The mechanism of the beta-lactam resistance in Staphylococcus aureus

金黄色葡萄球菌β-内酰胺耐药机制

• 批准号: 10227664
• 负责人: Taeok Bae
• 金额: $ 39.63万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227664
• 关键词: ATP-Dependent Proteases; Affect; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Binding Proteins; Biological Assay; Cell Wall; Cell membrane; Chromosomes; Development; Drug resistance; Enzymes; Genes; Genome; Goals; Hypersensitivity; Infection; Lactams; Mediating; Membrane; Methicillin Resistance; Molecular; Monobactams; Mutagenesis; Mutation; Pathway interactions; Penicillin-Binding Proteins; Peptide Hydrolases; Peptidoglycan; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Pneumonia; Production; Resistance; Resistance development; Role; Sepsis; Skin Tissue; Soft Tissue Infections; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus aureus infection; Suppressor Genes; Suppressor Mutations; Testing; Therapeutic Agents; Toxic Shock Syndrome; base; beta-Lactam Resistance; beta-Lactams; design; enzyme activity; genome sequencing; infectious disease treatment; lipoteichoic acid; methicillin resistant Staphylococcus aureus; minimal risk; mutant; novel; novel therapeutics; overexpression; pathogenic bacteria; protein function; resistance mechanism; resistance mutation; resistant strain; skin disorder; transposon sequencing; yeast two hybrid system

Project Summary Cell-wall targeting beta-lactam antibiotics are the largest group of antibacterial agents and have been the mainstay for treatment of bacterial infections. However, most beta-lactam antibiotics cannot be used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections because MRSA produces a cell- wall synthesis enzyme PBP2A, which most beta-lactams cannot inactivate. Although PBP2A is the primary determinant of MRSA beta-lactam resistance, the overall beta-lactam resistance of MRSA is affected by additional bacterial factors such as FtsH, a protease in the cell membrane. Overexpression of FtsH makes MRSA sensitive specifically to beta-lactams. The beta-lactam-sensitizing effect of FtsH is due to its degradation of YpfP, an enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). The YpfP- degradation by FtsH causes the production of abnormally large LTA. On the other hand, increased production of normal LTA by deletion of the ftsH gene makes MRSA more resistant to beta-lactams. When the production of normal LTA is suppressed by inhibiting the production of the LTA synthesis enzyme LtaS, MRSA becomes hypersensitive to beta-lactams. Based on these results, this study hypothesizes that the large LTA lowers MRSA beta-lactam resistance by suppressing the enzyme activities of the cell-wall synthesis enzymes whereas the normal LTA is required for them. Even under the beta-lactam sensitizing conditions, MRSA can regain resistance to beta-lactams. Genome-sequencing of 26 such mutants showed that 16 genes are critical for MRSA beta-lactam resistance. The long-term goal of this project is to develop novel β-lactam potentiators against MRSA. The objective of this study is to understand how FtsH and the disturbance in LTA synthesis sensitize MRSA to β-lactams, and how MRSA regains β-lactam resistance. In aim 1, with the MRSA strains producing either the large LTA or reduced amount of the normal LTA, the cell wall synthesis steps affected by the LTA molecules will be determined. The effect of LTA on cell wall synthesis will be directly tested by a peptidoglycan-synthesis assay. Also, the bacterial two-hybrid analysis will determine whether the degradation of YpfP is modulated by FtsH-binding proteins. In aim 2, by generating each of the resistance mutations in the chromosome of MRSA, the mutations critical for MRSA β-lactam resistance will be determined. Also, transposon-mediated mutagenesis will identify the genes essential for MRSA to regain the β-lactam resistance. Finally, to test whether the roles of the 16 genes are universally conserved among S. aureus strains, resistant mutants will be identified from two more MRSA strains, and their genomes will be sequenced. Completion of this study will reveal the intricate network of the beta-lactam resistance regulators in MRSA and open the door to developing novel beta-lactam potentiators with minimal risk of resistance development in the treatment of MRSA infections. Such drugs are expected to reduce the burdens of antibiotic resistance.

项目概要 细胞壁靶向β-内酰胺抗生素是最大的一类抗菌药物，并且已成为最重要的抗菌药物。 治疗细菌感染的主要药物。然而，大多数 β-内酰胺类抗生素不能用于 治疗耐甲氧西林金黄色葡萄球菌 (MRSA) 感染，因为 MRSA 产生一种细胞- 壁合成酶 PBP2A，大多数 β-内酰胺无法使其失活。虽然 PBP2A 是主要的 MRSA β-内酰胺耐药性的决定因素，MRSA 的总体 β-内酰胺耐药性受以下因素影响： 其他细菌因子，例如 FtsH（细胞膜中的一种蛋白酶）。 FtsH 的过度表达使得 MRSA 对 β-内酰胺特别敏感。 FtsH 的 β-内酰胺增敏作用是由于其 YpfP 的降解，YpfP 是一种合成脂磷壁酸 (LTA) 锚分子的酶。 YpfP- FtsH 的降解导致异常大的 LTA 的产生。另一方面，产量增加 通过删除 ftsH 基因来消除正常 LTA 使 MRSA 对 β-内酰胺具有更强的抵抗力。生产时 通过抑制 LTA 合成酶 LtaS 的产生，抑制正常 LTA 的产生，MRSA 变成 对β-内酰胺过敏。基于这些结果，本研究假设较大的 LTA 会降低 通过抑制细胞壁合成酶的酶活性来抵抗 MRSA β-内酰胺 而他们则需要普通的 LTA。即使在 β-内酰胺致敏条件下，MRSA 也能 恢复对β-内酰胺的抵抗力。对 26 个此类突变体的基因组测序表明，16 个基因至关重要 MRSA β-内酰胺耐药性。该项目的长期目标是开发新型β-内酰胺增效剂 对抗 MRSA。本研究的目的是了解 FtsH 和 LTA 合成中的干扰如何 使 MRSA 对 β-内酰胺敏感，以及 MRSA 如何重新获得 β-内酰胺耐药性。目标 1，针对 MRSA 菌株 产生大量 LTA 或减少量的正常 LTA，细胞壁合成步骤受 LTA 分子将被确定。 LTA对细胞壁合成的影响将通过 肽聚糖合成测定。此外，细菌双杂交分析将确定降解是否 YpfP 的表达受 FtsH 结合蛋白的调节。在目标 2 中，通过在 MRSA 的染色体，将确定对 MRSA β-内酰胺耐药性至关重要的突变。还， 转座子介导的诱变将鉴定 MRSA 恢复 β-内酰胺耐药性所必需的基因。 最后，为了测试这 16 个基因的作用在金黄色葡萄球菌菌株中是否普遍保守，耐药菌株 将从另外两种 MRSA 菌株中鉴定出突变体，并对它们的基因组进行测序。完成 这项研究将揭示 MRSA 中 β-内酰胺耐药调节因子的复杂网络，并打开大门 开发新型 β-内酰胺增效剂，在治疗中将产生耐药性的风险降至最低 MRSA 感染。此类药物有望减轻抗生素耐药性的负担。