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

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肠道疾病最常见的细菌原因。空肠弯曲菌是造成80%以上人类病例的原因，其症状通常包括出血性腹泻、发烧和腹痛。腹泻的一个关键前兆是当C.空肠弯曲菌侵入人肠上皮细胞（IEC），导致组织损伤和疾病。该细胞已经开发出一种用于维持ER转录保真度的系统-未折叠蛋白反应（UPR）。虽然它主要作用于未折叠蛋白质的体内平衡的不平衡，但已显示几种细菌物种如弯曲杆菌激活该途径以增加细胞内存活。来自Gong等人的未发表数据证明弯曲杆菌通过PERK和IRE 1臂介导UPR激活。然而，这篇论文没有确定激活UPR不同臂的分子驱动因素。活性氧物质（ROS）是一组具有奇数电子的氧基化学中间体。通常，ROS的产生是由一氧化二氮（NOX）途径的激活介导的，并被几种防御机制所抵消，如抗氧化剂的释放和平衡，即“氧化还原”状态。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产生途径组分以帮助细胞内存活和增殖：空肠差异调节人T84和Caco-2细胞的细胞内和细胞外ROS产生。C. jejuni下调烟酰胺腺嘌呤二核苷酸磷酸（NAPDH）氧化酶（NOX 1）的转录和翻译，NOX 1是IEC中的关键ROS生成酶以及抗氧化防御基因CAT和SOD 1。此外，二苯碘鎓（DPI）和siRNA抑制NOX 1可降低C.空肠在T84和Caco-2细胞内相互作用、侵入和存活的能力。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，但易受细菌影响。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-在激活之前，期间和之后。将开发缺乏某些毒力因子的弯曲杆菌突变体，并评估其侵入宿主细胞以及激活UPR和NOX途径的能力。在入侵测定期间对细菌和宿主细胞的转录分析将提供对入侵周期期间两种试剂的基因组变化的更好理解，并且细菌突变体将用于确定毒力因子转录的变化以及宿主细胞防御机制。将特别注意转录组的变化，相应的增加炎症分子，如白细胞介素。将使用不同的空肠弯曲菌菌株和不同的动物细胞系来研究文献中观察到的疾病状态的差异。将研究两种信号通路的生化基础，并使用诸如质谱、ELISA和下拉分析等技术来鉴定UPR和ROS通路激活的通路的单个组分。一旦完成了项目的核心方面，该项目变得更加广泛;它将结合联合收割机的Omic技术，以更好地系统了解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，以更好地再现体内环境。

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 Unpublished data from Gong et al.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.