Aggregatibacter actinomycetemcomitans Dispersin B: The Quintessential Antibiofilm Enzyme.

The extracellular matrix of most bacterial biofilms contains polysaccharides, proteins, and nucleic acids. These biopolymers have been shown to mediate fundamental biofilm-related phenotypes including surface attachment, intercellular adhesion, and biocide resistance. Enzymes that degrade polymeric biofilm matrix components, including glycoside hydrolases, proteases, and nucleases, are useful tools for studying the structure and function of biofilm matrix components and are also being investigated as potential antibiofilm agents for clinical use. Dispersin B is a well-studied, broad-spectrum antibiofilm glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans. Dispersin B degrades poly-N-acetylglucosamine, a biofilm matrix polysaccharide that mediates biofilm formation, stress tolerance, and biocide resistance in numerous Gram-negative and Gram-positive pathogens. Dispersin B has been shown to inhibit biofilm and pellicle formation; detach preformed biofilms; disaggregate bacterial flocs; sensitize preformed biofilms to detachment by enzymes, detergents, and metal chelators; and sensitize preformed biofilms to killing by antiseptics, antibiotics, bacteriophages, macrophages, and predatory bacteria. This review summarizes the results of nearly 100 in vitro and in vivo studies that have been carried out on dispersin B since its discovery 20 years ago. These include investigations into the biological function of the enzyme, its structure and mechanism of action, and its in vitro and in vivo antibiofilm activities against numerous bacterial species. Also discussed are potential clinical applications of dispersin B.

A high-throughput sequencing approach identifies immunotherapeutic targets for bacterial meningitis in neonates.

BACKGROUND: Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. METHODS: Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization. RESULTS: We selected for further study the conserved surface polysaccharide Poly-ß-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen. INTERPRETATION: Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. FUNDINGS: ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.

The endoglycosidase activity of Dispersin B is mediated through electrostatic interactions with cationic poly-ß-(1→6)-N-acetylglucosamine.

Bacterial biofilms consist of bacterial cells embedded within a self-produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 ß-hexosaminidase enzyme that catalyses the hydrolysis of poly-N-acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm-forming bacteria. Native PNAG is partially de-N-acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo- and exo-glycosidic bond cleavage of PNAG depending on the de-N-acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site-directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo-glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the -2 binding site. A DspBD242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspBD242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild-type DspB. These results suggest that the de-N-acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

Applications of an inactive Dispersin B probe to monitor biofilm polysaccharide production.

Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.

Experimental Polymorphism Survey in Intergenic Regions of the icaADBCR Locus in Staphylococcus aureus Isolates from Periprosthetic Joint Infections.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI.

Non-deacetylated poly-N-acetylglucosamine-hyperproducing Staphylococcus aureus undergoes immediate autoaggregation upon vortexing.

Biofilms are microbial communities of cells embedded in a matrix of extracellular polymeric substances generated and adhering to each other or to a surface. Cell aggregates formed in the absence of a surface and floating pellicles that form biofilms at the air-liquid interface are also considered to be a type of biofilm. Staphylococcus aureus is a well-known cause of biofilm infections and high-molecular-weight polysaccharides, poly-N-acetylglucosamine (PNAG) is a main constituent of the biofilm. An icaADBC operon comprises major machinery to synthesize and extracellularly secrete PNAG. Extracellular PNAG is partially deacetylated by IcaB deacetylase, and the positively charged PNAG hence interacts with negatively charged cell surface to form the major component of biofilm. We previously reported a new regulator of biofilm (Rob) and demonstrated that Rob binds to a unique 5-bp motif, TATTT, present in intergenic region between icaADBC operon and its repressor gene icaR in Yu et al. The deletion of the 5-bp motif induces excessive adherent biofilm formation. The real function of the 5-bp motif is still unknown. In an attempt to isolate the 5-bp motif deletion mutant, we isolated several non-adherent mutants. They grew normally in turbid broth shaking culture but immediately auto-aggregated upon weak vortexing and sedimented as a lump resulting in a clear supernatant. Whole genome sequencing of the mutants identified they all carried mutations in icaB in addition to deletion of the 5-bp motif. Purification and molecular characterization of auto-aggregating factor in the culture supernatant of the mutant identified that the factor was a massively produced non-deacetylated PNAG. Therefore, we created a double deficient strain of biofilm inhibitory factors (5-bp motif, icaR, rob) and icaB to confirm the aggregation phenomenon. This peculiar phenomenon was only observed in Δ5bpΔicaB double mutant but not in ΔicaR ΔicaB or ΔrobΔicaB mutant. This study explains large amount of extracellularly produced non-deacetylated PNAG by Δ5bpΔicaB double mutation induced rapid auto-aggregation of S. aureus cells by vortexing. This phenomenon indicated that Staphylococcus aureus may form biofilms that do not adhere to solid surfaces and we propose this as a new mechanism of non-adherent biofilm formation of S. aureus.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge.

