Deciphering the role of flavodoxins in the intestinal pathogen Clostridioides difficile

严格的厌氧菌和肠道病原体艰难梭状杆菌导致了当今最严重的医院感染之一。患有艰难梭状芽胞杆菌感染(CDI)的患者表现出从轻度腹泻到危及生命的肠道上皮炎症等各种症状。CDI治疗最具挑战性的是病原体形成内孢子的能力，内孢子是一种高度耐药的生命形式，即使在抗生素治疗后也能幸存下来，这也解释了伴随疾病的高复发率。在宿主内，艰难梭菌受到低水平氧气和免疫系统产生的不同活性氧种的压力。尽管这种病原体的生活方式是厌氧的，但它可以在宿主体内存活，甚至可以加速感染。一些艰难梭菌菌株具有极高的耐氧性。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控和启动应激反应的详细分子知识尚不清楚。含有黄素辅因子的细胞结构经常参与细菌的氧化应激反应。我们发现艰难梭菌中编码黄毒素的基因数量远远高于平均水平。黄毒素是一种特定折叠的小蛋白，与辅因子黄素单核苷酸(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。到目前为止，艰难梭菌的8种推测的黄毒素中没有一种与确切的功能有关。因此，它们参与细菌的新陈代谢或氧化应激反应是完全未知的。然而，几个事实使它们成为新的抗菌疗法的非常合适的潜在候选者。首先，它们不会出现在较高级的真核生物中。其次，黄毒素在许多其他细菌中被赋予了重要的细胞功能，最后，它们被描述为在几种病原体的毒力中发挥关键作用。该项目旨在对艰难梭菌的8种可能的黄毒素进行功能表征，以确定对该细菌的生存和在宿主中引起疾病至关重要的那些黄毒素。为了进行全面的表征，将在电子计算机上对黄毒素进行深入的研究，记录它们的基因表达谱，并在基因敲除实验中检验它们的重要性。一种包括各种生理条件和黄毒素敲除突变体的全球蛋白质组学方法将被应用于精确定位相互作用伙伴和黄毒素相互作用网络。最终，通过使用链霉菌标记的黄毒素作为诱饵，黄毒素的直接结合伙伴将在体外被拉出。这项研究的发现不仅将进一步揭示黄毒素在细菌中的作用，而且可能支持在CDI治疗中识别新的靶点。

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.