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

感染性腹泻是一个全球性的问题，弯曲杆菌是最常见的细菌原因。尽管它很重要，但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疾病最常见的细菌原因。超过80%的人类病例是由空肠弯曲杆菌引起的，其典型症状包括血性腹泻、发烧和腹痛。腹泻的一个关键先兆是当空肠弯曲菌入侵人类肠道上皮细胞(IECS)时引发的炎症，导致组织损伤和疾病。该细胞已经开发出一种维持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动态平衡失衡，但一些细菌物种，如弯曲杆菌，已被证明激活这一途径以增加细胞内的存活率。Gong等人未发表的数据。展示弯曲杆菌通过PERK和IRE1臂介导的UPR激活。然而，这篇论文没有确定激活UPR不同手臂的分子驱动因素。活性氧物种(ROS)是一组含氧的化学中间体，其电子数不均匀。正常情况下，ROS的产生是通过一氧化二氮(NOX)途径的激活来调节的，并被几种防御机制所抵消，如抗氧化剂的释放和维持氧化还原状态的平衡。有趣的是，几篇手稿已经确立了ROS的抗菌作用。以往的研究表明，空肠弯曲菌可以调节ROS的产生途径成分，以帮助细胞内的生存和增殖：空肠弯曲菌对人T84和Caco-2细胞的细胞内和细胞外ROS的产生具有差异性调节作用。空肠弯曲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IECS中关键的ROS产生酶，以及抗氧化防御基因CAT和SOD1。此外，二苯基碘(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、侵袭和存活的能力。UPR和NOX通路被激活，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UPR和ROS在弯曲杆菌介导的感染中-激活前、激活期间和激活后-之间的相互作用。缺乏某些毒力因子的弯曲杆菌突变株将被开发出来，并将评估它们入侵宿主细胞以及激活UPR和NOX途径的能力。在入侵试验中对细菌和宿主细胞进行转录分析将更好地了解这两种病原体在入侵周期中的基因组变化，细菌突变将用于确定毒力因子转录的变化，以及宿主细胞的防御机制。将特别注意转录组中与白细胞介素类等炎症分子增加相对应的变化。不同的空肠弯曲菌菌株和不同的动物细胞株将被用来研究文献中观察到的疾病状态的差异。将研究这两个信号通路的生化基础，并将使用质谱仪、ELISA和下拉分析等技术来鉴定UPR和ROS途径激活的单独成分。一旦该项目的核心方面完成，该项目将变得更加广泛；它将结合组学技术，在对空肠弯曲菌感染做出反应时，更好地系统地了解UPR和ROS系统，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，以更好地概括体内环境。

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 Unpublished data from Gong et al.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.