S2: Molecular Mechanisms Underlying Fluoroquinolone Susceptibility and Resistance

 描述(申请人提供)：抗生素耐药性是一个主要的世界性问题。氟喹诺酮类药物具有良好的疗效、生物利用度和最小的毒性，是一类非常重要的药物。Zechiedrich实验室的长期目标是确定革兰氏阴性细菌如何对抗菌剂做出反应并产生抗药性，并利用这一知识改进对抗生素耐药细菌感染的诊断，延长现有药物的用途，并帮助设计新的治疗方式。在之前的资助期间，我们构建了一个不断发展的符合HIPAA的甲骨文数据库，该数据库结合了自1999年以来收集的革兰氏阴性临床分离株的患者、医院和微生物学数据。我们发现，已知的氟喹诺酮耐药机制可解释大约一半的耐氟喹诺酮类临床分离株的耐药表型；因此，必须存在其他未知的耐药机制。为了揭示这些机制，我们开发了一种独特的方法，结合了基因组池、测序和SNP分析。我们发现了极具问题的新型临床分离株(除了两种抗生素外，对所有抗生素都有抗药性，而且对氟喹诺酮类药物的最低抑菌浓度非常高)。这些分离物的基因组偏离了参考基因组，无法定位到参考基因组。在所有其他氟喹诺酮耐药库中，我们在gyrA靶基因中发现了众所周知的SNP，并发现了三个与药物敏感的参考基因组相关的新SNP。我们还在173个基因中发现了283个SNPs，这些基因与氟喹诺酮类药物耐药和对其他特定抗菌药的耐药有关。相对于氟喹诺酮耐药的参考基因组，我们发现了35个与氟喹诺酮敏感表型相关的新SNPs。该提案的目的是：预测已识别的基因变体如何改变编码的蛋白质结构和功能，目标2：定义已发现的基因变体的功能，目标3：确定已发现的蛋白质变体的分子机制，以及目标4：继续发现与氟喹诺酮类药物敏感性或氟喹诺酮耐药表型相关的基因变体。我们的团队位于德克萨斯州休斯敦的德克萨斯医学中心，由一名分子微生物学家(Zechiedrich)、一名内科科学家(Hamill)、一名基因组学专家(Sucgang)、一名计算生物学家(Barth)和一名结晶学家(Lee)组成。在确定新药或新药的新靶点之前，需要在实现这些目标方面获得基础知识。更好地理解阻力是如何产生的，将为如何避免阻力提供重要线索。此外，识别与氟喹诺酮耐药或敏感性相关的SNPs将为医生提供工具，以开出有效的抗生素，从而挽救生命、节省资金，并保留目前的抗生素武器库。

 DESCRIPTION (provided by applicant): Antibiotic resistance is a major worldwide problem. With excellent efficacy, bioavailability, and minimal toxicity, the frequently prescribed fluoroquinolones are an extremely important drug class. The long-term goals of the Zechiedrich laboratory are to determine how gram-negative bacteria respond to and develop resistance to antimicrobial agents and to use this knowledge to improve diagnosis of antibiotic-resistant bacterial infections, to prolong the usefulness of current drugs, and to aid in the design of new therapeutic modalities. In previous funding periods, we constructed an evolving HIPAA compliant Oracle database that combines patient, hospital, and microbiological data from gram-negative clinical isolates collected since 1999. We discovered that known fluoroquinolone resistance mechanisms account for the resistance phenotype in approximately half of fluoroquinolone-resistant E. coli clinical isolates; therefore, additional unknown resistance mechanisms must exist. To uncover these mechanisms, we developed a unique approach that incorporates genome pooling, sequencing, and SNP analysis. We uncovered extremely problematic novel clinical isolates (resistant to all but two antibiotics and with very high fluoroquinolone MICs). The genomes of these isolates diverged from and could not be mapped to reference genomes. Conserved among all other fluoroquinolone-resistant pools, we found the well-known SNP in the gyrA target gene and three novel SNPs relative to drug-susceptible reference genomes. We also found 283 SNPs in 173 genes associated with fluoroquinolone resistance and resistance to specific additional antimicrobial agents. Relative to a fluoroquinolone-resistant reference genome, we found 35 novel SNPs associated with a fluoroquinolone-susceptible phenotype. The aims of this proposal are Aim 1: Predict how Identified Genetic Variants Alter the Encoded Protein Structure and Function, Aim 2: Define Function of Discovered Genetic Variants, Aim 3: Determine the Molecular Mechanisms of Discovered Protein Variants, and Aim 4: Continue to Discover Genetic Variants Associated with Fluoroquinolone Susceptibility or Fluoroquinolone Resistance Phenotypes. Our team, in the Texas Medical Center in Houston, Texas, is composed of a molecular microbiologist (Zechiedrich), a physician scientist (Hamill), a genomics expert (Sucgang), a computational biologist (Barth), and a crystallographer (Lee). The foundational knowledge gained in achieving these goals is required before new drugs or new targets for new drugs can be identified. A better understanding of how resistance arises will provide vital clues to how it may be avoided. Moreover, the identification of SNPs associated with fluoroquinolone resistance or susceptibility will provide physicians tools to prescribe antibiotics that will be effective, thus saving lives, money, and preserving the current arsenal of antibiotics.