Bap, a Staphylococcus aureus surface protein involved in biofilm formation

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.     

Identification and characterization of an Acinetobacter baumannii biofilm-associated protein

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm.     

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+- independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.     

Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.     

Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that Bap_MS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bap_MS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.     

Staphylococcus and biofilms

The genetic and molecular basis of biofilm formation in staphylococci is multifaceted. The ability to form a biofilm affords at least two properties: the adherence of cells to a surface and accumulation to form multilayered cell clusters. A trademark is the production of the slime substance PIA, a polysaccharide composed of beta-1,6-linked N-acetylglucosamines with partly deacetylated residues, in which the cells are embedded and protected against the host's immune defence and antibiotic treatment. Mutations in the corresponding biosynthesis genes (ica operon) lead to a pleiotropic phenotype; the cells are biofilm and haemagglutination negative, less virulent and less adhesive on hydrophilic surfaces. ica expression is modulated by various environmental conditions, appears to be controlled by SigB and can be turned on and off by insertion sequence (IS) elements. A number of biofilm-negative mutants have been isolated in which polysaccharide intercellular adhesin (PIA) production appears to be unaffected. Two of the characterized mutants are affected in the major autolysin (atlE) and in D-alanine esterification of teichoic acids (dltA). Proteins have been identified that are also involved in biofilm formation, such as the accumulation-associated protein (AAP), the clumping factor A (ClfA), the staphylococcal surface protein (SSP1) and the biofilm-associated protein (Bap). Concepts for the prevention of obstinate polymer-associated infections include the search for new anti-infectives active in biofilms and new biocompatible materials that complicate biofilm formation and the development of vaccines.     

Aloe-emodin inhibits Staphylococcus aureus biofilms and extracellular protein production at the initial adhesion stage of biofilm development

Staphylococcus aureus (S. aureus) biofilms are clinically serious and play a critical role in the persistence of chronic infections due to their ability to resist antibiotics. The inhibition of biofilm formation is viewed as a new strategy for the prevention of S. aureus infections. Here, we demonstrated that minimum inhibitory concentrations (MICs) of aloe-emodin exhibited no bactericidal activity against S. aureus but affected S. aureus biofilm development in a dose-dependent manner. Further studies indicated that aloe-emodin specifically inhibits the initial adhesion and proliferation stages of S. aureus biofilm development. Scanning electron microscopy (SEM) indicated that the S. aureus ATCC29213 biofilm extracellular matrix is mainly composed of protein. Laser scanning confocal microscope assays revealed that aloe-emodin treatment primarily inhibited extracellular protein production. Moreover, the Congo red assay showed that aloe-emodin also reduced the accumulation of polysaccharide intercellular adhesin (PIA) on the cell surface. These findings will provide new insights into the mode of action of aloe-emodin in the treatment of infections by S. aureus biofilms.     

Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility

Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl(2). The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 μM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant (fimA pilO) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I (fimA) or type IV (pilB) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures.     

Biofilm Formation and the Presence of the Intercellular Adhesion Locus ica among Staphylococci from Food and Food Processing Environments

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.     

The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched beta-1,6-N-acetyl-D-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-beta-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria.     

In silico analysis of antibody triggering biofilm associated protein in Acinetobacter baumannii

Acinetobacter baumannii surface protein, commonly known as biofilm associated protein (Bap), is involved in biofilm formation. A high propensity among the clinical isolates to form biofilm and a significant association of biofilms with multiple drug resistance has been demonstrated. Production of antibodies can be used for inhibition of biofilm and control of the diseases caused by A. baumannii. Large molecular mass of Bap justifies an approach to identifying A. baumannii effective antigens. It has a core domain of seven repeat modules A-G. With the large number of available biofilm gene sequences, bioinformatic tools are needed to identify the genes encoding the antigens. Proteins containing these tandem repeats of Bap domains have high propensities to attach to each other to form biofilm. We hypothesized that conserved and functional domains of tandem repeat could be identified with a search and alignment of the repeats for evaluation of antigenic determinants. Here we demonstrate the results of bioinformatics screening and gene scan of the gene sequence database of homolog sequences to identify conserved domains. Higher scoring hits were found in repeat modules mostly D, B, C and A, respectively. Upon the analysis four regions of highly structural and functional conserved regions from Bap sequence of A. baumannii were selected. 3D structure, antigenicity and solubility predictions revealed that these regions were appropriate candidates for antibody production.     

