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

Abstract

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 E. coli 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 csgD gene (csgD-KD). The qRT-PCR was performed to assess the expression of the csgD gene in csgD-KD cells. The csgD-KD cells were also evaluated for the expression of csgA, csgB, fimA, fimH, ompR, luxS, and bolA genes. The gene expression data obtained was further confirmed by spectroscopic, microscopic, and other assays to validate our study.

Results: CRISPRi-mediated knockdown of csgD gene shows reduction in curli amyloid formation, biofilm formation, and suppression of genes (csgA, csgB, fimA, fimH, ompR, bolA, and luxS) 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 E. coli. Hence, curli amyloid inhibition through the csgD gene may be used to improve the EcN and different probiotic strains by suppressing virulence factors.

Keywords: CRISPRi; E. coli; bacterial amyloids; biofilm; csgD; curli; dCas9; probiotic.

Figure 1

(A) csgD gene downregulation quantified by quantitative real-time PCR in E. coli Nissle 1917 (EcN) and K12 strain. The csgD-KD mutant A showed a 65.8% suppression in EcN (TEA) and in K12 cells and 63.8% reduction (TKA) as compared to the control cells (cells having no aTc induction but have both plasmids). The csgD-KD mutant B showed a 35.4% reduction in EcN (TEB) and 41.0% reduction in K12 (TKB). (B–H) Downregulation of the different genes in csgD-KD cells of EcN (TEA and TEB) and K12 (TKA and TKB). (B) Downregulation of csgA gene. (C) Downregulation of csgB gene. (D) Downregulation of ompR gene. (E) Downregulation of fimH gene. (F) Downregulation of fimA gene. (G) Downregulation of luxS gene. (H) Downregulation of bolA gene. The gene expression data was represented in fold change. One-way ANOVA analysis was done, and the *p-value < 0.05,**p-value < 0.005 was considered statistically significant. The 16s rRNA gene was used as endogenous control.

Figure 2

(A,B) Biofilm reduction quantification was done through CV assay in csgD-KD cells. (A) TKA showed 61.47% and TKB 48.09% biofilm reduction, respectively, compared to the control cells. (B) The EcN csgD-KD mutants TEA and TEB showed 75.6% and 64.7% biofilm reduction. (C,D) XTT reduction assay for cell viability in csgD csgD-KD cells. (C) Cell viability of csgD-KD cells of K12 (TKA and TKB) (D) Cell viability of csgD-KD cells of EcN (TEA and TEB). The data shown here represent an average value of triplicate experiments ± S (*P < 0.05, t-test, two-sided), (**P < 0.005, t-test, two-sided).

Figure 3

(A,B) The spectra showing Congo red fluorescence assay of csgD-KD cells. The control cell showed a higher fluorescence intensity as compared to the csgD-KD cells (A) Congo red fluorescence assay of csgD-KD cells of K12 (TKA and TKB) (B) Congo red fluorescence assay of csgD-KD cells of EcN (TEA and TEB). (C,D) The spectra are showing ThT (thioflavin) fluorescence assay in csgD-KD cells. The decline in fluorescence spectra intensity suggests lower curli amyloid production in csgD-KD cells, (C) ThT fluorescence assay of TKA and TKB, (D) ThT fluorescence assay of TEA and TEB.

Figure 4

(A–D) Congo red agar plate assay of EcN and K12 cells with csgD knockdown. (A,B) show csgD-KD cells of EcN and K12, respectively, while (C,D) show control cells of EcN and K12. The Congo red agar plates show a yellowish-white colony in mutated cells, suggesting a decrease in the curli production.

Figure 5

(A–D) Biofilm cell adherence assay through CSLM. (A,B) show control EcN and K12 cells while the (C,D) show csgD-KD cells of EcN and K12. The control cells (A,B) robust biofilm and aggregated cells have high EPS content, while the csgD-KD cells show a reduction in the EPS contents, as well, as the cells seem to be dispersed. The images were taken on a 20-μm scale. (E–H) 3D confocal images are showing biofilm thickness. (E,F) show control EcN and K12 cells, while G,H show csgD-KD cells of EcN and K12. The csgD-KD cells show a reduction in the biofilm thickness as compared to the control cells. In this figure and 6 images were taken at a 20-μm scale and on 63X magnification.

Figure 6

TEM images of csgD-KD cells of EcN and K12. (A–D) shows control of TEA, TKA, TEB, and TKB, respectively, while (E–H) represents the csgD-KD cells of TEA, TKA, TEB, and TKB. The control cells show a large number of fimbriae, curli amyloid fibers, and other cell surface extremities, while the csgD-KD cells show no fimbriae on their cell surface. All images were taken on a 500-nm scale.

Figure 7

String image showing CsgD and its interaction with CsgA, CsgB, OmpR, and other proteins.

Figure 8

A comprehensive overview of the study.