The importance of L-form switching in antibiotic evasion and recurrence of bacterial infection

抗菌素耐药性(AMR)被世界卫生组织视为人类面临的十大全球公共卫生威胁之一。据估计，全球每年有70万人死于耐药感染，预计到2050年，这一数字将上升至1000万，同时累计成本将达到100万亿美元。人类健康、动物健康以及食品和环境安全是相互关联的，因此需要采取跨部门的方法，以加快抗药性微生物斗争的进展。准确了解抗菌素在人体或动物体内的作用方式，对于开发新的治疗方法并确保现有药物的最佳使用是必要的。我的研究重点是一种未被充分研究的抗生素逃避机制，称为L形式转换。几乎所有的细菌都被一种叫做细胞壁的结构包围着，这种结构保护它们免受环境压力的影响，并有助于正常的分裂。我们最常用的许多抗生素，如青霉素，都针对这种结构。在使用这些抗生素治疗期间，细菌通常会爆发并死亡，但如果周围环境保护它们不会破裂，一些细菌可以在一种被称为L形式的无壁状态下存活。没有这堵墙，细菌就会变得脆弱，生长缓慢，但对针对这种结构的所有类型的抗生素都有耐药性。一旦抗生素治疗完成，以L形式生存的细菌可以重建墙壁并开始快速分裂，有可能导致反复感染。重要的是，包括大肠杆菌和金黄色葡萄球菌在内的许多病原体都可以经历L形式的转换，多年来一直有人推测，宿主可以为这一过程提供支持的环境。L-形状转换与人和动物的几种反复发作的疾病有关，如败血症、乳房炎、尿路和胃肠道感染。然而，L型的脆弱性质以及它们在宿主体内的数量很少，使得研究它们变得困难，而且历史上关于它们在疾病中作用的令人信服的证据出现得很慢。我利用尖端技术开发了新的方法来研究L型转换。之前，我使用先进的荧光显微镜，证明了反复尿路感染患者的尿液中存在细胞壁缺陷细菌，而且矛盾的是，它们可以在我们免疫系统的细胞内存活。在这些重大发现之后，迫切需要进一步的工作来回答关于L的许多问题--仍然存在的形式。宿主中哪些确切的位置有利于L型的生存，它们是否为L型提供了额外的保护，使其免受环境变化的影响？L型更喜欢细胞内还是细胞外？最重要的是，如何根除它们？我提出的奖学金计划的目的是克服这一进步的主要障碍，将显微镜和遗传学相结合，利用包括有机体在内的各种模型来研究L的形态转换。这些“微型器官”可以很容易地生长，并模仿真实的器官结构，如膀胱或肠道。我将跟踪L形式的各种细菌物种的转换，并在特定组织的背景下测试它们对抗生素治疗的敏感性。我将使用突变技术来鉴定细菌基因，这些基因可以让L高效地从宿主中切换过来。我还将把我目前对人类健康的关注扩展到动物健康，并研究L型转换是否也在农场动物疾病中发挥作用，如牛乳房炎。我的研究将为我们对L型反复感染的理解提供一个转变。虽然研究寄主中的L形式转换是具有挑战性的，但它为细菌致病性带来了新的见解。我的研究有可能影响如何更好地使用现有的治疗方法，以及开发应对感染的新策略。

Antimicrobial resistance (AMR) is considered by the World Health Organization as one of the top 10 global public health threats facing humanity. It is estimated that there are 700,000 annual global deaths due to resistant infections, which are predicted to rise to 10 million, alongside a cumulative cost of $100 trillion by 2050. Human health, animal health, as well as food and environment security are interlinked, therefore cross-sector approaches are needed to accelerate progress in the battle against organisms resistant to antimicrobials. Understanding precisely how antimicrobials work inside the human or animal body is necessary to develop novel treatments and to make sure the existing ones are used optimally.My research focuses on an understudied mechanism of antibiotic evasion, called L-form switching. Almost all bacteria are surrounded by a structure called the cell wall, which protects them against environmental stresses and helps with regular division. Many of our most commonly used antibiotics, such as penicillin, target this structure. During treatment with these antibiotics, bacteria usually burst and die, but some can survive in a wall-less state referred to as an L-form, if the surrounding environment protects them from bursting. Without the wall, bacteria are fragile and slow growing, but resistant to all types of antibiotics that target this structure. Once antibiotic treatment is finished, bacteria that survived in an L-form state can rebuild the wall and start dividing rapidly, potentially contributing to recurrent infections.Importantly, many pathogens, including E. coli and S. aureus, can undergo L-form switching and over the years it has been speculated that the host can provide a supportive environment for the process. L-form switching has been implicated in several recurrent diseases in human and animals, such as sepsis, mastitis, urinary tract and gastrointestinal infections. However, the fragile nature of L-forms and their low numbers in the host's body make them difficult to study, and convincing evidence for their role in disease was historically slow to emerge.I have developed novel approaches using cutting-edge technologies to study L-form switching. Using advanced fluorescent microscopy, I previously demonstrated the presence of cell wall-deficient bacteria in the urine of patients with recurrent urinary tract infections and that, paradoxically, they can survive inside the cells of our immune system. Following on from these significant discoveries, further work is urgently needed to answer the many questions about L-forms that remain. What are the precise locations within a host that are favourable for L-form survival, and do they provide additional protection for L-forms against environmental changes? Do L-forms prefer intracellular or extracellular locations? And most importantly, how can they be eradicated?The aim of my fellowship proposal is to overcome this major impediment to progress by combining microscopy and genetics to study L-form switching using various models, including organoids. These 'mini-organs' can be readily grown and mimic real organ structures, such as the bladder or intestine. I will follow L-form switching of various bacterial species and test their susceptibility to antibiotic treatment in the context of a specific tissue. I will use mutagenesis to identify bacterial genes that allow efficient L-from switching in the host. I will also expand my current focus on human health to animal health and study if L-form switching also plays a role in farm animal disease, such as bovine mastitis.My study will provide a transformation in our understanding of recurrent infection by L-forms. Although studying L-form switching in the host is challenging, it brings novel insights into bacterial pathogenicity. My research has the potential to influence how to use currently available treatments better, as well as develop novel strategies for tackling infection.