A role for amyloid in cell aggregation and biofilm formation

Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5p(V326N) cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5p(WT) cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.     

Genes involved in formation of structured multicellular communities by Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.     

Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.     

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.     

The influence of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis>-derived surfactants on staphylococcal adhesion and biofilm formation

The ability of surfactants obtained from three Lactobacillus acidophilus strains to inhibit Staphylococcus aureus and S. epidermidis biofilms was evaluated. Their influence was determined on bacterial initial adhesion, biofilm formation and dispersal using MTT-reduction assay, confocal laser scanning microscopy and image PHLIP analysis. The number of adhering S. aureus and S. epidermidis cells after a 3-h co-incubation with biosurfactants was reduced by 5-56 % in a strain-and dose-dependent manner. S. epidermidis-and, to a lower extent, in S. aureus-biofilm formation was also inhibited in the presence of the tested surfactants. The addition of surfactants to preformed mature biofilms accelerated their dispersal, and changed the parameters of biofilm morphology. The L. acidophilus-derived surfactants inhibit bacterial deposition rate and biofilm development (and also its maturation) without affecting cell growth probably due to the influence on the cell-surface hydrophobicity of staphylococci.     

A communal bacterial adhesin anchors biofilm and bystander cells to surfaces

While the exopolysaccharide component of the biofilm matrix has been intensively studied, much less is known about matrix-associated proteins. To better understand the role of these proteins, we undertook a proteomic analysis of the V. cholerae biofilm matrix. Here we show that the two matrix-associated proteins, Bap1 and RbmA, perform distinct roles in the biofilm matrix. RbmA strengthens intercellular attachments. In contrast, Bap1 is concentrated on surfaces where it serves to anchor the biofilm and recruit cells not yet committed to the sessile lifestyle. This is the first example of a biofilm-derived, communally synthesized conditioning film that stabilizes the association of multilayer biofilms with a surface and facilitates recruitment of planktonic bystanders to the substratum. These studies define a novel paradigm for spatial and functional differentiation of proteins in the biofilm matrix and provide evidence for bacterial cooperation in maintenance and expansion of the multilayer biofilm.     

Pattern formation in Pseudomonas aeruginosa biofilms

Bacteria are capable of forming elaborate multicellular communities called biofilms. Pattern formation in biofilms depends on cell proliferation and cellular migration in response to the available nutrients and other external cues, as well as on self-generated intercellular signal molecules and the production of an extracellular matrix that serves as a structural 'scaffolding' for the biofilm cells. Pattern formation in biofilms allows cells to position themselves favorably within nutrient gradients and enables buildup and maintenance of physiologically distinct subpopulations, which facilitates survival of one or more subpopulations upon environmental insult, and therefore plays an important role in the innate tolerance displayed by biofilms toward adverse conditions.     

Structural Basis for Biofilm Formation via the Vibrio cholerae Matrix Protein RbmA

During the transition from a free-swimming, single-cell lifestyle to a sessile, multicellular state called a biofilm, bacteria produce and secrete an extracellular matrix comprised of nucleic acids, exopolysaccharides, and adhesion proteins. The Vibrio cholerae biofilm matrix contains three major protein components, RbmA, Bap1, and RbmC, which are unique to Vibrio cholerae and appear to support biofilm formation at particular steps in the process. Here, we focus on RbmA, a structural protein with an unknown fold. RbmA participates in the early cell-cell adhesion events and is found throughout the biofilm where it localizes to cell-cell contact sites. We determined crystal structures of RbmA and revealed that the protein folds into tandem fibronectin type III (FnIII) folds. The protein is dimeric in solution and in crystals, with the dimer interface displaying a surface groove that is lined with several positively charged residues. Structure-guided mutagenesis studies establish a crucial role for this surface patch for RbmA function. On the basis of the structure, we hypothesize that RbmA serves as a tether by maintaining flexible linkages between cells and the extracellular matrix.