Overcoming antibiotic resistance by studying antibiotic hypersensitivity

自从抗生素被发现以来，它已经拯救了数百万人的生命。抗生素通过靶向一个必要的代谢过程来杀死致病菌。致病菌可以通过改变或提供不再与抗生素结合的新靶标、破坏抗生素或将抗生素从靶标中抽离来保护自己免受抗生素的侵害。抗生素耐药性现在是治疗由致病菌引起的疾病的一个严重问题，在欧盟，每年有25 000人死于无法治愈的感染。全球每年有44万多药耐药结核病新发病例，造成15万人死亡。目前正在几个方面寻求解决抗生素耐药细菌问题的办法，包括更好地控制现有抗生素的使用，发现和开发新的抗生素和不依赖于噬菌体疗法、细菌素、抗菌肽和疫苗等抗生素的抗菌战略。迄今为止尚未开发但可能令人兴奋的方法是使用联合治疗，即同时使用两种或两种以上的药物并协同作用以杀死抗生素耐药细菌。这种方法用于防治艾滋病毒和结核病已有一段时间了。然而，联合治疗并不总是需要两种抗生素；所使用的一种药物本身可能没有抗微生物活性，但可以增强抗生素的活性。众所周知，大多数人服用的增效剂是克拉维酸，它是一种酶-内酰胺酶的抑制剂，可以破坏-内酰胺类抗生素，如青霉素。有多少个增强剂目标，我们如何找到它们？有证据表明，细菌中有数百种不同功效的增强剂靶点，它们可能对不同类型的抗生素起作用。这一证据来自对具有单基因突变的细菌的抗生素敏感性的测量；与具有完整基因（亲本）的菌株相比，那些对抗生素更敏感的突变体表明，突变基因或其对代谢过程的影响是一个增强剂靶标。我们已经分离出一种细菌的突变体，即对一类抗生素过敏的彩链霉菌（结核分枝杆菌的亲戚），其中包括两种抗生素，这两种抗生素是某些致病菌的所谓“最后手段”抗生素。突变存在于修饰蛋白质所需的酶中，这些蛋白质与糖一起被定位在细胞外部。敲除这个修饰系统可能会对代谢过程产生各种各样的后果，目前还不清楚。我们假设，如果我们在代谢水平上了解这些后果，我们就可以确定增强剂的合理目标，并在某些情况下破坏它们的抗性机制。我们的第一个目标是询问相关细菌中蛋白质修饰系统的突变是否也具有超敏性，以确定我们的观察结果是否具有普遍性，并与NovaBiotics有限公司和阿伯丁海洋生物发现中心合作启动增效剂化学物质的筛选。其次，我们计划通过研究可能受细胞表面修饰影响的蛋白质和测量基因表达的变化，来确定突变体与亲本菌株相比发生了哪些主要的代谢变化。第三，我们计划确定哪些基因变化需要发生在过敏菌株上，使它们再次具有抗性，这将指出对过敏的解释以及对增强剂的抗性是如何产生的。在这个项目结束时，我们希望能够开始筛选与抗生素一起使用的增强剂，以对抗结核分枝杆菌和一些万古霉素耐药病原体。

Antibiotics have saved millions of lives since their discovery. Antibiotics kill pathogenic bacteria by targeting an essential metabolic process. Pathogenic bacteria can protect themselves from antibiotics by altering or providing a new target that no longer binds the antibiotic, destroying the antibiotic or pumping the antibiotic away from the target. Antibiotic resistance is now a serious problem in treating diseases caused by pathogenic bacteria such that in the EU 25,000 people die annually from untreatable infections. Globally there are 440,000 new cases annually of multidrug resistant tuberculosis resulting in 150,000 deaths. Solutions to the problem of antibiotic resistant bacteria are being sought on several fronts including better control over the use of existing antibiotics, the discovery and development of new antibiotics and antibacterial strategies that do not rely on antibiotics such as phage therapy, bacteriocins, antibacterial peptides and vaccines. A hitherto underexplored but potentially exciting approach is to use combination therapy in which two or more drugs are used simultaneously and act synergistically to kill antibiotic resistant bacteria. This approach has been used for combating HIV and tuberculosis for some time. However combination therapy need not always involve two antibiotics; one of the drugs used may not itself have anti-microbial activity but potentiates the activity of the antibiotic. A well-known potentiator that has been taken by most people is clavulanic acid, an inhibitor of the enzyme beta-lactamase that destroys beta-lactam antibiotics such as penicillin. How many potentiator targets are there and how do we find them? There is evidence that there are hundreds of potentiator targets in bacteria of varying efficacy and that might act against different types of antibiotics. This evidence comes from measuring the antibiotic sensitivity in bacteria that have single gene mutations; those mutants with greater sensitivity to an antibiotic compared to a strain with an intact gene (the parent) indicate that the mutated gene or its consequences on metabolic processes is a potentiator target. We have isolated mutants in a bacterium, Streptomyces coelicolor (a relative of Mycobacterium tuberculosis) that are hypersensitive to a subset of antibiotics including two antibiotics that are so-called 'last resort' antibiotics for some pathogenic bacteria. The mutations lie in enzymes required to modify proteins being localised to the outside of the cell with sugars. Knocking out this modification system may have a variety of consequences on metabolic processes, all unknown at present. We hypothesise that if we understand what these consequences are at the metabolic level, we can identify rational targets for potentiators and, in some cases, undermine their resistance mechanisms. Our first objective is to ask whether mutations in the protein modification system in related bacteria are also hypersensitive to establish whether our observations are general, and to initiate screens for potentiator chemicals in collaboration with NovaBiotics Ltd and the Marine Biodiscovery Centre in Aberdeen. Second we plan to determine what major metabolic changes have occurred in the mutants compared to the parent strain by studying the proteins that might be affected by modification in the cell surface and by measuring changes in gene expression. Third we plan to identify what genetic changes need to happen to the hypersensitive strains to make them resistant again and this will point to both an explanation of the hypersensitivity and how resistance to potentiators might arise. At the end of this project we hope to be in a position where we can start screening for potentiators for use with antibiotics that act against Mycobacterium tuberculosis and some vancomycin resistant pathogens.