Evolution and acquistion of drug resistance in MRSA

MRSA耐药性的进化和获得

• 批准号: 7156971
• 负责人: ALEXANDER TOMASZ
• 金额: $ 51.83万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7156971
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Chromosomes; Binding Sites; Biochemical; Biochemical Genetics; Biological; Biology; California; Cell Wall; Cells; Cellular biology; Clinical; Collaborations; Communities; Complex; Computer Analysis; Confocal Microscopy; DNA Microarray Chip; DNA Microarray format; DNA Sequence; DNA Sequence Rearrangement; Data Analyses; Development; Devices; Drug resistance; Evaluation; Evolution; Follow-Up Studies; Gene Expression; Gene Mutation; Genes; Genetic Crosses; Genetic Determinism; Genetic Transcription; Genomics; Homologous Gene; Hospitals; Human; Institutes; Investigation; Joints; Laboratories; Lactams; Lead; Left; Methods; Microarray Analysis; Molecular Profiling; Monobactams; Morphology; Mutation; Nature; Numbers; Open Reading Frames; Pathogenesis; Pathway interactions; Patients; Pattern; Peptidyltransferase; Phenotype; Point Mutation; Process; Proteins; Purpose; RNA; Rate; Recruitment Activity; Regulation; Regulator Genes; Regulon; Relative (related person); Research; Research Personnel; Research Project Grants; Research Proposals; Resistance; Rosa; Series; Site; Source; Staging; Staining method; Stains; Staphylococcus aureus; Structure; Techniques; Testing; Universities; Vancomycin; Vancomycin Resistance; Vancomycin-resistant S. aureus; analytical tool; antimicrobial drug; base; beta-Lactam Resistance; biomathematics; chemotherapy; commensal animal; genome sequencing; in vivo; insight; methicillin resistant Staphylococcus aureus; microbial; mutant; pathogen; pressure; programs; reconstruction; research study; resistance mechanism; synthetic enzyme

DESCRIPTION (provided by applicant): Bacterial pathogens rose to the challenge of antimicrobial agents by developing drug resistance through two types of mechanisms (i) an adaptive mechanism in which the bacterial pathogen "left to it's own devices" undergoes genetic changes (point mutations, regulatory changes, rearrangements etc.) under the selective pressure of the antibiotic. One may refer to this process as evolution of drug resistance. The second type of mechanism involves acquisition of resistance determinants from heterologous sources and incorporation of these into the regulatory circuitry of the particular pathogen. This research proposal will dissect genetic, biochemical, regulatory and cell biological aspects of resistance mechanisms in S. aureus concentrating on two of the most important antimicrobial agents used against this bacteria: b-lactam antibiotics and vancomycin. Both beta-lactam antibiotics and vancomycin target the bacterial cell wall. Therefore, elucidation of the mechanism of drug resistance should also provide important insights into the mechanism and organization of cell wall biosynthesis and assembly. Project A: Mechanism of VISA type resistance in S. aureus. An evolutionary type of vancomycin resistance will be studied in a series of isogenic MRSA isolates that developed vancomycin resistance during chemotherapy of a patient. Complete DNA sequencing of the susceptible parental and the vancomycin resistant mutant isolate; computational analysis of sequence differences; characterization of gene expression profiles by DNA microarrays, localization of cell wall synthetic sites and genetic crosses should lead to a better understanding of the mechanism of resistance and allow reconstruction of stages in the evolutionary pathway leading to the vancomycin resistant phenotype. Project B: Expression of the van A gene complex in S. aureus. The mechanism of acquired vancomycin resistance will be examined by genetic, biochemical and RNA profiling experiments in a VRSA strain which carries both the enterococcal vanA gene complex and the b-lactam resistance gene mecA. Project C: Integration and functioning of the acquired mecA gene in the S. aureus host will be studied following up the finding of functional cooperation between the resistance protein PBP2A and the native PBP2 of S. aureus: Direct interaction between the two proteins; their co-localization at subcellular sites of wall biosynthesis and the possible co-regulation of expression of pbpB and the mecA determinant will be tested. Project D: From resistance gene to resistant phenotype. An evolutionary type of process appears to be critical for the "grafting" of the b-lactam resistance gene mecA into the S. aureus cells and for the capacity of the bacteria to express high level resistance - as manifested in the unique heterogeneous phenotype of many MRSA clinical isolates. Attempts will be made to identify the number, nature and mode of action of domestic genes which are recruited in this process - using genetic crosses and microarray profiling combined with data analysis by modern biomathematical approaches.

描述（由申请人提供）：细菌病原体通过两种机制产生耐药性，从而应对抗菌药物的挑战：（i）一种适应性机制，其中细菌病原体“自行其是”在抗生素的选择压力下经历基因变化（点突变、调节变化、重排等）。人们可以将这一过程称为耐药性的进化。第二种类型的机制涉及从异源来源获取抗性决定簇并将其纳入特定病原体的调节回路中。该研究计划将剖析金黄色葡萄球菌耐药机制的遗传、生化、调节和细胞生物学方面，重点关注针对该细菌的两种最重要的抗菌药物：β-内酰胺抗生素和万古霉素。 β-内酰胺抗生素和万古霉素都针对细菌细胞壁。因此，阐明耐药机制也应该为细胞壁生物合成和组装的机制和组织提供重要的见解。项目A：金黄色葡萄球菌VISA型耐药机制。将在一系列在患者化疗期间产生万古霉素耐药性的同基因 MRSA 分离株中研究万古霉素耐药性的进化类型。对易感亲本和万古霉素耐药突变株进行完整的 DNA 测序；序列差异的计算分析；通过DNA微阵列对基因表达谱进行表征、细胞壁合成位点的定位和遗传杂交应该可以更好地理解耐药机制，并允许重建导致万古霉素耐药表型的进化途径的各个阶段。项目 B：van A 基因复合物在金黄色葡萄球菌中的表达。将通过携带肠球菌 vanA 基因复合物和 b-内酰胺抗性基因 mecA 的 VRSA 菌株中的遗传、生化和 RNA 分析实验来检查获得性万古霉素抗性的机制。项目C：在发现金黄色葡萄球菌的抗性蛋白PBP2A和天然PBP2之间的功能合作之后，将研究获得的mecA基因在金黄色葡萄球菌宿主中的整合和功能：两种蛋白质之间的直接相互作用；将测试它们在壁生物合成的亚细胞位点的共定位以及 pbpB 和 mecA 决定簇表达的可能共同调节。项目D：从抗性基因到抗性表型。进化类型的过程似乎对于将β-内酰胺抗性基因mecA“移植”到金黄色葡萄球菌细胞中以及细菌表达高水平抗性的能力至关重要——正如许多MRSA临床分离株独特的异质表型所证明的那样。将尝试利用基因杂交和微阵列分析并结合现代生物数学方法的数据分析来确定在此过程中招募的国内基因的数量、性质和作用方式。