GPVI

In a comparative study, researchers describe how two notable pathogens -- Escherichia coli and Acinetobacter baumannii -- employ distinctly different tools to fend off antibiotic attack by two different drugs. Antimicrobial resistance is a story of constantly moving parts and players. With every new or tweaked antibiotic or antimicrobial drug, the targeted pathogens begin the evolutionary dance of acquiring resistance, prompting researchers to constantly develop workarounds or entirely new classes of medicine. Understanding the underlying mechanisms of acquired antimicrobial resistance is critical to the fight, a case of knowing one's enemy. In a new paper published March 2, 2024 in npj Antimicrobials and Resistance, part of the Nature Portfolio, researchers at Sanford Burnham Prebys, working with Roche Pharma Research and Early Development, describe how two notable pathogens -- Escherichia coli and Acinetobacter baumannii -- employ distinctly different tools to fend off antibiotic attack. "This work was conceived as a comparative study of the mechanisms and dynamics of resistance acquisition for two drugs and two bugs," said senior author Andrei L. Osterman, Ph.D., at Sanford Burnham Prebys. "Comparing mutational landscapes triggered by the same drug in two distinct bugs allows us to deduce both shared and unique evolutionary trajectories toward resistance. A comparison of two drugs in the same bug reveals shared and unique mechanistic features of fundamental and translational importance, from drug discovery to rational optimization of treatment regimens." Both pathogens studied are gram-negative bacteria with shared characteristics, but also notable differences. For the most part, E. coli is a bacterium routinely found in the guts of humans and animals, where it resides with no ill effect. Some strains, however, do cause harm, everything from mild gastroenterological distress to urinary tract infections, respiratory illness and pneumonia. Acinetobacter baumannii is more problematic, particularly in clinical settings where it can cause severe infections, some life-threatening. Both bacteria have developed resistance to most current antibiotic treatments. In their paper, Osterman and colleagues combined experimental evolution in a continuous culturing device (morbidostat) with whole genome sequencing of evolving cultures to track how E. coli and A. baumannii acquired drug resistance against a pair of antibiotics that inhibit DNA gyrase, an essential enzyme in bacteria. Inhibiting the enzyme disrupts DNA synthesis and, subsequently, causes the bacteria to die. One antibiotic -- ciprofloxacin -- has been in active clinical use since 1987; the other -- GP6 -- is experimental. The researchers found that pathogenic bacteria with acquired resistance to ciprofloxacin remained susceptible to effective antimicrobial treatment by GP6 drug. However, the opposite was not true: evolution of resistance to GP6 also triggered resistance to ciprofloxacin. These findings underscored that E. coli and A. baumannii employ shared and unique mechanisms to acquire resistance to these two types of drugs. "Bacteria acquire resistance as a result of random mutational events that happen in the DNA replication as uncorrected 'typos'," said Osterman. "These spontaneously emerge in a handful of drug-resistant variants/strains out of godzillions of neutral mutations) under selective pressure." (A godzillion is a descriptive term, not a precise unit of measurement. It refers to a number of enormous size. In this case a number ranging between 108 to 109 neutral mutations.) These harmful mutations may underlie several types of resistance mechanisms, including modifying