The biofilm formation in bacterial cells is a complex phenomenon. To understand the mechanism of change in state of bacterial cells from planktonic to sessile or biofilm, is a fascinating area of research. The biofilm formation in bacterial cells (e.g. E. coli) is controlled by several factors including genes, transcription regulators, two-component systems (EnvZ/OmpR and CpxA/CpxR), bacterial secondary messenger (e.g. c-di-GMP) and chemical molecules like in quorum sensing [14, 15].
The BolA protein is found to be a transcriptional switch that regulates the transition of planktonic to the biofilm stage in bacterial cells. The BolA protein has pleiotropic effects and it regulates the flagellar gene expression and biofilm formation. In addition to the BolA, a common bacterial secondary messenger c-di-GMP also regulates the bacterial cell motility, cell cycle regulation, biofilm formation and virulence. The overexpression of the bolA gene reduces the swimming motility in E. coli by regulating its flagellar assembly and that eventually leads an increase in the biofilm formation [1]. It is also reported that alteration in the bolA gene expression affects the outer membrane properties and also beta-lactamase AmpC, PBP5, PBP6 and carboxypeptidases. The overexpression of bolA causes a reduction in membrane permeability, that ultimately leads to a decrease in the penetration of the high molecular mass antibiotics into the cells [18]. In view of these facts, we have initiated this study to explore the role of bolA in the biofilm formation and expression of curli amyloid and fimbriae (fimH) like associated virulence factors.
Curli proteins are secreted through the type VIII secretion system (T8SS). In E. coli, curli synthesis involves mainly seven genes from csgA to csgG. The curli biogenesis is controlled by two different operons namely csgBAC and csgDEFG. The CsgA and CsgB are major and minor subunits, respectively, and form the structural components of the curli fiber. The CsgC is the periplasmic chaperone whereas, CsgE, CsgF and CsgG form the secretion machinery of the curli. The csgD gene is the master regulator of curli biogenesis, biofilm production, cellulose formation and it controls the csgBAC operon at the transcriptional level [15, 19]. The CsgD also controls the adrA which regulates cellulose biosynthesis, post-transcriptionally and encodes another common biofilm matrix component [20]. The activity of the CsgD is under the control of several regulatory systems that respond to several environmental factors like temperature, osmolality, pH and nutrient conditions. Curli fibers help bacterial cells to adhere to biotic (e.g. host proteins) and abiotic surfaces that in turn, help in the biofilm formation and pathogenicity [20].
The bolA gene of the E. coli K-12 MG1655 cells was suppressed using CRISPRi technology. The gene expression data showed the downregulation in the mRNA expression level by 74.4%. bol-KD cells were further explored for the expression of the major curli subunit csgA gene and the master regulator of biofilm and curli synthesis genes csgD, whereas, both csgA and csgD genes in bolA knockdown cells were found downregulated. The csgA and csgD genes show suppression by 43.6% and 43.4%, respectively. In E. coli, Type I fimbriae play a role in urinary tract infections that help in the adhesion to the host cells e.g. to the mannose expressing receptors on uroepithelium and promotes the intracellular bacterial communities formation. These adhesin proteins are encoded by fim genes, having two transcription units, working independently. These units encode a polycistronic operon that forms the structural components (FimA, FimF, FimG, and FimH), recombinase FimB and FimE and pilus assembly system (FimC and FimD) [21]. Further, we have checked the expression level of another adhesion protein FimH of fimbriae which is encoded by the fimH gene. The FimH is a mannose-binding protein and acts as an adhesive protein to help bacterial cells to attach on cells surfaces and it occurs at the tip of the fimbriae [22]. The gene expression data showed 79.5% suppression of the fimH gene in bol-KD cells, which is very high as compared to the suppression of curli genes (Fig. 2). It was conclude from the gene expression data that bolA gene is somehow affecting the expression of curli and fimbriae gene in E. coli biofilm formation.
The functional amyloids form a structural framework of the extracellular matrix in the biofilm. Amyloid formation is associated with several incurable neurodegenerative diseases in the human being that ranges from Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease, and prion diseases to Spinal muscular atrophy (SMA). The amyloid structure is highly stable and is generally resistant to the harsh denaturing conditions. General proteases are even, unable to degrade it [20].
