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novel therapeutics; pathogen; release factor; resistant strain; rhomboid; screening; stem; tool; transposon sequencing; unpublished works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.

项目概要/摘要 在美国，抗生素耐药性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。许多用于治疗金黄色葡萄球菌的抗生素（包括 β-内酰胺类）均以生物发生为目标 主要通过抑制 PG 合酶来破坏必需肽聚糖 (PG) 细胞壁。作为β-内酰胺 随着耐药性的蔓延，确定新的抗生素靶标非常重要。其他参与构建 PG 的酶， 包括 PG 水解酶在内，由于其对于适应性、毒力和活性的重要性，成为有希望的候选者。 抗生素耐药性。鉴于水解酶具有潜在的破坏性，必须小心使用它们 受监管；破坏它们的调节是另一种抗生素策略。水解酶的调节机制是 开始被理解。我们的实验室最近在 S. 中鉴定出两种水解酶的直接蛋白质调节剂。 金黄色葡萄球菌。这些复合物的突变株都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-内酰胺再敏化的潜在目标 代理。第一个确定的调节因子是 ActH，它激活酰胺酶 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。这 第二个是 SpdC，控制氨基葡萄糖苷酶 SagB 的产物分布。在未发表的作品中，我们 提出 SagB-SpdC 作为 PG 释放因子，裂解新生 PG 链，将其与 膜并允许它们掺入细胞壁基质中。这些监管者都是他们的第一个 种类，初步的生物信息分析表明其他细菌中也存在类似的复合物。此外， ActH 和 SpdC 分别类似于菱形和 CAAX 蛋白酶，但它们的水解酶调节 功能不需要蛋白酶活性。这些调节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其机制 这些调节剂发挥作用并识别充当肽聚糖释放因子的其他酶。这些 进展将揭示杀死耐药细菌的新治疗途径。目标1将揭示其机制 ActH 如何激活 LytH。 LytH-ActH 络合和活性所需的最小结构域为 使用截断突变体确定。为了促进这些研究并以我们现有的化学工具为基础 实验室将开发一种连续、高通量的酰胺酶活性测定方法；该测定还将能够 未来筛选酰胺酶抑制剂。目标 2 将描述 SagB-SpdC 活性对 PG 底物的脂质并使用基于生物层干涉测量法识别 SpdC 上的脂质结合位点 底物结合测定以及底物与 SpdC 之间的交联实验。最后，目标 3 将 采用功能基因组学方法来识别其他可以在缺乏 PG 链的情况下释放 PG 链的酶 SagB-SpdC。这项工作将揭示 SagB-SpdC 如何在功能上连接到其他细胞过程， 揭示了金黄色葡萄球菌中可用于治疗的新漏洞。