Abstract
BackgroundBiofilm formation is a complex phenomenon of bacterial cells, involved in several human infections. Its formation is regulated and controlled by several protein factors. The BolA-like proteins (bolA gene) are conserved in both prokaryotes and eukaryotes. The BolA protein is a transcription factor involved in bacterial cell motility and biofilm formation. This study was initiated to elucidate the role of the bolA gene in the curli biogenesis and amyloid production as well as to observe changes in the expression of fimH, a fimbriae gene.MethodsKnockdown mutants of Escherichia coli MG1655 bolA gene (bolA-KD) were generated using CRISPR interference. The results obtained, were validated through gene expression using RT-PCR, microscopic analysis and different biofilm and amyloid assays.ResultsThe bolA knockdown mutants showed a decrement in curli amyloid fibers, in fimbriae production and biofilm formation. We have also observed a reduction in EPS formation, eDNA production and extracellular protein content. Gene expression data showed that bolA downregulation caused the suppression of csgA and csgD of curli that led to the reduction in curli fiber and the amyloid formation and also the suppression of fimH, leading to the loss of fimbriae.ConclusionsCurli fibers and fimbriae are found to be involved in biofilm formation leading to the pathogenicity of the bacterial cell. BolA is a conserved protein and is found to play a significant role in curli and fimbriae formation in E. coli. This study further proved that CRISPRi mediated suppression of the bolA gene leads to inhibition of biofilm formation through curli and fimbriae inhibition. Hence, it may be proposed as a possible target for intervention of biofilm mediated infections.
Highlights
Biofilm formation is a complex phenomenon of bacterial cells, involved in several human infections
Bacterial cell culture Escherichia coli MG1655 was used for biofilm formation and other phenotypic studies
Suppression of bolA gene through CRISPRi The bolA mRNA expression level was checked by performing real-time PCR
Summary
Biofilm formation is a complex phenomenon of bacterial cells, involved in several human infections. Its formation is regulated and controlled by several protein factors. The BolA-like proteins (bolA gene) are conserved in both prokaryotes and eukaryotes. The BolA protein is a transcription factor involved in bacterial cell motility and biofilm formation. Escherichia coli BolA and its homologs established a widely conserved protein family from prokaryotes to eukaryotes, called the BolA-like protein family. Being a DNA-binding regulator, BolA promotes spherical morphology on its overexpression in E. coli and is one of the newly discovered stress regulator proteins [1]. BolA gene has pleiotropic effects and controls a variety of phenotypes like biofilm production, biofilm regulation, bacterial morphology, fimbria-like adhesins, curli fiber formation, membrane permeability, and flagella formation (Fig. 1) [1, 2]. Its overexpression leads to a short spherical morphology which decreases the surface to volume ratio, causing a reduction in the exposed surface to unfavourable environmental conditions [3]
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