Characterization of mobile genetic elements from clostridium difficile and development of molecular tools for genetic manipulations

艰难梭菌目前被认为是导致北美和欧洲医院感染的最重要病原体之一，尤其是在老年人中。艰难梭菌的毒力主要依赖于两种毒素（TcdA和TcdB）以及产孢和在环境中持续存在的能力。最近艰难梭菌毒力菌株的基因组测序揭示了几个可移动的遗传元件，包括一个质粒和两个完整的噬菌体。因此，水平基因转移很可能是这种病原体进化的一个重要机制。然而，迄今为止对艰难梭菌的噬菌体和质粒的研究很少，根据它们在其他病原体的毒力中的作用，有必要更好地表征它们并评估它们对艰难梭菌的生物学和进化的影响。此外，用于艰难梭菌遗传操作的有效分子工具仍然非常有限，这阻碍了进一步的基础研究。我们的长期目标是表征艰难梭菌的噬菌体和质粒，并开发用于其遗传操作的分子工具，以深入了解这种细菌的生态，多样性，毒力和进化。将利用分子技术对约1000株菌株进行分类，并在微生物学和分子水平上对这些菌株基因组内的噬菌体进行鉴定和表征，包括全基因组测序。质粒也将被分离、测序和充分表征。在我们收集的艰难梭菌中，噬菌体和质粒的分布将被确定，这将有望为我们提供有关它们在艰难梭菌生态和进化中的作用和影响的线索。最后，这些质粒将用于开发分子工具（如载体）来操纵艰难梭菌基因组。这项研究将首次提供大量艰难梭菌菌株中噬菌体多样性的数据。我们还将获得有关艰难梭菌质粒及其潜在功能的基本数据。在这些可移动的遗传元件中有望发现新的毒力或适合度因子，这将有助于我们更好地了解艰难梭菌的微生物多样性。

Clostridium difficile is now considered as one of the most important pathogen responsible for nosocomial infections in North America and Europe, especially among the elderly. Virulence of C. difficile relies mainly on two toxins (TcdA and TcdB) and on the ability to sporulate and persist in the environment. The recent genome sequencing from a virulent strain of C. difficile revealed several mobile genetic elements, including a plasmid and two complete prophages. Thus, horizontal gene transfer is most likely an important mechanism for the evolution of this pathogen. However, prophages and plasmids of C. difficile have only been poorly studied so far and according to their role in virulence of other pathogens, there is a need to better characterize them and assess their impact on the biology and evolution of C. difficile. In addition, efficient molecular tools for the genetic manipulation of C. difficile remain very limited, which blocks further fundamental studies. Our long-term objective is to characterize prophages and plasmids from C. difficile and to develop molecular tools for its genetic manipulation in order to get insight into the ecology, diversity, virulence and evolution of this bacterium. A collection of ~1,000 strains will be classified by molecular techniques, and prophages within the genome of these strains will be identified and characterized at the microbiological and molecular level, including whole genome sequencing. Plasmids will also be isolated, sequenced and fully characterized. The distribution of prophages and plasmids across our C. difficile collection will be determined, which will hopefully give us clues about their role and impact on C. difficile ecology and evolution. Finally, these plasmids will be used to develop molecular tools (e.g. vectors) to manipulate the C. difficile genome. For the first time, this study will provide data about prophage diversity in a large collection of C. difficile strains. We will also gain fundamental data regarding C. difficile plasmids and their potential function(s). New virulence or fitness factors are expected to be found within these mobile genetic elements, which could help us better understand the microbial diversity of C. difficile.