In E. coli and Salmonella sp., the Congo red (CR) is frequently used to assess the curli production. The CR dye does not inhibit the growth of cells and binds to curliated whole cells and can be used to quantify the curliation in whole cells. When E. coli cells were grown in CR containing agar plates, there is depletion in the CR in the underlying agar of growth which suggests the curli production [22, 23]. The bol-KD cells were found to reduce curli amyloid formation on CR agar plates as shown in Fig. 3b. To further validate the study, we have performed the fluorescence spectroscopic assay of the biofilm cells with ThT and CR. ThT and CR dyes are the most commonly used indicator dyes that bind to curli amyloid fibrils and show increased fluorescence upon binding [24, 25]. The fluorescence spectra of both ThT and CR showed a sharp and very high fluorescence intensity in control cells while the bol-KD cells showed the least fluorescence intensity as compared to the control cells (Fig. 3c, d, respectively). The fluorescence data suggests that there is a reduction in the curli amyloid production in the bol-KD cells.
Toluidine blue O (TBO) is a cationic dye, and it is known to bind with the negative charged cell exterior EPS. In a very similar experiment to the ThT and Congo red fluorescence, we have assessed the binding of negatively charged EPS content of the biofilm cells with the cationic dye, toluidine blue oxide (TBO) with the help of fluorescence spectroscopy [26, 27]. The bol-KD biofilm cells show a decrease in the TBO-EPS fluorescence as compared to the control cells (Fig. 3e). This TBO fluorescence data suggest the reduction in the negatively charges extracellular content of the biofilm. But there is one difference that we have found in case of ThT and CR fluorescence spectra, that the change in fluorescence intensity is comparable between control and bol-KD cells in ThT and CR assay while in case of TBO the change in fluorescence spectra is negligible.
To visualize the curli and other cell surface extracellular extremities production in the biofilm cells, we have performed the TEM analysis. Our TEM data showed that the biofilm cells in the bol-kD cells appeared smooth with no or very low curli and fimbriae content on their cell surface (Fig. 5a–d, TEM images). The control cells in TEM images appeared to be very robust cells having thick cluster of curli, fimbriae on their surface in comparison to bolA knockdown cells. Further bolA knockdown cells appeared as elongated cells while the control cells remained spherical. We had already discussed that bolA is a morphogene and its overexpression causes the spherical cell morphology. The exact mechanism of how bolA controls the cell morphology change is not very well understood, although some reports showed that bolA overexpression causes a reduction in the MreB protein [28]. The MreB protein is required for the maintenance of the rod shape of the cells and its polymerization is critical for the bacterial cell cytoskeleton [28].
The crystal violet (CV) assay was also performed for the assessment of biofilm formation in bolA gene knockdown cells. The CV assay of bol-KD cells having InvF1 and InvF2 plasmids showed 67.8% and 61.1% biofilm reduction, respectively (Fig. 3a). This reduction in biofilm formation indicates the reduction in the fimbriae and curli fibers which helps in the different stages of biofilm formation [10, 14]. Further, we had confirmed the adherence and biofilm reduction with the help of a confocal microscope (CLSM) and found a decrease in the biofilm cell adherence, thickness, and cell aggregate formation in the bol-KD cells (Fig. 5 confocal images e-g). Both biofilm formation assay with CV and CLSM images, further validated that the bol-KD cells showed reduced and disintegrated biofilm.
Biofilm cells can be a thousand times more resistant to antimicrobial treatment than the planktonic cells [3]. In biofilms, the bacterial cells are surrounded with extracellular polymeric substance (EPS) and appear as microcolonies, comprised of DNA, RNA, proteins, polysaccharides, signal molecules, etc. [29]. The above-mentioned components of the EPS help to encase the cells in the biofilm. We have quantified the eDNA, extracellular protein and sugar content in the extracellular matrix of the biofilm and found a decrease in all of the above materials in the bol-KD cells as shown in Fig. 4a–c, respectively. We also accessed the eDNA production in the TBO agar plates, resulting a significant difference in darkness of the colonies colour in bol-KD and control plates, the control plate has thickened and protruded growth while the bol-KD cells show thin and flat growth (Additional file 1: Figure S3).
The XTT reduction assay was performed to know whether the CRISPRi mediated knockdown of the bolA gene is affecting the cell viability or not (Fig. 4d)? The reduction of the tetrazolium salts (XTT) from pale or light-colored to a brightly colored product known as formazans is the basis of the cell viability assay [30]. The XTT reduction data showed that 76.7% cells of InvF1 and 92% cells of InvF2 are viable in bolA knockdown cells which suggests that most of the bol-KD cells are viable and metabolically active [31]. A model has been proposed to show the functioning of bolA gene downregulation which affects the curli fibers, fimbriae and biofilm formation (Fig. 6).