"Molecular characterization of a novel host cell binding and invasion factor by the major foodborne pathogen Campylobacter jejuni"

“主要食源性病原体空肠弯曲菌的新型宿主细胞结合和入侵因子的分子特征”

• 批准号: 290441008
• 负责人: Dr. Manja Böhm
• 金额: --
• 依托单位: Max-Delbrück-Centrum für Molekulare Medizin (MDC) in der Helmholtz-Gemeinschaft
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/290441008?language=en
• 关键词: Molecular; characterization; novel; host; cell

Food-borne infections by Campylobacter jejuni are the major cause of human bacterial gastroenteritis, and may be responsible for as many as 400-500 million cases worldwide every year. Disease outcomes vary from mild, non-inflammatory, self-limiting diarrhea to severe, inflammatory, bloody diarrhea and abdominal pain lasting for several weeks, but C. jejuni is also associated with more severe sequelae such as the development of reactive arthritis and peripheral neuropathies, the Miller-Fisher and Guillain-Barrè syndromes, in some individuals. Major challenges are the lack of effective control schemes for C. jejuni in the food chain and a relatively poor understanding of basic mechanisms underlying host-pathogen interactions. Despite the array of bacterial virulence factors reported in the past, their potential role in pathogenesis is often controverse in the literature and still not fully clear. For example, it remained widely unknown which of the proposed C. jejuni factors are typical adhesins or not, and which disease-related signaling pathways are regulated by these factors. Recently, we have identified a novel C. jejuni virulence factor directly interacting with host cells, high temperature requirement A (HtrA), a surface exposed serine protease. Having established a series of important molecular tools and methods in our previous studies, this proposal is designed to gain a detailed understanding of the molecular mechanisms underlying specific HtrA functions in cell adhesion, invasion and host signaling during infection with C. jejuni in vitro and in vivo. In particular, we have identified an arginine-glycine-aspartate (RGD) motif in HtrA, and our preliminary data indicate that this motif is surface-exposed and crucial for cell binding and invasion by C. jejuni. We therefore propose that HtrA and its RGD-motif serve as recognition site for the host cell receptor integrin-b1. We generated C. jejuni strains expressing HtrA variants with point mutations in the RGD-motif or protease-inactive S197A, which will be further analysed using in vitro binding and cell invasion assays, quantitative immunoprecipitation combined with knockdown (QUICK), signaling methods, confocal microscopy and live cell imaging as well as short- and longterm mouse infections experiments. This approach will allow us to reconstruct the sequence of events occurring at the pathogen-host cell interface, and to identify novel key pathogenicity determinants. Further progress both in understanding of the biological significance of bacterial virulence factors and the nature of bacterial interactions with its host will lead to new strategies for detecting, controlling, and reducing Campylobacter infections. In a longterm perspective, this could help both to decrease the burden of Campylobacter-induced human illness and to reduce clinical expenses.

通过食物传播的空肠弯曲菌感染是人类细菌性胃肠炎的主要原因，每年全球可能有多达4亿至5亿病例。疾病的结果各不相同，从轻微的非炎症性自限性腹泻到持续数周的严重炎症性血性腹泻和腹痛，但空肠弯曲菌也与更严重的后遗症有关，如在某些人中发展为反应性关节炎和周围神经病变，米勒-费舍尔综合征和格林-巴利综合征。主要挑战是缺乏食物链中空肠弯曲菌的有效控制方案，以及对宿主-病原体相互作用的基本机制了解相对较差。尽管过去报道了一系列细菌毒力因子，但它们在发病机制中的潜在作用在文献中往往是有争议的，并且仍然不完全清楚。例如，目前尚不清楚所提出的空肠弯曲菌因子中哪些是典型的粘附素，以及哪些与疾病相关的信号通路受这些因子的调控。最近，我们发现了一种新的与宿主细胞直接相互作用的空肠弯曲菌毒力因子，高温要求A(HtrA)，一种表面暴露的丝氨酸蛋白酶。本研究在建立了一系列重要的分子工具和方法的基础上，对空肠弯曲菌在体内外感染过程中HtrA在细胞黏附、侵袭和宿主信号转导中的作用机制进行了详细的研究。特别是，我们在HtrA中发现了一个精氨酸-甘氨酸-天冬氨酸(RGD)基序，我们的初步数据表明，这个基序是表面暴露的，对空肠弯曲菌的细胞结合和入侵至关重要。因此，我们认为HtrA及其RGD基序可以作为宿主细胞受体整合素-b1的识别位点。我们获得了表达HtrA变异体的空肠弯曲菌菌株，这些菌株在RGD基序或蛋白酶非活性S197A上发生了点突变，将通过体外结合和细胞侵袭试验、定量免疫沉淀结合基因敲除(QUICK)、信号方法、共聚焦显微镜和活细胞成像以及短期和长期小鼠感染实验来进一步分析。这种方法将使我们能够重建发生在病原体-宿主细胞界面上的事件序列，并识别新的关键致病决定因素。在了解细菌毒力因子的生物学意义和细菌与宿主相互作用的性质方面的进一步进展将导致检测、控制和减少弯曲菌感染的新策略。从长远来看，这可能有助于减轻弯曲杆菌引起的人类疾病的负担，并减少临床费用。

项目成果

Expression of Helicobacter pylori Serine Protease HtrA in Campylobacter jejuni Reveals a Crucial Function in Oxygen Stress Resistance, Heat Tolerance and Epithelial Barrier Disruption

幽门螺杆菌丝氨酸蛋白酶 HtrA 在空肠弯曲菌中的表达揭示了其在氧应激抵抗、耐热性和上皮屏障破坏中的关键功能

• DOI: 10.15436/2471-5891.17.009
• 发表时间: 2017
• 作者: Boehm M;Tegtmeyer N;Harrer A;Skórko-Glonek J;Backert S
• 通讯作者: Backert S

HtrA‐dependent adherence and invasion of Campylobacter jejuni in human vs avian cells

空肠弯曲杆菌在人类和禽类细胞中依赖于 HtrA 的粘附和侵袭

• DOI: 10.1111/lam.13277
• 发表时间: 2019
• 期刊: Letters in Applied Microbiology
• 影响因子: 2.4
• 作者: Simson D;Boehm M;Backert S
• 通讯作者: Backert S

Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice

• DOI: 10.3389/fcimb.2019.00079
• 发表时间: 2019-03
• 期刊: Frontiers in Cellular and Infection Microbiology
• 影响因子: 5.7
• 作者: Anna-Maria Schmidt;U. Escher;S. Mousavi;M. Boehm;S. Backert;S. Bereswill;M. Heimesaat

Immunopathological properties of the Campylobacter jejuni flagellins and the adhesin CadF as assessed in a clinical murine infection model

• DOI: 10.1186/s13099-019-0306-9
• 发表时间: 2019-05-17
• 期刊: GUT PATHOGENS
• 影响因子: 4.2
• 作者: Schmidt, Anna-Maria;Escher, Ulrike;Heimesaat, Markus M.
• 通讯作者: Heimesaat, Markus M.