The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σE‐dependent sRNA RybB

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self‐produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ^E cell envelope stress response and thereby reducing the expression of CsgD – a crucial activator of curli and cellulose biosynthesis – due to csgD mRNA targeting by the σ^E‐dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm‐associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.     

The influence of CsgD on the expression of genes of folate metabolism and hmp in Escherichia coli K-12

The csgD gene codes for the regulatory protein CsgD. CsgD upregulates the synthesis of the adhesive fimbriae, curli, that are important for biofilm formation and downregulates flagellar synthesis. We compared the expression of genes involved in folate metabolism and a gene (hmp) in strains with an intact csgD gene and with a deletion in csgD. The hmp gene codes a flavohemoglobin that inactivates nitric oxide. Expression was monitored by measuring light production from single copy lux operon fusions. At late times of growth, expression of genes responsible for methylene tetrahydrofolate synthesis (glyA and gcvTHP) and formyltetrahydrofolate recycling (purU) was higher in cells with CsgD than those without. In contrast, expression of hmp was lower in the presence of CsgD throughout the period monitored. We used a novel defined medium which should assist in defining nutritional factors that contribute to curli formation.     

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA⁻ and OSA⁻ cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA⁻ cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.     

Cyclic di-GMP signalling controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining

Cyclic di-GMP (c-di-GMP), a novel secondary signalling molecule present in most bacteria, controls transition between motility and sessility. In Salmonella enterica serovar Typhimurium ( S. typhimurium ) high c-di-GMP concentrations favour the expression of a biofilm state through expression of the master regulator CsgD. In this work, we investigate the effect of c-di-GMP signalling on virulence phenotypes of S. typhimurium. After saturation of the cell with c-di-GMP by overexpression of a di-guanylate cyclase, we studied invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Elevated c-di-GMP had a profound effect on invasion into and IL-8 production by the gastrointestinal epithelial cell line HT-29. Invasion was mainly inhibited through CsgD and the extracellular matrix component cellulose, while inhibition of the pro-inflammatory response occurred through CsgD, which inhibited the secretion of monomeric flagellin. Our results suggest that transition between biofilm formation and virulence in S. typhimurium at the epithelial cell lining is mediated by c-di-GMP signalling through CsgD and cellulose expression.     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

Small regulatory RNAs control the multi-cellular adhesive lifestyle of Escherichia coli

Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single‐cell motile lifestyle and a multi‐cellular, sessile and adhesive state that enables biofilm formation on surfaces. For this, the bacterium needs to reprogramme its gene expression, and in many E. coli and Salmonella strains the lifestyle shift relies on control cascades that inhibit flagellar expression and activate the synthesis of curli, extracellular adhesive fibres important for co‐aggregation of cells and adhesion to biotic and abiotic surfaces. By combining bioinformatics, genetic and biochemical analysis we identified three small RNAs that act by an antisense mechanism to downregulate translation of CsgD, the master regulator of curli synthesis. Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

PslG,a self-produced glycosyl hydrolase,triggers biofilm disassembly by disrupting exopolysaccharide matrix

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.     

Matrix Production and Sporulation in Bacillus subtilis Biofilms Localize to Propagating Wave Fronts

Bacterial biofilms are surface-attached microbial communities encased in self-produced extracellular polymeric substances. Here we demonstrate that during the development of Bacillus subtilis biofilms, matrix production is localized to an annular front propagating at the periphery and sporulation to a second front at a fixed distance at the interior. We show that within these fronts, cells switch off matrix production and transition to sporulation after a set time delay of ～100 min. Correlation analyses of fluctuations in fluorescence reporter activity reveal that the fronts emerge from a pair of gene-expression waves of matrix production and sporulation. The localized expression waves travel across cells that are immobilized in the biofilm matrix in contrast to active cell migration or horizontal colony spreading. Our results suggest that front propagation arises via a local developmental program occurring at the level of individual bacterial cells, likely driven by nutrient depletion and metabolic by-product accumulation. A single-length scale and timescale couples the spatiotemporal propagation of both fronts throughout development. As a result, gene expression patterns within the advancing fronts collapse to self-similar expression profiles. Our findings highlight the key role of the localized cellular developmental program associated with the propagating front in describing biofilm growth.