Quorum sensing in Pseudomonas aeruginosa biofilms

In nature, the bulk of bacterial biomass is believed to exist as an adherent community of cells called a biofilm. Pseudomonas aeruginosa has become a model organism for studying this mode of growth. Over the past decade, significant strides have been made towards understanding biofilm development in P. aeruginosa and we now have a clearer picture of the mechanisms involved. Available evidence suggests that construction of these sessile communities proceeds by many different pathways, rather than a specific programme of biofilm development. A cell-to-cell communication mechanism known as quorum sensing (QS) has been found to play a role in P. aeruginosa biofilm formation. Because both QS and biofilms are impacted by the surrounding environment, understanding the full involvement of cell-to-cell signalling in establishing these complex communities represents a challenge. Nevertheless, under set conditions, several links between QS and biofilm formation have been recognized, which is the focus of this review. A role for antibiotics as alternative QS signalling molecules influencing biofilm development is also discussed.     

Biofilm development on Caenorhabditis elegans by Yersinia is facilitated by quorum sensing-dependent repression of type III secretion

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.     

The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.     

Flagellar motility is critical for Listeria monocytogenes biofilm formation

The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.     

Three-Dimensional Numerical Simulations of Biofilm Dynamics with Quorum Sensing in a Flow Cell

We develop a multiphasic hydrodynamic theory for biofilms taking into account interactions among various bacterial phenotypes, extracellular polymeric substance (EPS), quorum sensing (QS) molecules, solvent, and antibiotics. In the model, bacteria are classified into down-regulated QS, up-regulated QS, and non-QS cells based on their QS ability. The model is first benchmarked against an experiment yielding an excellent fit to experimental measurements on the concentration of QS molecules and the cell density during biofilm development. It is then applied to study development of heterogeneous structures in biofilms due to interactions of QS regulation, hydrodynamics, and antimicrobial treatment. Our 3D numerical simulations have confirmed that (i). QS is beneficial for biofilm development in a long run by building a robust EPS population to protect the biofilm; (ii). biofilms located upstream can induce QS downstream when the colonies are close enough spatially; (iii). QS induction may not be fully operational and can even be compromised in strong laminar flows; (v). the hydrodynamic stress alters the biofilm morphology. Through further numerical investigations, our model suggests that (i). QS-regulated EPS production contributes to the structural formation of heterogeneous biofilms; (ii) QS down-regulated cells tend to grow at the surface of the biofilm while QS up-regulated ones tend to grow in the bulk; (iii) when nutrient supply is sufficient, QS induction might be more effective upstream than downstream; (iv) QS may be of little benefit in a short timescale in term of fighting against invading strain/species; (v) the material properties of biomass (bacteria and EPS) have strong impact on the dilution of QS molecules under strong shear flow. In addition, with this modeling framework, hydrodynamic details and rheological quantities associated with biofilm formation under QS regulation can be resolved.     

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.     

The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal

BFP, a plasmid-encoded type IV bundle-forming pilus produced by enteropathogenic Escherichia coli (EPEC), has recently been shown to be associated with the aggregation of bacteria and dispersal of bacteria from bacterial microcolonies. In standard 3 h HEp-2 cell assays, EPEC adhere in localized microcolonies; after 6 h, bacterial microcolonies are no longer present, indicating that bacterial aggregation and dispersal occurs in vitro during EPEC adhesion to cultured epithelial cells. To examine the role of BFP in EPEC aggregation and dispersal, we examined HEp-2 cell adhesion of strain E2348/69 and defined E2348/69 mutants by immunofluorescence and immunoelectron microscopy. BFP was expressed initially as approximately 40 nm diameter pilus bundles that promoted bacteria-bacteria interaction and microcolony formation. BFP subsequently underwent a striking alteration in structural organization with the formation of much longer and thicker ( approximately 100 nm diameter) pilus bundles, which frequently aggregated laterally to form even thicker bundles often arranged in a loose three-dimensional network; EPEC dispersal from bacterial microcolonies was associated with this transformation of BFP from thin to thick bundles. Bacterial dispersal and transformation of BFP from thin to thick bundles did not occur with a bfpF mutant of strain E2348/69. It is concluded that BFP promotes both the formation and the dispersal of EPEC microcolonies, that the dispersal phase requires BfpF and that dispersal is associated with dramatic alterations in the structure of BFP bundles.     

Quorum sensing in streptococcal biofilm formation

Bacteria in their natural ecosystems preferentially grow as polysaccharide-encased biofilms attached to surfaces. Although quorum-sensing (QS) systems directing the 'biofilm phenotype' have been extensively described in Gram-negative bacteria, there is little understanding of the importance of these systems in Gram-positive biofilm formation. Streptococci are a diverse group of Gram-positive bacteria that colonize epithelial, mucosal and tooth surfaces of humans. In several streptococci, competence-stimulating peptide (CSP)-mediated QS has been connected with competence development for genetic transformation. Recent work, especially with bacteria that inhabit the biofilm of dental plaque, has linked CSP stimuli to other cell-density adaptations, such as biofilm formation.     

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.     

Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.     

Function of quorum sensing and cell signaling in the formation of aerobic granular sludge

Aerobic granular sludge (AGS) has recently attracted attention because of its excellent settling ability and treatment efficiency compared with traditional activated sludge. This review provides recent advances on the formation process of AGS and mainly analyzes the function of quorum sensing (QS) and cell signaling during AGS formation. QS and cell signaling play important roles in the formation of AGS. QS can accelerate the synthesis of extracellular polymeric substance (EPS) and increase microbial adhesion to the surface of AGS. Cell signaling can also promote the secretion of EPS and influence biofilm formation. Cyclic diguanylate (c-di-GMP), as a second messenger, acts an important role in granulation. C-di-GMP causes bacteria to adhere to each other and form a biofilm. Adding Ca²⁺ benefits bacterial growth and promotes c-di-GMP secretion. Adding Mn²⁺ reduces c-di-GMP content and triggers AGS disintegration. Finally, the review discusses the possible trends of AGS: QS and cell signaling can lay a theoretical foundation for the formation mechanism of AGS and would be of practical significance for its application in the future.     

Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation

Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.     

Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Protozoan grazing is a major mortality factor faced by bacteria in the environment. Vibrio cholerae, the causative agent of the disease cholera, is a natural inhabitant of aquatic ecosystems, and its survival depends on its ability to respond to stresses, such as predation by heterotrophic protists. Previous results show that grazing pressure induces biofilm formation and enhances a smooth to rugose morphotypic shift, due to increased expression of Vibrio polysaccharide (VPS). In addition to negatively controlling vps genes, the global quorum sensing (QS) regulator, HapR, plays a role in grazing resistance as the ΔhapR strain is efficiently consumed while the wild type (WT) is not. Here, the relative and combined contributions of VPS and QS to grazing resistance were investigated by exposing VPS and HapR mutants and double mutants in VPS and HapR encoding genes at different phases of biofilm development to amoeboid and flagellate grazers. Data show that the WT biofilms were grazing resistant, the VPS mutants were less resistant than the WT strain, but more resistant than the QS mutant strain, and that QS contributes to grazing resistance mainly in mature biofilms. In addition, grazing effects on biofilms of mixed WT and QS mutant strains were investigated. The competitive fitness of each strain in mixed biofilms was determined by CFU and microscopy. Data show that protozoa selectively grazed the QS mutant in mixed biofilms, resulting in changes in the composition of the mixed community. A small proportion of QS mutant cells which comprised 4% of the mixed biofilm biovolume were embedded in grazing resistant WT microcolonies and shielded from predation, indicating the existence of associational protection in mixed biofilms.