the protein targets of a given drug, the ability of bacterial cells to expel compounds (drugs) before they cause harm (efflux) and special bacterial enzymes that inactivate active drug compounds. Osterman said the work advances progress toward developing "resistibility profiles" of established and novel antimicrobial drugs, which would help set forth "boundaries for possible combinational treatment, including clinically relevant multidrug resistant strains. Our acquired knowledge provides crucial guidelines for all these translational activities." The findings are not limited to E. coli and A. baumannii. They have potential to at least partially predict resistance drivers across other closely related species posing significant health risks, including Pseudomonas, Salmonella and Klebsiella spp. all difficult-to-treat bacterial pathogens linked to serious and sometimes deadly infections. Additional authors on the study include Semen A. Leyn, James E. Kent, Jaime E. Zlamal and Marinela L. Elane, all at Sanford Burnham Prebys; and Maarten Vercruysse, Rocha Pharma. The study was supported by F. Hoffmann-La Roche Ltd and the National Institute of Allergy and Infectious Diseases (grant 5R01AI167977). The study's DOI is 10.1038/s44259-024-00021-y

背景：根据尾巴的形态，噬菌体通常分为足病毒科(Podoviridae)、虹膜病毒科(Siphoviridae)和肌病毒科(Myoviridae)三个科，Myophage的收缩尾巴是最复杂的，它在附着到宿主细胞上时经历了一个实质性的构象转换，形成通道，使噬菌体基因组能够注射到宿主细胞质中。最近，在中国巢湖分离出一种名为Pam3的肌病毒科噬藻体，它能特异性感染宿主蓝藻假藻(Pseudanabaena mucicola)。与T4噬菌体相比，Pam3拥有一个更简单的基板，这些组成较简单的噬菌体基板与已知结构的CISs相似，这表明它们在进化和工作模式上具有相似性。在这里，作者报道了其完整结构，并揭示了各组件的组装模式，这些精细的结构提供了对最小噬菌体的组装模式和感染模型的见解。结果：1.Pam3的整体结构为解析Pam3的基本保守模块及组装机制，作者首先纯化了Pam3颗粒，通过低温电镜得到其完整结构（图1）。图1 Pam3病毒粒子的整体结构Pam3的结构基因聚集在基因组的2272~19635 bp内，包含4个操纵子，编码头、颈、尾和底板组装的蛋白质。    Cryo-EM map显示Pam3有一个直径~680 Å的二十面体，由gp7，gp6，gp3等组成，gp9、gp11和gp12构成颈，壳内有54.5 kb基因组，然后是~840 Å收缩尾，基板非常简化，由wedge、其包围的hub-spike复合体和12个尾部纤维组成。2.Caspid与portal对称不匹配装配Portal vertex作为DNA转运马达组装的平台，作者首先解析了Caspid与Portal的结构与组装机制，发现他们以一种对称不匹配的机制相互作用：12重对称的portal十二聚体与5个具有5重对称的六邻体接触（图2）。另外，caspid和portal内有一些特殊结构稳定构型（图3,4），保持噬菌体的稳定性。          图2 Pam3的Caspid和Portal不对称组装图3 caspid的结构和组装  图4 portal vertex的结构和组装gp7是主要的caspid蛋白，外周节段变化使其聚成两种形式的衣壳——六方体和五方体，进一步组装成二十面体衣壳；gp6形成三聚体与相邻caspid的N端臂相互作用，增强衣壳结构的稳定性。portal的gp3十二聚体有一个中心通道，内有DNA链；每个gp3亚基由桶、冠、翼、茎和夹状物组成，最后由10个gp7亚基包围；在portal和caspid之间形成了一个套索结构使DNA固定在portal上。3.Adaptor、connector和terminator依次互锁成颈颈部与portal相连，在中心形成DNA注射通道，作者通过解析颈部结构的组成，揭示了Pam3颈部的连接机制（图5）。    图5 Pam3颈部的装配与连接portal与颈相连，颈包括adaptor/gp9十二聚体、connector/gp11六聚体和terminator/gp12六聚体，中心形成DNA通道，DNA末端被tape measure/gp16(TMP)所堵塞。颈通过portal和gp9的C端直接相互作用连接；gp9停靠在gp11上，被其α1螺旋包围；gp11的β发夹夹住gp12的N端环，这些颈组件之间的互补性使得颈高效连接和组装。4.尾部是一个三层的DNA注射机，由一个收缩鞘和围绕TMP的刚性管组成与T4相比，Pam3具有更短的收缩尾末端，作者解析Pam3尾部的冷冻电镜图谱，尾部的结构组成是由sheath、tube和TMP组成的三层结构。尾部的长度是由TMP决定的，作者发现Pam3的TMP采用了六倍螺旋束结构，为尾部组装提供支架；另外，gp12也对阻止尾部tube和sheath的生长起到重要作用（图6）。图6 Pam3尾部的结构Cryo-EM图谱显示，Pam3尾部包括sheath gp13六聚体、tube gp14六聚体、sheath initiator gp21六聚体和tube initiator gp17六聚体，并通过gp12与颈部相连。gp14围绕TMP组成一个六入口螺旋，gp14内部的β片与gp12的中心β片相似，两者形成直径~40 Å的通道；gp13通过交错的四链β片稳定前后六聚体。    gp12与tube的环作用，破坏tube的间六聚体界面并终止其生长，同时改变sheath的六聚体间结构，最终终止尾部sheath的生长；tube initiator gp17和sheath initiator gp21在基板上启动生长。5.Pam3有由六个基本组件组成的最小基板     Pam3拥有非常小的基板且正常行使功能，这是非常值得深入研究的。作者得到基板的完整结构，很大程度上帮助了对简单噬菌体组装模式和尾鞘收缩机制的理解。Pam3基板包含六种成分：三聚体gp20 spike、三聚体gp19 hub、baseplate wedge (异三聚体gp22-gp23)、6个单体gp18 plug和12个三聚体gp24 纤维。作者解析了Pam3穿透宿主细胞包膜的独特机制以及稳定各结构组分的独特方式。    图7 Pam3基板的结构gp20与gp19对接，形成hub-spike复合体。hub的三个亚基形成枢纽，与tube initiator相互作用。值得注意的是，spike的C端螺旋形成远端O型螺旋束，这是先前噬菌体研究中未发现过的。异质三联体形成一个核心的六螺旋束和一个三分叉单元，与T4 wedge不同，Pam3更简单，且缺乏额外插入域。wedge通过gp21和gp18锚定在hub-spike复合体上，每个plug单体嵌入tube initiator，将wedge的螺旋束固定在hub上。6.12个尾部纤维通过二硫键在上下交替中锚定在wedge上在wedge周围，12根尾纤维交替排列，α-螺旋结构域将纤维连接到wedge上，而其他两个结构域指向外，这可能有助于识别宿主受体。