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（由申请人提供）：金黄色葡萄球菌是一种高毒力且广泛成功的病原体，据推测是人类疾病的最常见原因。目前，它是全球社区和医院获得性感染的主要病原体，在宿主体内的众多生态位中引起多种疾病。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，迫切需要彻底探索该生物体的致病机制。我们的小组之前发表了许多报告，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影响毒力的实体。在我们实验室对此类酶的影响进行筛选时，我们发现了细胞内亮氨酸氨肽酶（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，我们使用实验室和临床菌株、局部和全身感染以及人类和小鼠疾病模型证明了 pepZ 突变体的显着减毒作用。这些发现不应该被低估：很少有细胞内氨肽酶被证明有助于细菌毒力；使得金黄色葡萄球菌 LAP 非常独特。重要的是，应该指出的是，观察到的衰减（i）并不是由简单的生长缺陷引起的； (ii) 不是金黄色葡萄球菌中其他氨肽酶的共同特征。通过酶促作用，氨肽酶将 N 端氨基酸从蛋白质底物上裂解下来。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标，导致适应性下降和发病机制。这得到了初步数据的支持，该数据揭示了 pepZ 破坏后细胞内和细胞外蛋白质组的变化。因此，我们将通过以下方式探索 LAP 对金黄色葡萄球菌疾病病因的贡献： 1. 识别金黄色葡萄球菌细胞内的 LAP 靶标：这将通过在使用蛋白质组学工具识别病理生理学底物之前绘制 LAP 的扩展底物特异性来实现。还将探讨 LAP 与已识别底物相互作用的亲和力和反应动力学。然后，我们将 2. 确定 LAP 和 PrsA 对毒力决定簇分泌的影响：初步数据表明，pepZ 突变体中的分泌组受到显着影响，这可能是由靶向 PrsA 折叠酶的 LAP 介导的。由于已知 PrsA 介导多种革兰氏阳性病原体的蛋白质分泌和毒力，因此在评估两种蛋白质在毒力决定子分泌中的作用之前，我们将探讨 LAP 对 PrsA 加工和运输的影响。我们认为，这项分析将为新型酶提供独特的见解，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。