Defining evolutionary trajectories: Molecular adaptation to antibiotic resistance

描述（由申请人提供）：美国疾病控制与预防中心估计，每年大约有200万例医院感染，导致8万多名患者死亡。抗生素耐药性是成功的药物治疗的进化结果，并突出了自然选择在形成导致耐药性的分子机制中的作用。为了从分子的细节上研究这些机制，我们正在对携带三种抗生素抗性基因之一的大量细菌进行持续的实验进化。通过改变适应过程中的选择条件，通过改变靶基因介导的抗生素耐药性将用于：1)确定种群内确定适应功能中间体的突变网络；2)确定导致适应度增加（即抗性）的蛋白质功能变化的物理化学基础；3)为使用相关符号上位模型成功建模成功的进化轨迹提供数据。研究的靶基因为E. faecalis Tn916 tetM（四环素核糖体保护基因）、Bacteroides Tn4400 tetX（四环素酶失活基因）和TnA TEM-1（内酰胺酶失活基因）。这些研究将提供广泛的数据和结果，包括：拟杆菌Tn4400 TetX和粪肠杆菌Tn916 TetM的高分辨率晶体结构，用于药物开发的特化扩展谱TetX和TetM突变文库，可扩展的高通量TetM活性测定，以及用于持续进化的稳健的浊度控制系统的开发。通过采用结合生物物理和种群策略的跨学科方法，我们可以将原子水平上蛋白质的变化与其对环境中生物体的影响联系起来，反之亦然。开发经过验证的分子适应模型是准确预测抗生素耐药性的重要一步。一旦完全实现，进化预测有望超越传统药物靶点的识别，发展新的临床策略，考虑适应的分子机制，以防止耐药性。公共卫生相关性：抗生素耐药性的上升是一个明显的健康威胁，需要立即和持续关注。随着耐药细菌的菌株继续蔓延到医院和社区，给社会造成的代价是惊人的。2004年的数据显示，尽管医学界尽了最大努力，但60%以上的ICU患者存在耐甲氧西林金黄色葡萄球菌（MRSA），近30%的ICU患者存在耐万古霉素肠球菌（VRE），而1995年这一比例分别为37%和14%，并且这一比例还在继续上升。儿童和老人尤其容易受到伤害。不幸的是，社区相关（CA）的MRSA暴发也在增加，这表明耐药菌株正在进入学校的更衣室和其他公共场所。除了临床环境中的人际传播外，农业和水产养殖中抗生素的广泛使用也导致耐药菌株的扩散。可移动的遗传元件，如转座子、共轭转座子和质粒，在微生物种群之间充当载体，并可将耐药性传播到农业或临床环境之外。因此，医院和农场可以成为将耐药基因转移到一般社区的繁殖地和储存库。虽然没有办法阻止进化，但更全面地了解抵抗分子适应的基本原理对科学家和临床医生来说都是一笔巨大的财富。这项工作是分子进化从分析过去事件的追溯姿态过渡到主动使用进化研究来预测耐药性，优化药物方案，并可能开发直接应用于药物的限制致病性适应的新试剂的重要一步。

DESCRIPTION (provided by applicant): Each year the CDC estimates that there are approximately two million cases of nosocomial infection that result in over 80,000 patient deaths. Antibiotic resistance is an evolutionary consequence of successful drug therapies and highlights the role of natural selection in shaping the molecular mechanisms leading to resistance. To examine these mechanisms in molecular detail, we are subjecting large populations of bacteria, carrying one of three antibiotic resistance genes, to continuous experimental evolution. By varying the conditions of selection during adaptation, antibiotic resistance mediated by changes to the target genes will be used to: 1) identify the network of mutations that define the functional intermediates to adaptation within the population; 2) determine the physicochemical basis for changes in protein function that lead to increased fitness (i.e. resistance) and 3) provide data for the successful modeling of successful evolutionary trajectories using correlated sign epistasis models. The target genes for study are E. faecalis Tn916 tetM (ribosomal protection against tetracyclines), Bacteroides Tn4400 tetX (enzymatic inactivation of tetracyclines) and TnA TEM-1 (enzymatic inactivation of (-lactams). These studies will provide a wide range of data and results including: high resolution crystallographic structures of Bacteroides Tn4400 TetX and E. faecalis Tn916 TetM, libraries of characterized expanded spectrum TetX and TetM mutants for drug development, a scalable high throughput TetM activity assay, and development of robust turbidostat systems for continuous evolution. By taking an interdisciplinary approach combining biophysical and population strategies, we can link changes in proteins at the atomic level to their consequences for the organism in its environment and vice versa. Developing validated models for molecular adaptation is an important step towards making accurate predictions of antibiotic resistance. Once fully realized, evolutionary forecasting holds the promise of going beyond the identification of traditional drug targets to the development of new clinical strategies that consider the molecular mechanisms of adaptation to prevent drug resistance. PUBLIC HEALTH RELEVANCE: The rise of antibiotic resistance is a clear health threat that requires immediate and continuing attention. As strains of drug resistant bacteria continue to spread into hospitals and communities the cost to society are staggering. Data from 2004 show that despite the best efforts of the medical community, methicillin resisistant Staphylococcus aureus (MRSA) were found in over 60% and vancomycin resistant Enterococci (VRE) in nearly 30% of ICU patients compared to 37% and 14% respectively in 1995 and continue to rise. Children and the elderly are particularly vulnerable. Unfortunately, community associated (CA) outbreaks of MRSA are also increasing suggesting that drug resistant strains are making their way into the locker rooms of schools and other public areas. In addition to human-to-human transmission within clinical settings, the widespread use of antibiotics in agriculture and aquaculture has also led to the proliferation of resistant strains. Mobile genetic elements such as transposons, conjugative transposons and plasmids act as vectors between microbial populations and can spread resistance beyond the agricultural or clinical environment. Hospitals and farms can therefore act as breeding grounds and reservoirs for the transfer of drug resistance genes into the general community. Although there is no way to stop evolution, a more complete understanding of the principles underlying molecular adaptation to resistance can be a powerful asset to both scientists and clinicians. The proposed work is an important step in the transition of molecular evolution from a retroactive posture that analyzes past events to one in which evolution research is used pro-actively for the prediction of drug resistance, optimization of drug regimens, and perhaps development of novel reagents that restrict pathogenic adaptation with direct application to medicine.