Evolution and acquistion of drug resistance in MRSA

描述(申请人提供)：细菌病原体通过两种机制产生抗药性，以应对抗菌剂的挑战：(I)一种适应机制，在这种机制中，细菌病原体“自行决定”经历基因变化(点突变、调节变化、重排等)。在抗生素的选择性压力下。人们可以将这一过程称为耐药性的进化。第二种机制包括从异源获得抗性决定因素，并将这些决定因素整合到特定病原体的调节电路中。这项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的问题，重点关注针对该细菌最重要的两种抗菌剂：B-内酰胺类抗生素和万古霉素。β-内酰胺类抗生素和万古霉素都以细菌细胞壁为靶标。因此，阐明耐药的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。对敏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；DNA微阵列对基因表达谱的表征、细胞壁合成位点和遗传杂交的定位应有助于更好地理解抗性机制，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：在金黄色葡萄球菌中表达van A基因复合体。获得性万古霉素耐药的机制将通过遗传、生化和RNA图谱实验在一株同时携带肠球菌vana基因复合体和β-内酰胺耐药基因mecA的VRSA菌株中进行。项目C：在发现金黄色葡萄球菌的抗性蛋白PBP2a和天然的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：两种蛋白之间的直接相互作用；它们在壁生物合成的亚细胞位置的共定位，以及PBPB和mecA决定簇表达的可能的共同调节。项目D：从抗性基因到抗性表型。一种进化类型的过程似乎对将β-内酰胺类耐药基因mecA“嫁接”到金黄色葡萄球菌细胞中以及细菌表达高水平耐药性的能力至关重要--许多MRSA临床分离株的独特异质表型就证明了这一点。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，并结合现代生物数学方法进行数据分析。

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.