High-throughput analysis of gene essentiality and sporulation in Clostridium difficile.
High-throughput analysis of gene essentiality and sporulation in Clostridium difficile.
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DOI：
10.1128/mbio.02383-14
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发表时间：
2015-02-24
期刊：
影响因子：
6.4
通讯作者：
Fagan RP
中科院分区：
文献类型：
--
作者：
Dembek M;Barquist L;Boinett CJ;Cain AK;Mayho M;Lawley TD;Fairweather NF;Fagan RP
艰难梭菌是抗生素相关肠道感染的最常见原因，也是发病和死亡的重要原因。感染艰难梭菌需要破坏肠道微生物群，最常见的是使用抗生素。治疗干预很大程度上依赖于少量广谱抗生素，这进一步加剧了肠道菌群失调，并使患者对再感染极为敏感。开发新型靶向治疗干预措施需要详细了解基本细胞过程（这些过程代表有吸引力的靶点）和物种特异性过程（例如细菌孢子形成）。由于缺乏遗传工具，我们对艰难梭菌感染遗传基础的了解受到了阻碍，尽管最近的进展在解决这一局限性方面取得了一些进展。在这里，我们描述了一种在临床重要的艰难梭菌菌株中快速产生大量转座子突变体的方法的开发。我们在艰难梭菌模型菌株中验证了我们的转座子诱变方法，然后在包含 70,000 多个独特突变体的高毒力流行菌株 R20291 (027/BI/NAP1) 中生成了综合转座子库。使用转座子定向插入位点测序 (TraDIS)，我们鉴定了体外生长所需的一组由 404 个必需基因组成的核心基因。然后，我们将该技术应用于孢子形成过程（这是艰难梭菌传播和发病机制的绝对要求），识别出 798 个可能影响孢子产生的基因。这项研究产生的数据将为社区提供宝贵的资源，并为未来对这种重要人类病原体的研究提供信息。艰难梭菌是医院患者（尤其是接受抗生素治疗的患者）潜在致命肠道感染的常见原因。由于缺乏解剖这种细菌的工具，我们对这种细菌的了解受到了阻碍。我们开发了一种同时研究细菌中每个基因功能的方法。使用此工具，我们确定了实验室细菌生长所需的一组 404 个基因。艰难梭菌还会产生高度耐药的孢子，可以在环境中长期存活，是细菌在患者之间传播的必要条件。我们已经应用我们的遗传工具来识别产生孢子所需的所有基因。所有这些基因都是治疗感染的新药的有吸引力的靶标。
Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.