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: 10323003
• 负责人: Thomas McCabe Bartlett
• 金额: $ 7.17万
• 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/10323003
• 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; crosslink; deep sequencing; design; drug resistant pathogen; experimental study; methicillin resistant Staphylococcus aureus; mutant; novel; novel therapeutics; opportunistic pathogen; 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致病菌，可引起危及生命的感染， 医院和社区一样。尤其值得关注的是耐甲氧西林的S。金黄色葡萄球菌（MRSA）菌株， 对靶向细胞壁合成的β-内酰胺类抗生素具有抗性。大多数MRSA菌株还携带额外的 耐药标记使他们对多种抗生素产生耐药性，因此治疗选择有限。因此，我们认为， 迫切需要开发新的抗链球菌有效的抗微生物疗法。金黄色。鉴于 细胞被膜是我们第一个也是最好的抗菌剂的目标，旨在了解机制的研究 负责其大会承诺，以发现新的弱点，可以针对未来的抗菌 治疗拟议的研究将解决两个基本领域的S。金黄色葡萄球菌细胞被膜组装体， 形态发生首先，我提出了耐甲氧西林因子PBP2a（一种B类青霉素， 结合蛋白或bPBP）与细胞壁合成机制的其余部分一起作用以促进β-内酰胺抗性。 β-内酰胺类如甲氧西林通常通过抑制bPBP的转肽酶活性起作用，这是必不可少的。 用于形成细胞壁交联和抵抗膨压。我所在实验室的最新研究， 其他人已经证明，bPBP与所谓的"分离、延伸、分裂和分裂"复合作用， 孢子形成"（SEDS）蛋白，将SEDS蛋白聚合的新肽聚糖交联到生长的 细胞壁我的初步结果表明，甲氧西林不敏感的PBP2a可能通过取代甲氧西林- 敏感的bPBP与SEDS蛋白FtsW复合（"伴侣交换"假说），恢复细胞壁 面临β-内酰胺挑战的合成。第二，我将设计和使用高通量细胞学筛选， 在S.金黄色。尽管有着巨大的重要性和密集的 S.金黄色葡萄球菌细胞包膜，到目前为止，它还没有任何这样的屏幕的主题。在这里，我利用荧光- 活化细胞分选（FACS）以筛选转座子文库的包膜生物发生缺陷，然后进行深度细胞分选。 分离的突变体的转座子-基因组连接的测序（Tn-seq）。这一方法发现了许多 具有增强的细胞裂解速率和细胞分离缺陷的新突变体。初步鉴定 表明这些新发现的因子在染色体分离、分裂位点的放置和 细胞壁合成对其中许多因素的详细描述可能会在本报告所述期间之后继续进行。 在我自己的研究实验室里形成基础项目，并提供潜在的目标， 用于未来的小分子抗菌治疗。F32应用的具体目标是： 目的1：确定PBP2a如何整合到天然细胞壁合成机制。 目的2：利用细胞学筛选鉴定在细胞包膜生物发生中起作用的新因子。