Understanding the Regulation and Biological Roles of Peptidoglycan Hydrolases in Staphylococcus aureus

了解金黄色葡萄球菌肽聚糖水解酶的调节和生物学作用

• 批准号: 10532762
• 负责人: Julia Elaine Page
• 金额: $ 5.27万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10532762
• 关键词: Affinity; Amino Acids; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biogenesis; Bioinformatics; Biological; Biological Assay; Biology; Catalytic Domain; Cell Wall; Cell physiology; Cells; Cessation of life; Chemicals; Complex; Crystallization; Data; Defect; Dependence; Enzymes; Fluorescence; Future; Genes; Genetic; Genomic approach; Glucosaminidase; Goals; Growth; Homologous Gene; Hydrolase; In Vitro; Infection; Interferometry; Length; Life; Lipid A; Lipid Binding; Lipids; Maps; Measures; Medicine; Membrane; Molecular; Molecular Conformation; N-Acetylmuramoyl-L-alanine Amidase; Nature; Oxacillin; Pathway interactions; Penicillin Resistance; Penicillins; Peptide Hydrolases; Peptides; Peptidoglycan; Phylogenetic Analysis; Physiology; Polymers; Polysaccharides; Positioning Attribute; Procedures; Process; Protein Family; Protein Region; Proteins; Regulation; Research; Resolution; Role; Series; Site; Staphylococcus aureus; Staphylococcus aureus infection; Structure; Tail; Testing; Therapeutic; Virulence; Work; World War II; amidase; bacterial resistance; beta-Lactam Resistance; beta-Lactams; candidate validation; crosslink; design; experimental study; fitness; functional genomics; high throughput screening; inhibitor; innovation; mutant; novel; novel therapeutics; pathogen; release factor; resistant strain; rhomboid; screening; stem; tool; transposon sequencing; unpublished works

PROJECT SUMMARY/ABSTRACT The leading cause of antibiotic resistance-associated death in the US is the Gram-positive pathogen Staphylococcus aureus. Many antibiotics used to treat S. aureus, including the beta-lactams, target biogenesis of the essential peptidoglycan (PG) cell wall, predominantly by inhibiting the PG synthases. As beta-lactam resistance spreads, it is important to identify new antibiotic targets. Other enzymes involved in building PG, including the PG hydrolases, serve as promising candidates due to their importance for fitness, virulence, and antibiotic resistance. Given the potentially destructive nature of hydrolytic enzymes, they must be carefully regulated; disrupting their regulation is another antibiotic strategy. Mechanisms of hydrolase regulation are just beginning to be understood. Our lab has recently identified two direct protein regulators of hydrolases in S. aureus. Mutant strains of either of these complexes have growth and virulence defects, and they are particularly sensitive to the beta-lactam oxacillin. They are thus potential targets for beta-lactam re-sensitizing agents. The first regulator identified is ActH, which activates the amidase LytH. LytH-ActH cleaves stem peptides to control availability of PG substrates, regulating where new PG is made around the cell. The second, SpdC, controls the product distribution of the glucosaminidase SagB. In unpublished work, we propose that SagB-SpdC acts as a PG release factor, cleaving nascent PG strands to separate them from the membrane and allow their incorporation into the cell wall matrix. These regulators are each the first of their kind, and preliminary bioinformatic analyses suggest similar complexes exist in other bacteria. Furthermore, ActH and SpdC resemble the rhomboid and CAAX proteases respectively, but their hydrolase-regulating functions do not require protease activity. These regulator roles are novel functions for these ubiquitous families of proteins. The overarching goal of the proposed research is to uncover the mechanisms by which these regulators act and to identify additional enzymes that function as peptidoglycan release factors. These advances will reveal new therapeutic avenues to kill resistant bacteria. Aim 1 will uncover the mechanism of how ActH activates LytH. The minimum domains required for LytH-ActH complexation and activity will be determined using truncation mutants. To facilitate these studies and build on existing chemical tools from our lab, a continuous, high-throughput assay for amidase activity will be developed; this assay will also enable future screening for amidase inhibitors. Aim 2 will characterize the dependence of SagB-SpdC activity on the lipid of a PG substrate and identify the lipid binding site on SpdC, using a biolayer interferometry-based substrate binding assay and crosslinking experiments between the substrate and SpdC. Finally, aim 3 will employ a functional genomics approach to identify other enzymes that can release PG strands in the absence of SagB-SpdC. This work will uncover how SagB-SpdC is functionally connected to other cellular processes, revealing new vulnerabilities in S. aureus that can be therapeutically exploited.

项目总结/摘要 在美国，抗生素耐药相关死亡的主要原因是革兰氏阳性病原体 金黄色葡萄球菌。许多抗生素用于治疗S。金黄色葡萄球菌，包括β-内酰胺，靶向生物合成 的必需肽聚糖（PG）的细胞壁，主要是通过抑制PG酶。作为β-内酰胺 随着耐药性的扩散，重要的是要确定新的抗生素靶标。其他参与构建PG的酶， 包括PG水解酶，由于它们对适应性、毒力和 抗生素耐药性由于水解酶具有潜在的破坏性，因此必须小心使用。 监管;扰乱他们的监管是另一种抗生素策略。水解酶的调节机制 开始被理解。本实验室最近鉴定了两种直接调节S. 金黄色。这些复合物中的任一种的突变株具有生长和毒力缺陷，并且它们是 对β-内酰胺苯唑西林特别敏感。因此，它们是β-内酰胺再致敏的潜在靶点 剂.确定的第一个调节剂是ActH，其激活酰胺酶LytH。LytH-ActH切割茎 肽来控制PG底物的可用性，调节细胞周围新PG的产生位置。的 第二，SpdC，控制氨基葡糖苷酶SagB的产物分布。在未出版的作品中，我们 SagB-SpdC作为PG释放因子，切割新生的PG链，将其从细胞中分离出来。 膜，并允许它们掺入细胞壁基质。这些监管机构都是他们的第一个 种类，初步的生物信息学分析表明，类似的复合物存在于其他细菌中。此外，委员会认为， ActH和SpdC分别类似于菱形蛋白酶和CAAX蛋白酶，但它们的水解酶调节活性与蛋白酶的活性相似。 这些功能不需要蛋白酶活性。这些调节器的作用是这些无处不在的新功能 蛋白质家族拟议研究的总体目标是揭示 这些调节剂起作用并鉴定起肽聚糖释放因子作用的其它酶。这些 这些进展将揭示新的治疗方法来杀死耐药细菌。目标1将揭示 ActH如何激活LytH LytH-ActH复合和活性所需的最小结构域为 使用截短突变体测定。为了促进这些研究，并利用我们现有的化学工具， 实验室，一个连续的，高通量的测定酰胺酶活性将被开发;这种测定方法也将使 未来筛选酰胺酶抑制剂。目的2将表征SagB-SpdC活性对 PG底物的脂质，并确定SpdC上的脂质结合位点，使用基于生物层干涉法的 底物结合测定和底物与SpdC之间的交联实验。最后，aim 3将 采用功能基因组学方法来鉴定其他可以在缺乏PG链的情况下释放PG链的酶， SagB-SpdC这项工作将揭示SagB-SpdC是如何在功能上与其他细胞过程联系在一起的， 揭示了S.金黄色葡萄球菌，可用于治疗。