CRISPRimediated suppression of Nissle 1917 virulence factors: A strategy for creating an engineered probiotic using gene suppression.

BACKGROUND

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 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.

METHODS

CRISPRi was used to create the knockdown mutants of the gene (-KD). The qRT-PCR was performed to assess the expression of the gene in -KD cells. The -KD cells were also evaluated for the expression of , and genes. The gene expression data obtained was further confirmed by spectroscopic, microscopic, and other assays to validate our study.

RESULTS

CRISPRi-mediated knockdown of gene shows reduction in curli amyloid formation, biofilm formation, and suppression of genes () involved in virulence factors production.

CONCLUSION

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 . Hence, curli amyloid inhibition through the gene may be used to improve the EcN and different probiotic strains by suppressing virulence factors.