AUTHOR=Azam Mohd W. , Khan Asad U. 

TITLE=CRISPRi-mediated suppression of E. coli Nissle 1917 virulence factors: A strategy for creating an engineered probiotic using csgD gene suppression

JOURNAL=Frontiers in Nutrition

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/nutrition/articles/10.3389/fnut.2022.938989

DOI=10.3389/fnut.2022.938989

ISSN=2296-861X

ABSTRACT=<sec><title>Background</title><p>Biofilm formation is a complex phenomenon, and it is the causative agent of several human infections. Bacterial amyloids are involved in biofilm formation leading to infection persistence. Due to antibiotic resistance, their treatment is a great challenge for physicians. Probiotics, especially <italic>E. coli</italic> Nissle 1917 (EcN), are used to treat human intestinal disorders and ulcerative colitis. It also expresses virulence factors associated with biofilm and amyloid formation. EcN produces biofilm equivalent to the pathogenic UPEC strains.</p></sec><sec><title>Methods</title><p>CRISPRi was used to create the knockdown mutants of the <italic>csgD</italic> gene (<italic>csgD</italic>-KD). The qRT-PCR was performed to assess the expression of the <italic>csgD</italic> gene in <italic>csgD</italic>-KD cells. The <italic>csgD</italic>-KD cells were also evaluated for the expression of <italic>csgA, csgB, fimA, fimH, ompR, luxS</italic>, and <italic>bolA</italic> genes. The gene expression data obtained was further confirmed by spectroscopic, microscopic, and other assays to validate our study.</p></sec><sec><title>Results</title><p>CRISPRi-mediated knockdown of <italic>csgD</italic> gene shows reduction in curli amyloid formation, biofilm formation, and suppression of genes (<italic>csgA, csgB, fimA, fimH, ompR, bolA, and luxS</italic>) involved in virulence factors production.</p></sec><sec><title>Conclusion</title><p>Curli amyloid fibers and fimbriae fibers play a critical role in biofilm formation leading to pathogenicity. CsgD protein is the master regulator of curli synthesis in <italic>E. coli</italic>. Hence, curli amyloid inhibition through the <italic>csgD</italic> gene may be used to improve the EcN and different probiotic strains by suppressing virulence factors.</p></sec>