作者对二硫键进行测试，发现二硫键对于尾部纤维的连接是必不可少的。    图8 尾部纤维通过二硫键连接到wedge上wedge的每个异质三联体包含两个富含Cys的区域，它们通过二硫键桥接向上和向下的纤维。通常，wedge上的两个富含半胱氨酸的区域提供两个三角形平台，以交替的向上和向下配置促进两个尾部纤维与wedge的对接。在DTT处理下，Pam3的几乎所有尾部纤维都从wedge中分离出来。此外，用DTT处理4小时，大部分Pam3粒子的尾部呈现后收缩状态。因此，作者认为这些二硫键对于稳定尾部纤维和wedge互作是必不可少的。结论：作者通过对Pam3的完整结构解析，揭示了一个最小的肌病毒科噬菌体的头、颈、尾、基板和尾部纤维的组装和连接，也为噬菌体感染期间鞘收缩的机制提供了见解。与其他噬菌体作对比，发现了其独特的特性与机制，具体的组装步骤如图9。图9 Pam3组装的假设模型总之，Pam3包含了构建有效和功能性的噬菌体所必需的必要的和保守的成分作者解决了淡水噬藻体的完整结构，为了解最小噬藻体的组装模式提供了基础。这些结构揭示了功能性肌噬体组装和作用所需的基本和高度保守的模块，这可能是合成生物学中应用的简化和用户友好的模型。              原文链接：https://doi-org.libproxy1.nus.edu.sg/10.1073/pnas.2213727120识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

PARIS--(BUSINESS WIRE)-- September 7th, 2023, the University of Birmingham and Acticor Biotech (Paris:ALACT) announce the full regulatory approval of LIBERATE clinical study. In 2021, the University of Birmingham and Acticor Biotech signed a partnership agreement to evaluate glenzocimab efficacy in myocardial infarction in a new clinical trial called LIBERATE. The University has received full regulatory approvals to initiate the study. This new clinical trial is based on a long-standing collaboration between Acticor Biotech and the University of Birmingham. The publication in August of a scientific paper from Dr Mark Thomas entitled: “Amplified inhibition of atherosclerotic plaque-induced platelet activation by glenzocimab with dual antiplatelet therapy “ (link to the publication) in the Journal of Thrombosis and Haemostasis, reinforced the mode of action of glenzocimab and its major role as an antithrombotic drug. The randomised, double-blind Phase 2b LIBERATE study will recruit more than 200 patients suffering from a ST-elevation myocardial infarction (STEMI) and planned for a percutaneous coronary intervention. The study aims to assess the safety and the efficacy of glenzocimab 1000 mg versus placebo to reduce the myocardial infarct size at Day 90 post-treatment. The trial will be conducted in two acute care hospitals in the UK: the Queen Elizabeth Hospital, Birmingham and the Northern General Hospital, Sheffield. Patient recruitment is expected to start by the end of 2023. Doctor Mark Thomas, Associate Professor of Cardiology at the University of Birmingham and Honorary Consultant Cardiologist, who designed the trial and led its development, said: “Our recent studies of glenzocimab at the University of Birmingham have just been published – these explain the cellular mechanisms for why it is highly effective at reducing “blood stickiness”, particularly when combined with existing medications. We are very pleased to receive regulatory approval to launch the LIBERATE clinical trial to investigate whether glenzocimab can reduce the type of blood clotting that causes heart damage during heart attacks. This exciting collaboration with Acticor has the potential to benefit our patients in Birmingham and Sheffield, and across the world.” Professor Jon Townend, Consultant Cardiologist at University Hospitals Birmingham, Honorary Professor of Cardiology in the Institute of Cardiovascular Sciences at the University of Birmingham, and Chief Investigator of the trial said: “This new drug looks extremely promising and we are excited to be within sight of beginning recruitment for this important trial” Yannick Pletan, Chief Medical Officer, and General Manager of Acticor Biotech declared: “Glenzocimab has already delivered very promising results in the treatment of acute ischemic stroke and we hope to confirm its therapeutics potential in other cardiovascular emergency indications. LIBERATE phase 2b study extends glenzocimab development programme to myocardial infarction. We are delighted