PASTA probes: Defining the role of the PASTA kinase Stk1 in S. aureus virulence gene expression using a chemical biology approach

PASTA 探针：使用化学生物学方法定义 PASTA 激酶 Stk1 在金黄色葡萄球菌毒力基因表达中的作用

• 批准号: 9813183
• 负责人: Meghan Scobee Blackledge
• 金额: $ 41.18万
• 依托单位: HIGH POINT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813183
• 关键词: Adjuvant; Affect; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Attenuated; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Basic Science; Binding; Binding Sites; Biological Assay; Biological Process; Biology; Carbazoles; Cell Wall; Chemicals; Chemosensitization; Data; Development; Drug Targeting; Evaluation; Gene Expression; Gene Expression Regulation; Gene Proteins; Genes; Genetic Transcription; Goals; Homeostasis; In Vitro; Infection; Knowledge; Lead; Maps; Measurable; Mediating; Medical; Microbial Biofilms; Mission; Modeling; Molecular; Monobactams; National Institute of General Medical Sciences; Outcome; Pathway interactions; Penicillin-Binding Proteins; Penicillins; Phenotype; Phosphorylation; Phosphotransferases; Predisposition; Prevention; Proteins; Proteomics; Public Health; Regulatory Pathway; Research; Role; Serine; Staphylococcus aureus; Staphylococcus aureus infection; Structure; Structure-Activity Relationship; Threonine; Toxin; Transcriptional Regulation; United States National Institutes of Health; Variant; Virulence; Virulent; advanced disease; base; beta-Lactams; combat; design; extracellular; in vivo; inhibitor/antagonist; insight; methicillin resistant Staphylococcus aureus; mutant; new therapeutic target; novel; novel therapeutics; prevent; resistant strain; screening; small molecule; therapeutic target; tool; transcriptomics

Project Summary The regulatory kinase Stk1 controls genes involved in antibiotic resistance, biofilm formation, and toxin expression in virulent strains of Staphylococcus aureus. Inhibition of Stk1 in vitro potentiates methicillin-resistant S. aureus (MRSA) to b-lactam antibiotics and inhibits biofilm formation, making it an attractive target for the development of novel antibiotic adjuvant and antibiofilm therapies. However, studies of stk1 deletion mutants in vivo have many raised questions about the role of Stk1 in infection. In some MRSA strains and infection models, deletion of stk1 attenuates infection, whereas in other models and strains the stk1 deletion mutant displays enhanced virulence. Because only a few substrates of Stk1 have been identified, it is difficult to predict the full consequences of Stk1 inhibition. Additionally, strain-specific differences in the downstream genes whose expression is affected by Stk1 phosphorylation has prevented comprehensive understanding of Stk1-mediated gene expression in S. aureus. Our long-term goal is to develop novel anti-virulence treatments to combat persistent and antibiotic resistant bacterial infections. Our overall objectives in this proposal are to develop Stk1 inhibitors as chemical probes that can identify strain-specific differences in Stk1 inhibition and expand our knowledge of Stk1-mediated virulence gene expression in medically-relevant strains of S. aureus. Specifically, we will use our designed chemical probes to interrogate the inhibitor binding site of Stk1 across several strains of S. aureus to elucidate strain-specific differences that could affect development of broadly active inhibitors. Lead probes will then be used to identify novel antibiotic classes whose susceptibility is affected by Stk1 activity. Finally, we will use a combined transcriptomic and proteomic approach to identify novel Stk1 substrates and map downstream genes whose expression is affected by Stk1. Together, this will enhance existing knowledge of virulence pathways and their regulatory mechanisms in S. aureus. These results will have a significant impact as they will expand our understanding of gene regulation in S. aureus and provide necessary information for evaluation of Stk1 as a therapeutic target, thereby providing crucial new information for the development of novel antibacterial therapies.

项目摘要 调节激酶Stk 1控制参与抗生素耐药性、生物膜形成和毒素的基因 在金黄色葡萄球菌的强毒株中表达。体外抑制Stk 1增强甲氧西林耐药性 S.金黄色葡萄球菌（MRSA）对b-内酰胺抗生素的敏感性，并抑制生物膜的形成，使其成为一个有吸引力的目标， 开发新型抗生素佐剂和抗真菌膜疗法。然而，stk 1缺失突变体的研究 在体内的研究中，对Stk 1在感染中的作用提出了许多问题。在某些MRSA菌株和感染中 在其他模型和菌株中，stk 1缺失突变体可减弱感染，而在其他模型和菌株中， 显示出增强的毒性。由于Stk 1的底物很少，因此很难预测 Stk 1抑制的全部后果。此外，下游基因的菌株特异性差异， 表达受Stk 1磷酸化的影响，这阻碍了对Stk 1介导的 基因在S.金黄色。我们的长期目标是开发新的抗病毒治疗方法， 持续性和抗生素耐药性细菌感染。我们在本建议书中的总体目标是开发Stk 1 抑制剂作为化学探针，可以识别Stk 1抑制的菌株特异性差异，并扩大我们的研究范围。 Stk 1介导的医学相关的S.金黄色。具体地说， 我们将使用我们设计的化学探针在几个菌株中询问Stk 1的抑制剂结合位点 色葡萄金黄色葡萄球菌，以阐明菌株特异性差异，可能会影响广泛活性抑制剂的发展。 然后将使用铅探针来鉴定新的抗生素类别，其敏感性受Stk 1活性的影响。 最后，我们将使用转录组学和蛋白质组学相结合的方法来鉴定新型Stk 1底物， 定位受Stk 1影响的下游基因。总之，这将增强现有的知识 S.的毒力途径及其调节机制。金黄色。这些结果将产生重大影响 因为它们将扩展我们对S.并提供必要的信息， 评估Stk 1作为治疗靶点，从而为开发 新的抗菌疗法。