Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai, India.
VIT Bhopal University, Sahore, India.
Gene. 2021 Nov 30;803:145890. doi: 10.1016/j.gene.2021.145890. Epub 2021 Aug 8.
Escherichia coli Nissle 1917 (EcN) is an efficient probiotic strain extensively used worldwide because of its several health benefits. Adhesion to the intestinal cells is one of the prerequisites for a probiotic strain. To identify the genes essential for the adhesion of EcN on the intestinal cells, we utilized a quantitative genetic footprinting approach called transposon insertion sequencing (INSeq). A transposon insertion mutant library of EcN comprising of ~17,000 mutants was used to screen the adherence to the intestinal epithelial cells, Caco-2. The transposon insertion sites were identified from the input and output population by employing next-generation sequencing using the Ion torrent platform. Based on the relative abundance of reads in the input and output pools, we identified 113 candidate genes that are essential for the fitness of EcN during the adhesion and colonization on the Caco-2 cells. Functional categorization revealed that these fitness genes are associated with carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, post-translational modification, stress response, motility and adhesion, and signal transduction. To further validate the genes identified in our INSeq analysis, we constructed individual knock-out mutants in five genes (cyclic di-GMP phosphodiesterase (gmp), hda, uidC, leuO, and hypothetical protein-coding gene). We investigated their ability to adhere to Caco-2 cells. Evaluation of these mutants showed reduced adhesion on Caco-2 cells, confirming their role in adhesion. Understanding the functions of these genes may provide novel insights into molecular regulation during colonization of probiotic bacteria to the intestinal cells, and useful to develop designer probiotic strains.
大肠杆菌 Nissle 1917（EcN）是一种高效的益生菌菌株，由于其多种健康益处，在全球范围内得到广泛应用。黏附于肠道细胞是益生菌菌株的前提之一。为了确定 EcN 黏附于肠道细胞的必需基因，我们利用了一种称为转座子插入测序（INSeq）的定量遗传足迹法。我们使用了一个包含约 17000 个突变体的 EcN 转座子插入突变体文库，来筛选对肠道上皮细胞 Caco-2 的黏附。通过使用 Ion torrent 平台的下一代测序，从输入和输出群体中鉴定转座子插入位点。根据输入和输出池中的读取相对丰度，我们鉴定了 113 个候选基因，这些基因对于 EcN 在 Caco-2 细胞上黏附和定植过程中的适应性是必需的。功能分类表明，这些适应性基因与碳水化合物运输和代谢、细胞壁/膜/包膜生物发生、翻译后修饰、应激反应、运动和黏附以及信号转导有关。为了进一步验证我们在 INSeq 分析中鉴定的基因，我们在五个基因（环二鸟苷酸磷酸二酯酶（gmp）、hda、uidC、leuO 和假定蛋白编码基因）中构建了单个敲除突变体。我们研究了它们黏附 Caco-2 细胞的能力。对这些突变体的评估表明，它们在 Caco-2 细胞上的黏附能力降低，证实了它们在黏附中的作用。了解这些基因的功能可能为理解益生菌细菌定植到肠道细胞过程中的分子调控提供新的见解，并有助于开发设计益生菌菌株。