to collaborate with all the teams involved, the University of Birmingham particularly, sponsor of this study, to extend the therapeutic field of glenzocimab.” ENDS Notes to editor: The University of Birmingham is ranked amongst the world’s top 100 institutions. Its work brings people from across the world to Birmingham, including researchers, teachers and more than 6,500 international students from over 150 countries. The University of Birmingham is a member of Birmingham Health Partners (BHP), a strategic alliance which transcends organisational boundaries to rapidly translate healthcare research findings into new diagnostics, drugs and devices for patients. Birmingham Health Partners is a strategic alliance between five organisations who collaborate to bring healthcare innovations through to clinical application: University of Birmingham University Hospitals Birmingham NHS Foundation Trust Birmingham Women's and Children's Hospitals NHS Foundation Trust Sandwell and West Birmingham Hospitals NHS Trust West Midlands Academic Health Science Network About ACTICOR BIOTECH Acticor Biotech is a clinical stage biopharmaceutical company, a spin-off from INSERM (the French National Institute of Health and Medical Research), which is aiming to develop an innovative treatment for cardiovascular emergencies, including ischemic stroke. Acticor Biotech is developing glenzocimab (ACT017), a humanized monoclonal antibody (mAb) fragment directed against a novel target of major interest, platelet glycoprotein VI (GPVI). Glenzocimab inhibits platelet binding to the thrombus without affecting physiological hemostasis, thereby limiting the bleeding risk, particularly in the brain. The positive results from its Phase 1b/2a study, ACTIMIS, confirmed the safety pro showed a reduction in mortality and intracerebral hemorrhage in the glenzocimab-treated group in patients with stroke. The efficacy of glenzocimab is now being evaluated in ACTISAVE, an international Phase 2/3 study. In July 2022, Acticor Biotech was granted "PRIME" status by the European Medicines Agency (EMA) for glenzocimab in the treatment of stroke. This designation will allow the company to strengthen its interactions and obtain early dialogues with regulatory authorities. Acticor Biotech is supported by a panel of European and international investors (Karista, Go Capital, Newton Biocapital, CMS Medical Venture Investment (HK) Limited, A&B (HK) Limited, Mirae Asset Capital, Anaxago, Primer Capital, Mediolanum farmaceutici and the Armesa foundation). Acticor Biotech is listed on Euronext Growth Paris since November 2021 (ISIN: FR0014005OJ5 – ALACT). For more information, visit: Disclaimer This press release contains certain forward-looking statements concerning Acticor Biotech and its business. Such forward-looking statements are based on assumptions that Acticor Biotech considers to be reasonable. However, there can be no assurance that such forward-looking statements will be verified, which statements are subject to numerous risks, including the risks set forth in the Document de référence registration document as approved by the Autorité des marchés financiers under number R. 22-011 on 26 April 2022 and to the development of economic conditions, financial markets and the markets in which Acticor Biotech operates. The forward-looking statements contained in this press release are also subject to risks not yet known to Acticor Biotech or not currently considered material by Acticor Biotech. The occurrence of all or part of such risks could cause actual results, financial conditions, performance or achievements of Acticor Biotech to be materially different from such forward-looking statements.