: Lindsey Neil Shaw
• 金额: $ 21.99万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827670
• 关键词: Abscess; Acute suppurative arthritis due to bacteria; Address; Affect; Affinity; Amino Acids; Aminopeptidase; Antibiotic Resistance; Attenuated; Bacteria; Biological Assay; Blood; Body Weight decreased; Cells; Cleaved cell; Clinical; Community Hospitals; Data; Defect; Development; Disease; Disease model; Enzymes; Etiology; Foundations; Growth; Health; Human; Infection; Investigation; Kinetics; Knowledge; Laboratories; Leucine Aminopeptidase; Literature; Maps; Mediating; Modeling; Molecular; Multi-Drug Resistance; Mus; N-terminal; Nature; Nosocomial Infections; Organism; Pathogenesis; Peptide Hydrolases; Phenotype; Physiology; Process; Property; Protein Secretion; Proteins; Proteome; Proteomics; Publishing; Reaction; Report (document); Resistance; Resort; Role; Sepsis; Severities; Specificity; Staphylococcus aureus; Substrate Specificity; Synovitis; Systemic infection; Therapeutic; Virulence; Virulence Factors; Virulent; Work; attenuation; bone erosion; cellular targeting; design; extracellular; fitness; human disease; insight; macrophage; methicillin resistant Staphylococcus aureus; mutant; novel; novel therapeutics; pathogen; tool; trafficking

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.

描述(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，被认为是人类疾病最常见的原因。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，在宿主内过多的生态位中造成各种疾病。随着抗生素耐药性的出现，以及对抗生素耐药的临床分离株的出现，迫切需要对这种细菌的致病机制进行彻底的探索。我们小组此前发表了一些报告，记录了分泌型蛋白酶在金黄色葡萄球菌感染中作为毒力因子的作用。然而，到目前为止，这种危险细菌的胞内蛋白水解酶还没有被探索为影响毒力的实体。在我们实验室对这种酶的影响进行筛选的过程中，我们发现了细胞内亮氨酸氨基肽酶(LAP，PepZ)中的一个突变，它强烈影响金黄色葡萄球菌的致病潜力。具体地说，我们使用实验室和临床菌株、局部和系统感染以及人类和小鼠疾病模型证明了PepZ突变的显著减弱。这些发现不应被低估：很少有细胞内氨基肽酶被证明对细菌的毒力有贡献；这使得金黄色葡萄球菌LAP非常独特。关键的是，应该注意到，观察到的衰减(I)不是简单的生长缺陷造成的；(Ii)不是金黄色葡萄球菌其他氨基肽酶的共同特征。在酶的作用下，氨基肽酶将N末端的氨基酸从蛋白质底物上分解出来。因此，我们的中心假设是PepZ突变细胞无法处理关键的细胞靶点，导致适应性降低，并导致发病。这得到了初步数据的支持，这些数据揭示了PepZ中断后细胞内和细胞外蛋白质组的变化。因此，我们将通过1.识别金黄色葡萄球菌细胞内的LAP靶标：在使用蛋白质组学工具识别病理生理底物之前，通过定位LAP的扩展底物专一性来实现LAP在金黄色葡萄球菌疾病发病中的作用。还将探讨LAP与已鉴定底物相互作用的亲和力和反应动力学。确定LAP和PRSA对毒力决定簇分泌的影响：初步数据表明，PepZ突变体的分泌体受到显著影响，这可能是通过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菌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，这一分析将为我们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。