Uncovering the mechanisms of ABC transporter glycoconjugate translocation in Mycobacterium tuberculosis and their roles in virulence.

揭示结核分枝杆菌中 ABC 转运蛋白糖复合物易位的机制及其在毒力中的作用。

基本信息

  • 批准号:
    BB/R017255/1
  • 负责人:
  • 金额:
    $ 65.42万
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Research Grant
  • 财政年份:
    2018
  • 资助国家:
    英国
  • 起止时间:
    2018 至 无数据
  • 项目状态:
    已结题

项目摘要

Tuberculosis and leprosy are ancient infectious diseases caused by intracellular bacteria of the genus Mycobacterium. They have affected humanity for thousands of years and such long-term 'success' is closely linked to how their cell envelope, which is reminiscent of a molecular fortress, is structured and interacts with host cells. Building a molecular fortress involves complex metabolic pathways and requires exquisite coordination between multi-enzyme factories that produce the parts inside the cell before being exported outside. Understanding these export processes in detail opens new options for therapeutic intervention. The mycobacterial cell envelope differs from that of many other bacteria (pathogenic or not) in that it includes several distinct molecular layers such as peptidoglycan, polysaccharides, unusually long fatty acids named mycolic acids as well as phospholipids, collectively providing a shield against the destructive forces of antibiotics or the human immune system. Past research has largely clarified the identity of the enzymatic factories and their final products, but surprisingly little is known of how intermediate products are 'moved' across the inner membrane, before being passed to external 'assembly lines'. IN PREVIOUS WORK, we have identified two mycobacterial proteins, Rv3781 (Wzm) and Rv3783 (Wzt) as being involved in moving an essential polysaccharide called galactan across the inner cytoplasmic membrane. Rv3781 and Rv3783 are part of a much larger family of mycobacterial proteins called ATP Binding Cassette (ABC) Transporters. These membrane bound proteins require the energy (in the form of ATP) to export molecules across the cytoplasmic membrane in which they 'sit'. By sequence analysis we have identified two proteins, Rv1272c and Rv1273c, that share features similar to a well characterised ABC transporter (MsbA) in Gram negative bacteria. We show that Rv1272c plays an important role in assembly of the mycobacterial inner cytoplasmic membrane and that it is likely 'supported' by its partner protein, Rv1273c.The HYPOTHESIS leading to this proposal is that mycobacteria orchestrate glyconjugate transport by utilising specific ABC transporter proteins to 'move' these important glycan structures across the inner cytoplasmic membrane. It is important to investigate these process because they play key roles in assembly of the mycobacterial cell wall core and produce key molecules that 'hijack' the host immune response.WE PROPOSE to systematically characterise the function of ABC transporter proteins in a multi-pronged approach. First, we will generate direct and/or conditional gene knock-outs (using a genetic method known as CESTET) and investigate the effect of deleting individual ABC transporter genes on mycobacterial survival and cell envelope composition. Secondly, we will comprehensively characterise three key ABC transporter systems, that we have previously identified from preliminary work, in terms of what physiological substrates they bind and how the expression of these genes interact with the global transcriptional profile of the organism. This effort will entail comprehensive biochemical analysis of the cell envelope using specific radio-labelled tracer molecules, techniques such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy to determine atomic structures and RNA-seq combined with RT-PCR to establish transcriptional networks associated with ABC transporter gene expression. We will also investigate the importance of ABC transporter function of how mycobacteria are engulfed by macrophages (immune cells responsible for eradicating microorganisms). Finally, by utilising a mouse model of acute TB infection through a collaborative partnership with Dr Joanne Bacon at Public Health England, we will probe the in vivo essentiality of ABC transporters involved in immune-regulation of the host.
结核病和麻风是由分枝杆菌属的胞内细菌引起的古老的传染病。它们影响人类已有数千年之久,这种长期的“成功”与它们的细胞膜密切相关,这种细胞膜让人想起分子堡垒,它的结构和与宿主细胞的相互作用。建立分子堡垒涉及复杂的新陈代谢途径,需要多酶工厂之间的精细协调,这些工厂在细胞内生产部分,然后再出口到外部。对这些输出过程的详细了解为治疗干预开辟了新的选择。分枝杆菌的细胞膜与许多其他细菌(无论是致病的还是非致病的)的不同之处在于,它包括几个不同的分子层,如肽聚糖、多糖、名为分枝杆菌酸的超长脂肪酸以及磷脂,共同提供了抵御抗生素或人类免疫系统破坏性力量的屏障。过去的研究在很大程度上澄清了酶工厂及其最终产品的身份,但令人惊讶的是,人们对中间产品在被传递到外部“装配线”之前是如何“移动”到内膜上的知之甚少。在以前的工作中,我们已经鉴定了两种分枝杆菌蛋白,Rv3781(WZM)和Rv3783(WZT),它们参与了一种名为半乳糖的必需多糖跨内细胞膜的运输。Rv3781和Rv3783是一个更大的分枝杆菌蛋白家族的一部分,称为ATP结合盒(ABC)转运蛋白。这些结合在细胞膜上的蛋白质需要能量(以三磷酸腺苷的形式)将分子输出到它们所在的细胞质膜上。通过序列分析,我们鉴定了两种蛋白质Rv1272c和Rv1273c,它们的特征与革兰氏阴性细菌中的ABC转运蛋白(MSBA)相似。我们发现Rv1272c在分枝杆菌内细胞膜的组装中起着重要的作用,并且可能受到其伴侣蛋白Rv1273c的支持。导致这一提议的假设是,分枝杆菌通过利用特定的ABC转运蛋白来协调糖共轭运输,从而将这些重要的糖链结构‘移动’到内细胞膜上。研究这些过程很重要,因为它们在分枝杆菌细胞壁核心的组装中发挥关键作用,并产生‘劫持’宿主免疫反应的关键分子。我们建议以一种多管齐下的方法系统地表征ABC转运蛋白的功能。首先,我们将产生直接和/或有条件的基因敲除(使用称为CESTET的遗传方法),并研究删除单个ABC转运蛋白基因对分枝杆菌存活和细胞膜成分的影响。其次,我们将综合表征三个关键的ABC转运蛋白系统,我们先前从初步工作中确定了它们结合了什么生理底物,以及这些基因的表达如何与生物体的全球转录谱相互作用。这项工作将需要使用特定的放射性标记示踪分子对细胞被膜进行全面的生化分析,使用诸如质谱学和核磁共振(核磁共振)光谱等技术来确定原子结构,并将RNA-SEQ与RT-PCR相结合来建立与ABC转运蛋白基因表达相关的转录网络。我们还将调查ABC转运蛋白功能在分枝杆菌如何被巨噬细胞(负责根除微生物的免疫细胞)吞噬中的重要性。最后,通过与英国公共卫生的乔安妮·培根博士合作,利用急性结核病感染的小鼠模型,我们将探索ABC转运蛋白在体内参与宿主免疫调节的重要性。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Mycobacterium tuberculosis modifies cell wall carbohydrates during biofilm growth with a concomitant reduction in complement activation
结核分枝杆菌在生物膜生长过程中改变细胞壁碳水化合物,同时减少补体激活
  • DOI:
    10.1101/2021.03.23.436651
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Keating T
  • 通讯作者:
    Keating T
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