Towards the identification of the common features of bacterial biofilm development.

Microorganisms can live and proliferate as individual cells swimming freely in the environment, or they can grow as highly organized, multicellular communities encased in a self-produced polymeric matrix in close association with surfaces and interfaces. This microbial lifestyle is referred to as biofilms. The intense search over the last few years for factors involved in biofilm development has revealed that distantly related bacterial species recurrently make use of the same elements to produce biofilms. These common elements include a group of proteins containing GGDEF/EAL domains, surface proteins homologous to Bap of Staphylococcus aureus, and some types of exopolysaccharides, such as cellulose and the poly-beta-1,6-N-acetylglucosamine. This review summarizes current knowledge about these three common elements and their role in biofilm development.     

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.     

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.     

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus

Recent progress in elucidating the role of the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) in staphylococcal biofilm development has in turn contributed significantly to our understanding of the pathogenesis of device-related infections. Nevertheless, our understanding of how the ica locus and PIA/PNAG biosynthesis are regulated is far from complete and many questions remain. Moreover, beyond ica, evidence is now emerging for the existence of ica-independent biofilm mechanisms in both Staphylococcus aureus and Staphylococcus epidermidis. Teichoic acids, which are a major carbohydrate component of the S. epidermidis biofilm matrix and the major cell wall autolysin, play an important role in the primary attachment phase of biofilm development, whereas the cell surface biofilm-associated protein and accumulation-associated protein are capable of mediating intercellular accumulation. These findings raise the exciting prospect that other surface proteins, which typically function as antigenic determinants or in binding to extracellular matrix proteins, may also act as biofilm adhesins. Given the impressive array of surface proteins expressed by S. aureus and S. epidermidis, future research into their potential role in biofilm development either independent of PIA/PNAG or in cooperation with PIA/PNAG will be of particular interest.     

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.     

Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus

Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.     

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.     

Contribution of the Staphylococcus aureus Atl AM and GL Murein Hydrolase Activities in Cell Division, Autolysis, and Biofilm Formation

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.     

Evaluation of Staphylococcus aureus and Escherichia coli biofilm formation on the surface of polypropylene mesh

A serious complication of hernioplasty with the use of a biomaterial implant is deep surgical site infection (SSI) encompassing the implant. Among the most common etiological factors of deep SSI in patients after hernioplasty are Staphylococcus aureus and Escherichia coli strains, which may create a biofilm on the surface of synthetic implants. The aim of this study was assessment of biofilm formation by S. aureus and E. coli on the surface ofpolypropylene mesh. The study included 108 strains (62 S. aureus and 46 E. coli) from the collection of Department of Microbiology Collegium Medicum im. L. Rydygier in Bydgoszcz, Nicolaus Copernicus University in Torun (CM UMK). Evaluation of biofilm formation was performed using the method of reduction of 2,3,5-triphenyltetrazolium chloride (TTC) and a scanning electron microscope. In the group of S. aureus strains, 88.7% isolates formed biofilm very strongly, 1.6% strongly, and 9.7% poor. Among E. coli strains, 54.3% isolates were characterized by very strong biofilm formation, while 45.7% strong biofilm formation. Strains ofS. aureus strongly than E. coli form a biofilm on the surface of monofilament polypropylene mesh.     

葡萄球菌生物膜形成机制与ica之间的关系

ica位点编码的胞外多糖(PIA/PNAG)对理解葡萄球菌生物膜相关感染病理学方面具有重要的意义.关于ica位点与PIA/PNAG之间如何调节的研究还不全面,另外一种独立于ica的生物膜形成机制存在于表皮葡萄球菌和金黄色葡萄球菌中;细胞表面相关蛋白也能调节生物膜的形成,这些发现为探究它们在生物膜形成机制的潜在作用提供了重要基础.     

The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.     

Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules

The assembly of class I MHC molecules and their export from the endoplasmic reticulum (ER) is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with yellow fluorescent protein-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ER/Golgi intermediate compartment, and second, by retrieving class I molecules that have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, because we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31.     

Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch

The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca²⁺]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation‐prone region of Staphylococcus aureus Bap which adopts a dumbbell‐shaped fold. The middle module (MM) connecting the N‐terminal and C‐terminal lobes consists of a tandem of novel double‐Ca²⁺‐binding motifs involved in cooperative interaction networks, which undergoes Ca²⁺‐dependent order–disorder conformational switches. The N‐terminal lobe is sufficient to mediate amyloid aggregation through liquid–liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca²⁺] but inhibited by ordered MM stabilized by Ca²⁺ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti‐biofilm drug design.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.