Evolution and acquistion of drug resistance in MRSA

描述（由申请人提供）：细菌病原体通过两种类型的机制产生耐药性，从而应对抗菌剂的挑战：（i）适应性机制，其中细菌病原体“听之任之”经历遗传变化（点突变、调控变化、重排等）。在抗生素的选择压力下人们可以将这一过程称为耐药性的演变。第二类机制涉及从异源来源获得抗性决定簇，并将这些抗性决定簇并入特定病原体的调节回路。本研究计划将从遗传、生化、调控和细胞生物学等方面剖析沙门氏菌耐药机制。金黄色葡萄球菌集中在两种最重要的抗微生物剂上：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靶向细菌细胞壁。因此，阐明耐药机制也应该提供重要的见解细胞壁的生物合成和组装的机制和组织。项目A：S.金黄色。将在一系列同基因MRSA分离株中研究万古霉素耐药性的进化类型，这些分离株在患者化疗期间产生万古霉素耐药性。敏感亲本和万古霉素耐药突变分离株的完整DNA测序;序列差异的计算分析;通过DNA微阵列表征基因表达谱，定位细胞壁合成位点和遗传杂交，应能更好地理解耐药机制，并重建导致万古霉素耐药表型的进化途径阶段。项目B：货车A基因复合物在沙门氏菌中的表达。金黄色。将通过在携带肠球菌vanA基因复合物和b-内酰胺耐药基因mecA的VRSA菌株中进行遗传、生化和RNA分析实验，检查获得性万古霉素耐药的机制。项目C：获得性mecA基因在S.金黄色葡萄球菌宿主的PBP 2A和天然PBP 2之间的功能合作的发现将被研究。金黄色：将测试两种蛋白质之间的直接相互作用;它们在壁生物合成的亚细胞位点处的共定位以及pbpB和mecA决定子的表达的可能的共调节。项目D：从抗性基因到抗性表型。一个进化类型的过程似乎是关键的“嫁接”的b-内酰胺耐药基因mecA到S。金黄色葡萄球菌细胞和细菌表达高水平耐药性的能力-如许多MRSA临床分离株的独特异质表型所示。将尝试确定在这一过程中招募的国内基因的数量，性质和作用模式-使用遗传杂交和微阵列分析结合现代生物数学方法的数据分析。

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.