CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in E. coli: An Approach to Inhibit Biofilm.

Azna Zuberi,Asad U Khan,Lama Misba

doi:10.3389/fcimb.2017.00214

Azna Zuberi, Asad U Khan + Show 1 more

Open Access

https://doi.org/10.3389/fcimb.2017.00214

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Abstract

Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, E. coli biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation. To implement CRISPRi system for luxS gene suppression, we have synthesized complementary sgRNA to target gene sequence and co-expressed with dCas9, a mutated form of an endonuclease. Suppression of luxS expression was confirmed through qRT-PCR. The effect of luxS gene on biofilm inhibition was studied through crystal violet assay, XTT reduction assay and scanning electron microscopy. We conclude that CRISPRi system could be a potential strategy to inhibit bacterial biofilm through mechanism base approach.

Highlights

Biofilm is the emergent form of bacterial life, described as the bacterial aggregates encased in self-producing matrix (Singh et al, 2000; Flemming et al, 2016) Bacteria in this type of sessile biofilm lifestyle develop resistance against the antimicrobial treatments (Whiteley et al, 2001)
The PCR products were purified, ligated and transformed in TOP 10, competent E. coli cells and the data was confirmed through sequencing of linearized DNA fragments containing target specific single guide RNA (sgRNA), generated through colony PCR with different set of primers (Supplementary Table 2)
After 24 h of growth, adherent biofilms were washed with 200 μl PBS solution to remove planktonic cells

Summary

Introduction

Biofilm is the emergent form of bacterial life, described as the bacterial aggregates encased in self-producing matrix (Singh et al, 2000; Flemming et al, 2016) Bacteria in this type of sessile biofilm lifestyle develop resistance against the antimicrobial treatments (Whiteley et al, 2001). One such Gram-negative anaerobe is E. coli, known for its intestinal (InPEC) and extra intestinal (ExPEC) infections due to the formation of aggressive and dense bacterial biofilms.

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