Charting the fitness landscape of a penicillin-binding protein

抗菌素耐药性(AMR)的迅速上升是影响世界的最紧迫问题之一。AMR每年直接导致130万人死亡，预计到2030年将使2400万人陷入极端贫困(世卫组织和联合国抗菌素耐药性机构间协调小组)。对抗AMR的需求是极其紧迫的。微生物进化AMR的一种常见方式是通过突变。例如，突变可以累积并影响介导抗生素耐药性的细菌基因的功能。β-内酰胺(BL)抗生素是最广泛使用的抗生素之一，它们通过结合和抑制青霉素结合蛋白来杀死细菌。因此，导致青霉素结合蛋白氨基酸替换的突变可以使细菌逃脱BL抗生素的杀灭作用。A组链球菌(GAS)是一种著名的人类病原体，被认为对BL抗生素高度敏感。然而，近年来临床分离的GAS被发现对BL抗生素不那么敏感，而且出现了一个新的担忧，即降低抗生素敏感性将为GAS全面产生抗生素耐药性铺平道路。GAS中对BL抗生素敏感性的下降主要是由一种名为PBP2x的特定青霉素结合蛋白的替换引起的。在缺乏先验知识的情况下，几乎不可能了解这些单个替换及其众多组合对PBP2x的影响以及细菌对BL抗生素的敏感性，更不用说预测哪些替换将导致对BL抗生素的进一步进化。我的研究将解决的问题是：PBP2x中的哪些替换将直接导致气体中BL抗生素耐药性的进化，哪些是多余的？哪些替代在进化上更容易获得？遗传和环境因素是如何调节或限制这些替换的获得的？在这里，我将使用实验和计算相结合的方法来剖析和预测PBP2x替换对细菌对BL抗生素的敏感性的影响。首先，我将全面量化pbp2x突变在三种BL抗生素和两种非BL抗生素的选择下的影响。这将使我能够同时捕捉数以千万计的pbp2x突变的突变影响，并绘制pbp2x突变与细菌对各种类型抗生素敏感性之间的关系。接下来，我还将研究这些pbp2x突变在具有不同遗传背景的细菌宿主中的突变效应。这一系列研究将揭示突变之间意想不到的基因相互作用，否则这些突变将很难预测。这种情况的一个例子是，pbp2x突变并不能使细菌对抗生素不那么敏感，但它的存在对于积累其他更有害的突变是绝对必要的。总体而言，这项研究的结果将使我们能够区分哪些pbp2x突变与BL型抗生素的GAS耐药性直接相关，和/或哪些突变对BL型抗生素耐药性的演变具有重要意义。这项研究得出的见解将有助于我们提前预测令人担忧的细菌变异，这可能导致对抗AMR的进一步影响。

The rapid rise of antimicrobial resistance (AMR) is one of the most pressing problems affecting the world. AMR is directly causing 1.3 million deaths a year, and is expected to plunge 24 million people into extreme poverty by 2030 (WHO and the UN Interagency Coordinating Group on Antimicrobial Resistance). The need to combat AMR is extremely urgent.A common way for microbes to evolve AMR is through mutations. For instance, mutations can accumulate and affect the function of bacterial genes that mediate antibiotic resistance. Beta-lactam (BL) antibiotics are one of the most widely prescribed antibiotics, and they kill bacteria by binding and inhibiting their penicillin-binding proteins. As such, mutations that cause amino acid substitutions in penicillin-binding proteins can allow a bacterium to escape the killing effect of BL antibiotics.Group A Streptococcus (GAS) is a notable human pathogen that was deemed highly susceptible to BL antibiotics. However, clinical isolates of GAS are found to be less susceptible to BL antibiotics in recent years, and there is an emerging concern that reduced antibiotic susceptibility will pave the way to full-blown antibiotic resistance in GAS. The decreasing susceptibility to BL antibiotics in GAS is primarily driven by substitutions in a particular penicillin-binding protein called PBP2x. In the absence of prior knowledge, it is nearly impossible to understand the impact of these individual substitutions and their numerous combinations on PBP2x and the bacterial susceptibility to BL antibiotics, let alone predict which substitutions will lead to further evolution of resistance to BL antibiotics.The questions that my study will address are: Which substitutions in PBP2x will contribute directly to the evolution of BL antibiotic resistance in GAS, which ones are redundant? Which substitutions are more evolutionarily accessible? How do genetic and environmental factors mediate or constrain the acquisition of these substitutions?Here, I will dissect and predict the impact of PBP2x substitutions on the bacterial susceptibility to BL antibiotics using a combination of experimental and computational approaches. First, I will quantify the effects of pbp2x mutations comprehensively under the selection of three types of BL antibiotics and two non-BL antibiotics. This will allow me to capture the mutational effects of thousands and millions of pbp2x mutations in parallel, and map the relationship between pbp2x mutations and bacterial susceptibilities to the various types of antibiotics. Next, I will also investigate the mutational effects of these pbp2x mutations in bacterial hosts with different genetic backgrounds. This line of study will reveal unexpected genetic interactions between mutations that would otherwise be difficult to predict. An example of such a scenario is when a pbp2x mutation does not make a bacterium less susceptible to an antibiotic, but its presence is absolutely essential for the accumulation of other more deleterious mutations. On the whole, the outcome of this research will allow us to distinguish which pbp2x mutations are directly relevant to GAS resistance to BL antibiotics, and/or which ones are important for the evolution of BL antibiotic resistance. Insights derived from this research will help us to predict bacterial variants of concern ahead of time, which can lead to further implications for combatting AMR.