我们拟回答"促旋酶突变上位相互作用影响氟喹诺酮类药物抗性的分子机理"这一科学问题。此前，我们从中国结核病患者样本中分离的耐氟喹诺酮类药物的结核分枝杆菌中发现了一些促旋酶单突变和双突变。含有这促解旋酶突变体的结核菌的耐药程度差异相当大,单突变菌株的耐药程度有时比含有这两个单突变的双突变菌株显著高。我们猜测：这可能涉及突变位点之间的上位相互作用，而上位相互作用可能影响解旋酶DNA超螺旋活性，也可能影响细菌的适应度等其他方面。具体涉及哪种分子机理则有待研究。我们将采用重组的含有突变的促旋酶进行体外酶活性、药物结合能力分析，以及含有突变酶的重组耻垢分枝杆菌及临床分离突变菌株分析上位相互作用影响耐药性是通促解旋酶活性还是影响适应度等更全面的效应。这将可能从生化层面揭示促旋酶的结构-功能关系，有助于筛选出耐药强弱诊断的新分子标记，指导临床个性化用药，也有助于指导新一代FQ药物的设计和优化。

The scientific issue to be explored in this proposal is: the molecular basis underlying the effect of gyrase mutations epistatic interactions on the Mycobacterium tuberculosis fluoroquinolone resistance.We have some interesting findings previously from the fluoroquinolones resistant clinical M.tuberculosis isolates:both single and double mutations can be involved in the resistance. However, the double mutants being coincidently the combination of single mutants sometimes demonstrated much lower resistance than either single mutation, which defying the explanation by conventional wisdom. We hypothesize there might be a role of epistatic interaction of the double mutations.The role of epistasis might be multiple: DNA gyrase activiy alone or more holistic effect such as fitness cost might be influenced. It is important to define the detailed role involved in the epistasis.Both purified recombinant mutated gyrases and intact clinical isolates or recombinant M.smegmatis will be employed to address this issue. The activity of gyrase, its fluoroquinolones binding capabilites will be tested to define whether the enzyme activity or more comprehensive effect such as fitness cost is engaged for this epistasis. The answer might reveal novel structure-activity relationships of gyrase biochemically,conducive to novel biomarker for the degree of drug resistance to serve the personalized medications. This will inform future optimization of fluoroquinolones.