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: 10452551
• 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/10452551
• 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.

项目摘要 针对β-内酰胺类抗生素的细胞壁是最大的一组抗菌剂，一直是 治疗细菌感染的中流砥柱。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-内酰胺类药物不能使其失活。尽管PBP2a是主要的 MRSA对β-内酰胺类抗生素耐药的决定因素，MRSA对β-内酰胺类抗生素的总体耐药性受 其他细菌因子，如细胞膜中的一种蛋白酶FtsH。FtsH的过度表达使 MRSA对β-内酰胺类药物特别敏感。FtsH的β-内酰胺增敏作用是由于其 YpfP的降解，这是一种合成脂磷壁酸(LTA)锚分子的酶。Ypfp- FtsH的降解会导致异常大的LTA的产生。另一方面，产量增加 FtsH基因缺失使MRSA对β-内酰胺类抗生素更具耐药性。当制作的时候 通过抑制LTA合成酶LTAS的产生而抑制正常LTA的产生，MRSA成为 对β-内酰胺类药物过敏。基于这些结果，本研究假设较大的LTA会降低 抑制细胞壁合成酶活性对耐甲氧西林金黄色葡萄球菌耐药 而它们需要正常的LTA。即使在β-内酰胺类致敏条件下，MRSA也可以 对β-内酰胺类药物重新产生抗药性。对26个这样的突变体进行的基因组测序显示，有16个基因是关键的 用于耐甲氧西林金黄色葡萄球菌。该项目的长期目标是开发新型β-内酰胺增强剂。 对抗耐甲氧西林金黄色葡萄球菌。本研究的目的是了解FtsH和干扰在LTA合成中的作用 使耐甲氧西林金黄色葡萄球菌对β-内酰胺类抗生素敏感，以及耐甲氧西林金黄色葡萄球菌如何恢复对β-内酰胺类抗生素的耐药性。在目标1中，使用MRSA菌株 产生大量的LTA或减少正常LTA的量，细胞壁合成步骤受到 LTA分子将被确定。LTA对细胞壁合成的影响将直接通过 肽多聚糖合成试验。此外，细菌双杂交分析将确定是否降解 YpfP的表达受FtsH结合蛋白调控。在目标2中，通过在 对耐甲氧西林金黄色葡萄球菌β-内酰胺类抗生素耐药的关键突变进行分析。另外， 转座子介导的突变将识别耐甲氧西林金黄色葡萄球菌重新获得β-内酰胺类耐药性所必需的基因。 最后，为了测试这16个基因的作用是否在耐药的金黄色葡萄球菌菌株中普遍保守 将从另外两个MRSA菌株中鉴定出突变株，并对它们的基因组进行测序。完成 这项研究将揭示耐甲氧西林金黄色葡萄球菌中β-内酰胺类耐药调节剂的复杂网络，并打开大门 开发新型β-内酰胺类增效剂，使治疗癌症的耐药性风险降至最低 MRSA感染。这类药物有望减轻抗生素耐药性的负担。