Cell envelope synthesis and antibiotic resistance in Staphylococcus aureus

金黄色葡萄球菌的细胞包膜合成和抗生素耐药性

• 批准号: 9907566
• 负责人: Thomas McCabe Bartlett
• 金额: $ 6.53万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907566
• 关键词: 3-Dimensional; Address; Affinity; Animal Model; Antibiotic Resistance; Antibiotics; Area; Bacillus subtilis; Bacteria; Biochemical; Biogenesis; Biological Assay; Biology; Cell Separation; Cell Wall; Cell division; Cells; Cellular Morphology; Cellular biology; Chimeric Proteins; Chromosome Segregation; Co-Immunoprecipitations; Communities; Complement; Complex; Containment; Coupled; Cytology; Cytolysis; Dangerousness; Defect; Development; Drug Targeting; Drug resistance; Ensure; Enzymes; Face; Fellowship; Fluorescence-Activated Cell Sorting; Foundations; Future; Genes; Genomics; Growth; Healthcare Systems; Hospitals; In Vitro; Infection; Interphase Cell; Knowledge; Laboratories; Laboratory Research; Lactams; Libraries; Life; Mass Spectrum Analysis; Methicillin; Methicillin Resistance; Microscopy; Modeling; Monobactams; Morphogenesis; Morphology; Pathogenesis; Pathway interactions; Penicillin-Binding Proteins; Peptidoglycan; Peptidyltransferase; Phenotype; Play; Polymers; Polysaccharides; Process; Proteins; Research; Resistance; Rod; Role; Rotation; Shapes; Site; Staphylococcus aureus; Staphylococcus aureus infection; Testing; Work; antimicrobial; base; beta-Lactam Resistance; beta-Lactams; cell envelope; combat; crosslink; deep sequencing; design; drug resistant pathogen; experimental study; methicillin resistant Staphylococcus aureus; mutant; novel; novel therapeutics; pathogen; pathogenic bacteria; personalized approach; pressure; protein crosslink; resistance factors; small molecule; transposon sequencing; virtual; yeast two hybrid system

PROJECT SUMMARY Staphylococcus aureus is a Gram-positive opportunistic pathogen responsible for life-threatening infections in hospitals and communities alike. Especially concerning are methicillin-resistant S. aureus (MRSA) strains, which are resistant to -lactam antibiotics that target cell wall synthesis. Most MRSA strains also carry additional resistance markers rendering them resistant to multiple antibiotics, so treatment options are limited. Therefore, there is an urgent need to develop new antimicrobial therapies that are effective against S. aureus. Given that the cell envelope is the target of our first and best antimicrobials, studies aimed at understanding the mechanisms responsible for its assembly promise to uncover new vulnerabilities that can be targeted by future antimicrobial therapies. The proposed research will address two fundamental areas of S. aureus cell envelope assembly and morphogenesis. First, I address the mechanism by which methicillin-resistance factor PBP2a (a class b penicillin- binding protein, or bPBP) works with the rest of the cell wall synthesis machinery to promote -lactam resistance. -lactams like methicillin normally function by inhibiting the transpeptidase activity of bPBPs, which are essential for forming cell wall crosslinks and resisting turgor pressure. Recent research from my host laboratories and others has demonstrated that bPBPs act in complex with so-called ‘separation, elongation, division, and sporulation’ (SEDS) proteins, crosslinking new peptidoglycan polymerized by SEDS proteins into the growing cell wall. My preliminary results indicate that methicillin-insensitive PBP2a may function by replacing a methicillin- sensitive bPBP in a complex with the SEDS protein FtsW (the “partner-swapping” hypothesis), restoring cell wall synthesis in the face of -lactam challenge. Second, I will design and employ high-throughput cytological screens to identify novel cell envelope biogenesis factors in S. aureus. In spite of the great importance and intensive study of S. aureus cell envelope, so far it has not been the subject of any such screen. Here, I utilize fluorescence- activated cell sorting (FACS) to screen a transposon library for envelope biogenesis defects, followed by deep sequencing of the transposon-genomic junctions of isolated mutants (Tn-seq). This approach has identified many novel mutants with an enhanced rate of cell lysis and with cell separation defects. Preliminary characterization suggests that these newly identified factors play roles in chromosome segregation, division site placement, and cell wall synthesis. The detailed characterization of many of these factors will likely continue beyond the period of this fellowship to form foundational projects in my own research laboratory, as well as provide potential targets for future small molecule antimicrobial therapies. The specific aims of this F32 application are to: AIM 1: Determine how PBP2a integrates into the native cell wall synthetic machinery. AIM 2: Identify novel factors that function in cell envelope biogenesis using cytological screens.

项目摘要 金黄色葡萄球菌是一种革兰氏阳性的机会性病原体，负责威胁生命的感染 医院和社区。特别是关于甲氧西林的金黄色葡萄球菌（MRSA）菌株的尤其是 对靶细胞壁合成的-内酰胺抗生素具有抗性。大多数MRSA菌株也携带更多 电阻标记物使其对多种抗生素具有抵抗力，因此治疗选择受到限制。所以， 迫切需要开发针对金黄色葡萄球菌有效的新抗菌疗法。鉴于 细胞包膜是我们第一个也是最好的抗菌剂的靶标，旨在了解机制 负责其集会承诺，揭示新的漏洞，这些漏洞可以由未来的抗菌剂来针对 疗法。拟议的研究将针对金黄色葡萄球菌的两个基本组装和 形态发生。首先，我解决了甲氧基耐药因子PBP2A的机制（A类B级青霉素 - 结合蛋白（BPBP）与其他细胞壁合成机制一起促进腹膜抗性。 -乳酰胺像甲氧西林通常通过抑制BPBP的转肽酶活性来起作用，这是必不可少的 用于形成细胞壁交联并抵抗thrgor压力。我的主人实验室的最新研究和 其他人已经证明，BPBP具有所谓的“分离，伸长，分裂和 孢子型蛋白（SED）蛋白质，通过SEDS蛋白聚合到生长 细胞壁。我的初步结果表明，对甲氧西林不敏感的PBP2A可能通过取代甲氧西林 - SEDS蛋白FTSW（“合作伙伴交换”假设）中的敏感BPBP，恢复细胞壁 面对lactam挑战的合成。第二，我将设计和员工高通量细胞学屏幕 鉴定金黄色葡萄球菌中的新细胞包膜生物发生因子。尽管非常重要和密集 对金黄色葡萄球菌细胞包膜的研究，到目前为止，这并不是任何此类屏幕的主题。在这里，我利用荧光 - 激活的细胞分选（FACS）以筛选转座子库，以解决包膜生物发生缺陷，然后深 分离突变体（TN-SEQ）的转座子基因组连接的测序。这种方法已经确定了许多 新型突变体具有增强的细胞裂解速率和细胞分离缺陷。初步表征 表明这些新确定的因素在染色体隔离，部门位置和 细胞壁合成。这些因素中许多因素的详细表征可能会持续到该时期之后 在我自己的研究实验室中构成基础项目的奖学金，并提供潜在的目标 对于将来的小分子抗菌疗法。该F32应用程序的具体目的是： AIM 1：确定PBP2A如何集成到天然细胞壁合成机械中。 目标2：确定使用细胞学筛选在细胞包膜生物发生中起作用的新因素。