Control of pathogen gene expression during symbiotic maintenance.

共生维持过程中病原体基因表达的控制。

• 批准号: MR/R021821/1
• 负责人: Betty Chung
• 金额: $ 150.12万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FR021821%2F1
• 关键词: Control; pathogen; gene; expression; during

Salmonella and Toxoplasma are widely spread intracellular pathogens. Salmonella typhi, the cause of typhoid fever, infects around 21.5 million people each year. Meanwhile around a third of the world's population is infected by Toxoplasma. Understanding host responses to pathogens is critical for developing effective intervention strategies. A major response of cells during pathogen infection is changes in gene expression, leading to changes in the proteins being produced in the cell. Proteins are essential biopolymers in all living organisms, playing roles as structural components of cells, enzymes, and immune response agents such as antibodies. Regulation of gene expression can occur at two levels: transcription (where mRNA is synthesised in the cell nucleus by the macromolecular machine RNA polymerase) and translation (where mRNA is decoded into proteins in the cell cytoplasm by the macromolecular machine known as the ribosome). When cells are stressed, specific gene expression pathways are activated (e.g. cytokines as part of the innate immune system). However, so far, very few studies have systematically studied these changes in gene expression at the level of translation. This is particularly true for bacterial and protozoan pathogens. The primary reason is due to technical difficulties with global monitoring of protein synthesis. Transcriptional regulation has been previously studied; however there is evidence that a significant amount of regulation also occurs at the translational level. This makes sense as direct modulation of protein synthesis provides a faster and more efficient response to pathogen infection, as it circumvents de novo mRNA transcription, processing and transport to the cell cytoplasm. Translational control is a highly dynamic process and global studies have only recently become possible with the advent of RiboSeq - a high-throughput technique that allows capturing the location and abundance of all ribosomes on mRNAs, allowing precise global measurement of real-time protein synthesis. I plan to carry out an integrated program of research aimed towards understanding the complex interplay of host and pathogen gene regulation, and its ultimate effect on the host proteome and host response to biotic stress. My research proposal will be executed in three stages. First, I will infect cells with either Salmonella or Toxoplasma and harvest samples at different time points for analysis of genome-wide responses at the level of RNA synthesis (using RNA sequencing, also known as RNASeq) as well as protein synthesis (using RiboSeq), permitting a sophisticated interrogation of host-pathogen interactions. By analysing global RNA and protein synthesis at different stages of infection, I will be able to distinguish genes that are activated at the early stage of infection at the level of protein synthesis, RNA synthesis, or both. Second, I hypothesise that there will be a number of genes activated at the level of protein synthesis during early infection that then go on to regulated the expression of other genes at the level of RNA synthesis (such genes are known as transcription factors and are common mediators of immune responses). This would provide the cell with a rapid response mechanism that initiates and shapes longer term anti-pathogen responses. I will use a technique known as chromatin-immunoprecipitation sequencing to capture and sequence the DNA bound by candidate transcription factors and compare the data with RNA expression profiles at later stages of infection to monitor their effects. Third, I will dissect the molecular mechanisms that drive switches in protein synthesis during early infection. Comparison between bacterial and eukaryotic pathogen infections will allow identification of common anti-pathogen pathways besides the responses that are specific for each pathogen. The results generated will be valuable for informing the development of intervention strategies.

沙门氏菌和弓形虫是广泛传播的细胞内病原体。伤寒沙门氏菌是伤寒的病因，每年感染约2150万人。与此同时，世界上大约三分之一的人口感染了弓形虫。了解宿主对病原体的反应对于制定有效的干预策略至关重要。病原体感染期间细胞的主要反应是基因表达的变化，导致细胞中产生的蛋白质的变化。蛋白质是所有生物体中必不可少的生物聚合物，作为细胞、酶和免疫应答剂（如抗体）的结构组分发挥作用。基因表达的调节可以在两个水平上发生：转录（其中mRNA在细胞核中由大分子机器RNA聚合酶合成）和翻译（其中mRNA在细胞质中由称为核糖体的大分子机器解码成蛋白质）。 当细胞受到应激时，特定的基因表达途径被激活（例如，作为先天免疫系统一部分的细胞因子）。然而，到目前为止，很少有研究在翻译水平上系统地研究基因表达的这些变化。对于细菌和原生动物病原体尤其如此。主要原因是全球监测蛋白质合成的技术困难。转录调控以前已经研究过;然而，有证据表明，大量的调控也发生在翻译水平。这是有意义的，因为蛋白质合成的直接调节提供了对病原体感染的更快和更有效的响应，因为它避免了从头mRNA转录、加工和运输到细胞质。翻译控制是一个高度动态的过程，全球研究直到最近才随着RiboSeq的出现而成为可能，RiboSeq是一种高通量技术，可以捕获mRNA上所有核糖体的位置和丰度，从而可以精确地实时全球测量蛋白质合成。我计划开展一项综合研究计划，旨在了解宿主和病原体基因调控的复杂相互作用，及其对宿主蛋白质组和宿主对生物胁迫的反应的最终影响。我的研究计划将分三个阶段执行。首先，我将用沙门氏菌或弓形虫感染细胞，并在不同的时间点收获样品，用于在RNA合成（使用RNA测序，也称为RNASeq）以及蛋白质合成（使用RiboSeq）水平上分析全基因组反应，从而对宿主-病原体相互作用进行复杂的询问。通过分析感染不同阶段的总体RNA和蛋白质合成，我将能够在蛋白质合成、RNA合成或两者的水平上区分感染早期被激活的基因。第二，我假设在感染早期，有许多基因在蛋白质合成水平上被激活，然后在RNA合成水平上调节其他基因的表达（这些基因被称为转录因子，是免疫反应的常见介质）。这将为细胞提供一种快速反应机制，启动并形成长期的抗病原体反应。我将使用一种称为染色质免疫沉淀测序的技术来捕获和测序候选转录因子结合的DNA，并将数据与感染后期的RNA表达谱进行比较，以监测它们的影响。第三，我将剖析在早期感染过程中驱动蛋白质合成开关的分子机制。细菌和真核病原体感染之间的比较将允许识别除了对每种病原体特异性的反应之外的共同抗病原体途径。所产生的结果将对制定干预战略具有参考价值。

项目成果

Chaperone-mediated coupling of subunit availability to activation of flagellar Type III secretion.

伴侣介导的亚基可用性与鞭毛 III 型分泌激活的耦合。

• DOI: 10.17863/cam.68599
• 发表时间: 2021
• 作者: Bryant O

The distinct translational landscapes of Gram-positive and Gram-negative bacteria

革兰氏阳性菌和革兰氏阴性菌的独特翻译景观

• DOI: 10.1101/2023.05.25.542305
• 发表时间: 2023
• 作者: Bryant O

Monitoring Real-time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

使用福斯特共振能量转移和圆二色性监测短 RNA 发夹的实时温度动态。

• DOI: 10.21769/bioprotoc.3950
• 发表时间: 2021
• 期刊: Bio-protocol
• 影响因子: 0.8
• 作者: Balcerowicz M

Coupling of subunit availability to activation of PMF-driven flagellar type III secretion

亚基可用性与 PMF 驱动的鞭毛 III 型分泌激活的耦合

• DOI: 10.1099/acmi.ac2019.po0216
• 发表时间: 2019
• 期刊: Access Microbiology
• 作者: Bryant O

Chaperone-mediated coupling of subunit availability to activation of flagellar Type III secretion

伴侣介导的亚基可用性与鞭毛 III 型分泌激活的耦合

• DOI: 10.17863/cam.71768
• 发表时间: 2021
• 作者: Bryant O