Deciphering the role of flavodoxins in the intestinal pathogen Clostridioides difficile

破译黄素氧还蛋白在肠道病原体艰难梭菌中的作用

• 批准号: 453440095
• 负责人: Dr. Susanne Sievers
• 金额: --
• 依托单位: Institut für Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/453440095?language=en
• 关键词: Deciphering; role; flavodoxins; intestinal; pathogen

The strictly anaerobic bacterium and intestinal pathogen Clostridioides difficile causes one of the most problematic nosocomial infections nowadays. Patients suffering from a Clostridioides difficile infection (CDI) show symptoms ranging from mild diarrhea to life-threatening inflammation of the intestinal epithelium. Most challenging for the treatment of a CDI is the pathogen’s ability to form endospores, a highly resistant life-form that even survives antibiotic treatment, which also explains the high relapse rate accompanying the disease. Within the host, C. difficile is stressed with low levels of oxygen and different reactive oxygen species produced by the immune system. Despite its anaerobic lifestyle, the pathogen can survive in the host and even expedite an infection. Some C. difficile strains are characterized by an extraordinary high oxygen tolerance. However, detailed molecular knowledge on how the pathogen senses oxidative stress, how the signal is transduced and a stress response eventually regulated and initiated is missing. Cellular structures containing a flavin cofactor are often involved in the oxidative stress response of bacteria. We identified a much higher-than-average number of genes encoding for putative flavodoxins in C. difficile. Flavodoxins are small proteins of a specific fold and bound to the cofactor flavin mononucleotide (FMN). The FMN cofactor is capable of redox reactions involving the transfer of single electrons meaning that it can generate or neutralize radicals. None of the 8 putative flavodoxins of C. difficile has been associated with an exact function so far. Thus, their involvement in the bacterium’s metabolism or oxidative stress response is completely unknown. However, several facts turn them into well suited potential candidates for new antimicrobial therapies. Firstly, they do not occur in higher developed eukaryotes. Secondly, an essential cellular function has been assigned to flavodoxins in many other bacteria and finally, they were described to take up a vital role in virulence in several pathogens. This project aims at the functional characterization of the 8 putative flavodoxins of C. difficile to pinpoint those that are essential for the bacterium to survive and cause disease in the host. For the comprehensive characterization, the flavodoxins will be thoroughly studied in-silico, their gene expression profile will be recorded and in gene knock-down experiments their essentiality will be tested. A global proteomics approach comprising various physiological conditions and flavodoxin knock-down mutants will be applied to pinpoint interaction partners and flavodoxin interaction networks. Eventually, direct binding partners of flavodoxins will be pulled out in-vitro by using strep-tagged flavodoxins as bait. Findings of this enterprise will not only further enlighten the role of flavodoxins in bacteria in general, but will possibly support the identification of novel targets in CDI therapy.

严格的厌氧菌和肠道病原体艰难梭菌导致当今最有问题的医院感染之一。患有艰难梭菌感染（CDI）的患者表现出从轻度腹泻到危及生命的肠上皮炎症的症状。治疗CDI最具挑战性的是病原体形成内生孢子的能力，内生孢子是一种高度耐药的生命形式，甚至可以在抗生素治疗中存活，这也解释了伴随疾病的高复发率。在宿主体内，C.艰难梭菌受到低水平的氧和由免疫系统产生的不同活性氧的压力。尽管它的厌氧生活方式，病原体可以在宿主中存活，甚至加速感染。一些C。艰难梭菌菌株的特征在于非常高的耐氧性。然而，关于病原体如何感知氧化应激，信号如何转导以及最终调节和启动应激反应的详细分子知识缺失。含有黄素辅因子的细胞结构通常参与细菌的氧化应激反应。我们在C.很难黄酮素是一种特定折叠的小蛋白质，与辅因子黄素单肽（FMN）结合。FMN辅因子能够进行涉及单电子转移的氧化还原反应，这意味着它可以产生或中和自由基。C.到目前为止，difficile与确切的功能相关联。因此，它们参与细菌的代谢或氧化应激反应是完全未知的。然而，有几个事实使它们成为新的抗菌疗法的合适的潜在候选者。首先，它们不存在于更高级的真核生物中。其次，在许多其他细菌中，黄酮素具有重要的细胞功能，最后，它们被描述为在几种病原体中的毒力中起重要作用。本项目旨在对C.很难确定那些细菌在宿主中生存和致病所必需的蛋白质。为了进行全面表征，将通过计算机模拟对黄酮素进行彻底研究，记录其基因表达谱，并在基因敲除实验中测试其必要性。一个全球蛋白质组学的方法，包括各种生理条件和flavodoxin敲低突变体将适用于查明相互作用的合作伙伴和flavodoxin相互作用网络。最终，通过使用链球菌标记的黄素毒素作为诱饵，黄素毒素的直接结合伴侣将在体外被拉出。这项研究的发现不仅将进一步揭示黄酮素在细菌中的作用，而且可能支持CDI治疗中新靶点的鉴定。