MICA: Mechanistic understanding of cell wall biosynthesis to combat antimicrobial resistance

MICA：了解细胞壁生物合成对抗抗菌素耐药性的机制

• 批准号: MR/N002679/1
• 负责人: Christopher Dowson
• 金额: $ 412.42万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN002679%2F1
• 关键词: MICA; Mechanistic; understanding; cell; wall

The discovery of the antibiotic penicillin opened the door to the treatment of a wide range of infections. It works by stopping bacteria from making the polymer in the cell wall (peptidoglycan, PG) that holds them together. This is assembled by specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) that either have the ability to stitch together both the sugar backbone and the peptides (these are known as bi-functional enzymes), or either just the sugar back bone or just the peptide-crosslinks (mono-functional enzymes). We know little about how the polymerization and cross linking activities are controlled or co-ordinated, or how they truly interact with their natural substrates. Furthermore, the construction of peptide cross-links by PBPs is famously the target inhibited by penicillin which stops cell wall construction and kills the bacterium. Penicillin has been an excellent antibiotic, not least because it targets multiple PBPs simultaneously within a bacterium and resistance rarely develops by altering the PBP target (with the notable exception of bacteria that can acquire altered PBP genes from other species that are poor targets for the antibiotic). Unfortunately, many bacteria have acquired resistance to penicillin by other mechanisms. Primarily this has been due to the acquisition of enzymes that degrade the antibiotic (beta-lactamases), or reduce penetration (influx) of antibiotic into the bacterium or increasing the rate of efflux out of the bacterium. We urgently need to fight back and the strategy of exploring PBPs to make better versions of current antibiotics that are more active, can evade beta-lactamases or resistance due to changing influx or efflux. Global pharmaceutical companies have a real interest in progressing such developments, however, they need better mechanistic insight into how PBPs work. We attend to address these fundamental gaps in our understanding. Why can we succeed where others have failed? 1. Progress in achieving this mechanistic insight has been hampered by past inability to routinely synthesise the key chemical components or precursors that make this polymer. From past MRC and BBSRC funding we can now make key chemical components at Warwick, and have developed an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide.2. Having studied how to synthesise all of the chemical precursors used by different PBPs we have developed completely new continuous assays that will now help us to understand how PBPs polymerise precursors or how they crosslink these. We have one assay to finalise, which would bring together our ability to study polymerization and crosslinking in one reaction. Alongside a continuous crosslinking assay, these new technologies represent a 70 year long breakthrough and world first. 3. Super high resolution imaging is now available so that we can see how PBPs work inside bacteria and in the test tube, how they interact with each other and other proteins or lipids within bacterial cells. We can also study PBP structure at ultra-high resolution to understand how PBPs interact at the molecular level with their natural substrates and different well known antibiotics. We also have access to new chemical approaches, which along with our assays and structural biology will help direct us to new ways to stop these enzymes.4. Finally, we have brought together international academic experts from across the UK with skills in microbiology, chemistry and physics to work in synchrony and closely with many industry experts and a wider scientific advisory panel. This concentration of effort across a wide skill base with new technology will help ensure rapid progress and results with broad application that will be valuable for future programs of antibiotic discovery and development.

抗生素青霉素的发现为治疗各种感染打开了大门。它的工作原理是阻止细菌在细胞壁中制造聚合物（肽聚糖，PG），将它们结合在一起。这是由专门的蛋白质（称为青霉素结合蛋白或PBP，存在于所有细菌中）组装的，这些蛋白质要么能够将糖骨架和肽（这些被称为双功能酶）缝合在一起，要么只有糖骨架或只有肽交联（单功能酶）。我们对聚合和交联活动是如何控制或协调的，或者它们是如何与天然底物相互作用的知之甚少。此外，通过PBPs构建肽交联是青霉素抑制的目标，青霉素阻止细胞壁构建并杀死细菌。青霉素一直是一种出色的抗生素，尤其是因为它同时靶向细菌内的多个PBP，并且很少通过改变PBP靶点而产生耐药性（值得注意的例外是，细菌可以从其他物种获得改变的PBP基因，而这些物种是抗生素的不良靶点））。不幸的是，许多细菌通过其他机制获得了对青霉素的耐药性。这主要是由于获得降解抗生素的酶（β-内酰胺酶），或减少抗生素进入细菌的渗透（流入）或增加细菌外排的速率。我们迫切需要反击和探索PBPs的策略，以使当前抗生素的更好版本更有活性，可以逃避β-内酰胺酶或由于改变流入或流出而产生的耐药性。全球制药公司对推进此类开发有真实的兴趣，然而，他们需要更好地了解PBPs如何工作。我们致力于解决我们理解中的这些根本差距。为什么我们能在别人失败的地方取得成功？1.在实现这一机制的洞察力的进展一直阻碍了过去无法常规合成的关键化学成分或前体，使这种聚合物。从过去的MRC和BBSRC的资金，我们现在可以使关键的化学成分在沃里克，并已开发了一个特殊的跟踪记录提供试剂，研究肽聚糖生物合成的学术界世界各地。在研究了如何合成不同PBPs使用的所有化学前体之后，我们开发了全新的连续测定法，现在将帮助我们了解PBPs如何聚合前体或如何交联这些前体。我们有一个试验要完成，这将使我们能够在一个反应中研究聚合和交联。除了连续交联分析，这些新技术代表了70年的突破和世界第一。3.超高分辨率成像现在可以使用，这样我们就可以看到PBPs如何在细菌内部和试管中工作，它们如何与细菌细胞内的其他蛋白质或脂质相互作用。我们还可以在超高分辨率下研究PBP的结构，以了解PBP如何在分子水平上与其天然底物和不同的已知抗生素相互作用。我们还可以使用新的化学方法，沿着我们的分析和结构生物学将帮助我们找到阻止这些酶的新方法。最后，我们汇集了来自英国各地的具有微生物学，化学和物理学技能的国际学术专家，与许多行业专家和更广泛的科学咨询小组同步密切合作。这种在广泛的技能基础上利用新技术的集中努力将有助于确保快速进展和广泛应用的结果，这对未来的抗生素发现和开发计划是有价值的。

项目成果

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage

SosA 抑制金黄色葡萄球菌响应 DNA 损伤的细胞分裂

• DOI: 10.1101/364299
• 发表时间: 2018
• 作者: Bojer M

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

• DOI: 10.1073/pnas.2018089118
• 发表时间: 2021-04-06
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Aggarwal SD;Lloyd AJ;Yerneni SS;Narciso AR;Shepherd J;Roper DI;Dowson CG;Filipe SR;Hiller NL
• 通讯作者: Hiller NL

Towards an automated analysis of bacterial peptidoglycan structure.

• DOI: 10.1007/s00216-016-9857-5
• 发表时间: 2017-01
• 期刊: ANALYTICAL AND BIOANALYTICAL CHEMISTRY
• 影响因子: 4.3
• 作者: Bern, Marshall;Beniston, Richard;Mesnage, Stephane
• 通讯作者: Mesnage, Stephane

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine.

• DOI: 10.1038/s41467-017-02118-7
• 发表时间: 2017-12-05
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Batson S;de Chiara C;Majce V;Lloyd AJ;Gobec S;Rea D;Fülöp V;Thoroughgood CW;Simmons KJ;Dowson CG;Fishwick CWG;de Carvalho LPS;Roper DI
• 通讯作者: Roper DI

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

• DOI: 10.1038/s41586-022-04555-x
• 发表时间: 2022-04
• 期刊: Nature
• 影响因子: 64.8