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度毒性且广泛成功的病原体，被认为是人类疾病的最常见原因。目前，它是全球社区和医院感染的领先特工，在宿主内部的许多生态壁ches中引起了各种各样的疾病。随着抗生素耐药性的出现，以及抗临床分离株对最后的度假抗生素的抗性，迫切需要对这种生物使用的致病机制进行彻底探索。我们的小组以前发表了许多报告，记录了分泌蛋白酶作为金黄色葡萄球菌感染中的毒力因素的作用。然而，到目前为止，尚未将这种危险细菌的细胞内蛋白水解酶视为影响实体的毒力。在实验室对这种酶影响的屏幕上，我们确定了细胞内亮氨酸氨基肽酶（LAP，PEPZ）中的突变体，该突变体强烈影响金黄色葡萄球菌的致病潜力。具体而言，我们证明了使用实验室和临床菌株，局部和全身感染以及人类和鼠类疾病模型对PEPZ突变体的显着衰减。这些发现不应不足：很少有细胞内氨基肽酶被证明会导致细菌毒力。使S. Aureus圈高度独特。至关重要的是，应注意的是，观察到的衰减（i）不是由于简单的生长缺陷而造成的。 （ii）不是金黄色葡萄球菌中其他氨基肽酶的共同特征。从酶上，氨基肽酶从蛋白质底物中裂解N末端氨基酸。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，导致适应性降低和发病机理。这是由初步数据支持的，该数据揭示了PEPZ破坏时细胞内和细胞外蛋白质组织的变化。因此，我们将通过1。识别金黄色葡萄球菌内的圈靶靶标对金黄色葡萄球菌因果关系的贡献：这将通过在识别蛋白质组学工具识别病理生理底物之前绘制膝盖的扩展底物特异性来实现。还将探索与已识别的底物相互作用的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对毒力决定性分泌的影响：初步数据表明，pepz突变体在PEPZ突变体中受到明显影响，并且这可能是通过靶向PRSA折叠酶的单圈介导的。由于已知PRSA在多种革兰氏阳性病原体中介导蛋白质的分泌和毒力，因此，在评估两种蛋白质在毒力确定性分泌中的作用之前，我们将探索LAP对PRSA加工和运输的影响。我们认为，该分析将为一种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性质，并可以帮助新的治疗疗法的合理发展。