Exploring susceptibility of L-form bacteria to antimicrobial peptides

抗菌素耐药性（AMR）被世界卫生组织视为全球十大公共卫生威胁之一。l型转换是一种机制，允许细菌（包括重要的病原体，如大肠杆菌（E. coli）、金黄色葡萄球菌（S. aureus）或假单胞菌）暂时失去细胞壁。因此，l型细菌对针对细胞壁的抗生素（如青霉素）完全耐药，并与许多传染病的复发有关，包括尿路感染（uti）、败血症或食源性感染。虽然对细胞壁靶向药物有耐受性，但l型应该对其他类型的抗生素保持敏感性，例如那些靶向膜的抗生素。抗菌肽是一种天然存在的小肽，是包括人类在内的多种生物免疫系统的一部分。这些肽最常见的目标是病原体膜，而抗菌肽如nisin在体外被证明对l型有效。在这个博士项目中，我们将研究大肠杆菌和金黄色葡萄球菌l型对各种抗菌肽的敏感性，并与它们的壁对应物进行比较。利用细胞培养感染模型（包括细胞系和类器官），我们将首次研究抗菌肽是否可以用于消除宿主组织中的l型细菌，从而可能有助于预防复发性感染。如果不采取行动，耐药性每年在全球造成约130万人死亡，估计到2050年这一数字将增加到1 000万人，累计费用将达到100万亿美元。这项工作将有助于更好地了解抗生素逃避的机制，更有效地利用现有的，以及潜在的开发新的治疗细菌感染的方法。各种抗菌肽（包括抗菌肽、防御素、抗菌肽或合成肽）对大肠杆菌和金黄色葡萄球菌l型的有效性将通过杀伤试验和高端显微镜进行测试。我们将系统地测试将细胞壁靶向抗生素与抗菌肽联合使用是否比单独使用每种药物更有效，以及每种药物的确切比例是最有效的。我们将使用细胞系如RAW 264.7， THP-1巨噬细胞和肠道小鼠类器官结合先进的荧光显微镜来测试AMPs在组织环境下对抗l型的有效性。该DTP的目标是通过建立协同效应、发展新的合作和开辟新的研究领域来增加价值。这个项目如何增加价值，超越标准学生所能达到的价值？这个研究项目是纽卡斯尔大学两个研究小组之间的新合作。一级导师Katarzyna Mickiewicz主要研究l型细菌在疾病中的作用，二级导师Henrik Strahl von Schulten主要研究细胞包膜靶向抗生素的作用方式。这是两个互补的专业领域，这将加速项目，并为学生提供交付项目的最佳培训机会，这是不可能仅由这些主管中的一个实现的。高级第二导师提供40%的指导和专业知识，这对初级初级导师（60%）来说是非常宝贵的。出版物

BACKGROUND Antimicrobial resistance (AMR) is considered by the World Health Organization as one of the top 10 global public health threats. L-form switching is a mechanism, that allows bacteria (including important pathogens such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas spp.) to temporarily lose their cell wall. L-form bacteria are, thus, completely resistant to antibiotics, which target cell wall, such as penicillins , and are implicated in recurrence of many infectious diseases including urinary tract infections (UTIs), sepsis or foodborne infections. While tollerant to cell wall-targeting agents, L-forms should retain susceptibility to other types on antibiotics, for example those targeting membranes. OBJECTIVES AND NOVELTYAntimicrobial peptides (AMPs) are naturally occurring small peptides, which are part of the immune system of various organisms, including humans. These peptides most commonly target pathogen membranes, and AMPs such as nisin were shown to be effective against L-forms in vitro. In this PhD project, we will investigate the susceptibility of E. coli and S. aureus L-forms to various antimicrobial peptides in comparison to their walled counterparts. Using cell culture infection models (including cell lines and organoids) we will investigate for the first time if antimicrobial peptides can be used to eliminate L-form bacteria in the context of host tissue, therefore potentially contributing to prevention of recurrent infections. TIMELINESSAMR contributes to ~1.3 million deaths per year globally, with this number estimated to raise to 10 million by 2050, alongside a cumulative cost of $100 trillion, if no action is taken. This work will contribute to a better understanding of mechanisms of antibiotic evasion, more efficient use of the existing, and potential development of new treatments against bacterial infections. EXPERIMENTAL APPROACHEffectiveness of various AMPs (including cathelicidins, defensins, cecropin or synthetic ones) against E. coli and S. aureus L-forms will be tested using killing assays and high-end microscopy. We will systematically test if combining cell wall-targeting antibiotics with antimicrobial peptides might constitute a more effective treatment than each of the agents used alone, and precisely what proportion of each of the agents is most effective. We will use cell lines such as RAW 264.7, THP-1 macrophages and intestinal murine organoids combined with advanced fluorescence microscopy to test the effectiveness of AMPs against L-forms in a context of a tissue.The aim of this DTP is to add value by building synergies, developing new collaborations, and opening up new areas of research. How does this project add value, over and above what could be achieved with a standard studentship? (Maximum 100 words) This research project is a new collaboration between two Newcastle University based research groups. The primary supervisor Katarzyna Mickiewicz focuses on the role of L-form bacteria in disease and the secondary supervisor Henrik Strahl von Schulten on the mode of action of cell envelope targeting antibiotics. These are two complementary areas of expertise, which will accelerate the project and provide optimal training opportunities for the student to deliver on the project, which would not be possible for only one of these supervisors to achieve. 40% contribution from the senior second supervisor who will provide mentorship as well as the expertise, will be invaluable to the junior primary supervisor (60% contribution). Publications