Campylobacter pathogenesis: the Unfolded Protein Response (UPR), inflammation and human disease

弯曲杆菌发病机制：未折叠蛋白反应 (UPR)、炎症和人类疾病

• 批准号: 2881697
• 金额: --
• 依托单位: London School of Hygiene & Tropical Medicine
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2881697
• 关键词: Campylobacter; pathogenesis; Unfolded; Protein; Response

Infectious diarrhoea is a global problem with Campylobacter being the most common bacterial cause. Despite its importance, the mechanisms by which Campylobacter infection promotes inflammation and disease in humans remain unclear. Campylobacter infection is the most common bacterial cause of human diarrhoeal disease. The species Campylobacter jejuni is responsible for over 80% of human cases with symptoms typically including bloody diarrhoea, fever and abdominal pains. A key precursor of diarrhoea is inflammation triggered when C. jejuni invades human intestinal epithelial cells (IECs), leading to tissue damage and disease. The cell has developed a system for maintaining transcriptional fidelity of the ER - the unfolded protein response (UPR). Although it mainly acts upon imbalances in the homeostasis of unfolded proteins, several bacterial species like Campylobacter have been shown to activate this pathway to increase intracellular survival. Unpublished data from Gong et al. demonstrate campylobacter mediated UPR activation through the PERK and IRE1 arms. The paper however stopped short of identifying the molecular drivers that activate the different arms of the UPR. Reactive oxygen species (ROS) are a group of oxygen-based chemical intermediaries with an uneven number of electrons. Normally, ROS production is mediated by the activation of the nitrous oxide (NOX) pathway and is counteracted by several defence mechanisms, such as antioxidant release and a balance, the 'Redox' state, is maintained. Interestingly, several manuscripts have established an antimicrobial role of ROS. Previous work demonstrate that Campylobacter can modulate ROS production pathway components to aid intracellular survival and proliferation: C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX1), a key ROS-generating enzyme in IECs and antioxidant defence genes CAT and SOD1. Furthermore, inhibition of NOX1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade, and survive within T84 and Caco-2 cells. The UPR and NOX pathways are activated to aid intracellular bacteria clearance and form part of the autonomous cell response but are susceptible to bacterial influence. The project will aim to investigate the interplay between the UPR and ROS in Campylobacter mediated infection - before, during and after activation. Campylobacter mutants lacking certain virulence factors will be developed and their ability to invade host cells, as well as to activate the UPR and NOX pathways will be assessed. Transcriptional analysis of both bacterial and host cells during invasion assays will provide a better understanding of the genomic changes of both agents during the invasion cycle, and bacterial mutants will be used will be used to determine changes to the transcription of virulence factors, as well as host cell defence mechanisms. Particular attention will be given to changes in the transcriptome corresponding to increased inflammatory molecules such as interleukins. Different strains of C jejuni and various animal cell lines will be used to investigate the discrepancies in disease states observed in literature.The biochemical basis of both signalling pathways will be investigated, and techniques such as Mass-spec, ELISA and pulldown assays will be used to identify individual components of the pathways of UPR and ROS pathway activation.Once the core aspects of the project have been completed, the project become more expansive; It will combine 'Omic technologies to gain a better systemic understanding of the UPR and ROS systems when reacting to C jejuni infection and will relate the data to inflammatory processes. Other aims of the project include the development of a 3D cell culture model to better recapitulate in vivo environments.

感染性腹泻是一个全球性问题，弯曲杆菌是最常见的细菌原因。尽管其重要性，弯曲杆菌感染促进人类炎症和疾病的机制仍不清楚。弯曲杆菌感染是人类腹泻病最常见的细菌原因。超过 80% 的人类病例是由空肠弯曲菌引起的，这些病例的症状通常包括血性腹泻、发烧和腹痛。腹泻的一个关键前兆是空肠弯曲菌侵入人肠上皮细胞（IEC）时引发炎症，导致组织损伤和疾病。该细胞开发了一种维持内质网转录保真度的系统——未折叠蛋白反应（UPR）。尽管它主要作用于未折叠蛋白质的稳态失衡，但一些细菌物种（如弯曲杆菌）已被证明可以激活该途径以增加细胞内的存活率。龚等人未发表的数据。证明弯曲杆菌通过 PERK 和 IRE1 臂介导 UPR 激活。然而，该论文未能确定激活 UPR 不同臂的分子驱动因素。活性氧（ROS）是一组具有不均匀电子数量的氧基化学中间体。通常，ROS 的产生是由一氧化二氮 (NOX) 途径的激活介导的，并受到多种防御机制的抵消，例如抗氧化剂的释放和平衡，即“氧化还原”状态的维持。有趣的是，一些手稿已经确定了 ROS 的抗菌作用。先前的工作表明，弯曲杆菌可以调节 ROS 产生途径成分，以帮助细胞内存活和增殖：空肠弯曲杆菌差异调节人 T84 和 Caco-2 细胞中细胞内和细胞外 ROS 的产生。空肠弯曲杆菌下调烟酰胺腺嘌呤二核苷酸磷酸 (NAPDH) 氧化酶 (NOX1) 的转录和翻译，NOX1 是 IEC 中关键的 ROS 生成酶以及抗氧化防御基因 CAT 和 SOD1。此外，二亚苯基碘 (DPI) 和 siRNA 对 NOX1 的抑制降低了空肠弯曲菌在 T84 和 Caco-2 细胞内相互作用、入侵和存活的能力。 UPR 和 NOX 途径被激活以帮助细胞内细菌清除并形成自主细胞反应的一部分，但容易受到细菌的影响。该项目旨在研究弯曲杆菌介导的感染中 UPR 和 ROS 之间的相互作用——激活前、激活期间和激活后。将开发缺乏某些毒力因子的弯曲杆菌突变体，并评估它们侵入宿主细胞以及激活 UPR 和 NOX 途径的能力。在入侵测定过程中对细菌和宿主细胞进行转录分析将有助于更好地了解两种病原体在入侵周期中的基因组变化，并且将使用细菌突变体来确定毒力因子转录的变化，以及宿主细胞防御机制。将特别关注与白介素等炎症分子增加相对应的转录组变化。将使用不同的空肠弯曲菌菌株和各种动物细胞系来研究文献中观察到的疾病状态的差异。将研究两条信号通路的生化基础，并使用 Mass-spec、ELISA 和 Pulldown 检测等技术确定 UPR 和 ROS 途径激活途径的各个组成部分。一旦项目的核心部分完成，该项目就会变得更加广泛；它将结合“Omic”技术，以便更好地系统了解 UPR 和 ROS 系统对空肠弯曲菌感染的反应，并将数据与炎症过程联系起来。该项目的其他目标包括开发 3D 细胞培养模型，以更好地重现体内环境。