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate.

Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly-N-acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c-di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.

Plasmid-Encoded H-NS Controls Extracellular Matrix Composition in a Modern Acinetobacter baumannii Urinary Isolate.

Acinetobacter baumannii is emerging as a multidrug-resistant (MDR) nosocomial pathogen of increasing threat to human health worldwide. The recent MDR urinary isolate UPAB1 carries the plasmid pAB5, a member of a family of large conjugative plasmids (LCPs). LCPs encode several antibiotic resistance genes and repress the type VI secretion system (T6SS) to enable their dissemination, employing two TetR transcriptional regulators. Furthermore, pAB5 controls the expression of additional chromosomally encoded genes, impacting UPAB1 virulence. Here, we show that a pAB5-encoded H-NS transcriptional regulator represses the synthesis of the exopolysaccharide PNAG and the expression of a previously uncharacterized three-gene cluster that encodes a protein belonging to the CsgG/HfaB family. Members of this protein family are involved in amyloid or polysaccharide formation in other species. Deletion of the CsgG homolog abrogated PNAG production and chaperone-usher pathway (CUP) pilus formation, resulting in a subsequent reduction in biofilm formation. Although this gene cluster is widely distributed in Gram-negative bacteria, it remains largely uninvestigated. Our results illustrate the complex cross-talks that take place between plasmids and the chromosomes of their bacterial host, which in this case can contribute to the pathogenesis of Acinetobacter. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii displays the highest reported rates of multidrug resistance among Gram-negative pathogens. Many A. baumannii strains carry large conjugative plasmids like pAB5. In recent years, we have witnessed an increase in knowledge about the regulatory cross-talks between plasmids and bacterial chromosomes. Here, we show that pAB5 controls the composition of the bacterial extracellular matrix, resulting in a drastic reduction in biofilm formation. The association between biofilm formation, virulence, and antibiotic resistance is well documented. Therefore, understanding the factors involved in the regulation of biofilm formation in Acinetobacter has remarkable therapeutic potential.

Anionic amino acids support hydrolysis of poly-ß-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B.

The exopolysaccharide poly-ß-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm-dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site-directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the -1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

The staphylococcal exopolysaccharide PIA - Biosynthesis and role in biofilm formation, colonization, and infection.

Exopolysaccharide is a key part of the extracellular matrix that contributes to important mechanisms of bacterial pathogenicity, most notably biofilm formation and immune evasion. In the human pathogens Staphylococcus aureus and S. epidermidis, as well as in many other staphylococcal species, the only exopolysaccharide is polysaccharide intercellular adhesin (PIA), a cationic, partially deacetylated homopolymer of N-acetylglucosamine, whose biosynthetic machinery is encoded in the ica locus. PIA production is strongly dependent on environmental conditions and controlled by many regulatory systems. PIA contributes significantly to staphylococcal biofilm formation and immune evasion mechanisms, such as resistance to antimicrobial peptides and ingestion and killing by phagocytes, and presence of the ica genes is associated with infectivity. Due to its role in pathogenesis, PIA has raised considerable interest as a potential vaccine component or target.

Glycomics Microarrays Reveal Differential In Situ Presentation of the Biofilm Polysaccharide Poly-N-acetylglucosamine on Acinetobacter baumannii and Staphylococcus aureus Cell Surfaces.

The biofilm component poly-N-acetylglucosamine (PNAG) is an important virulence determinant in medical-device-related infections caused by ESKAPE group pathogens including Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii. PNAG presentation on bacterial cell surfaces and its accessibility for host interactions are not fully understood. We employed a lectin microarray to examine PNAG surface presentation and interactions on methicillin-sensitive (MSSA) and methicillin-resistant S. aureus (MRSA) and a clinical A. baumannii isolate. Purified PNAG bound to wheatgerm agglutinin (WGA) and succinylated WGA (sWGA) lectins only. PNAG was the main accessible surface component on MSSA but was relatively inaccessible on the A. baumannii surface, where it modulated the presentation of other surface molecules. Carbohydrate microarrays demonstrated similar specificities of S. aureus and A. baumannii for their most intensely binding carbohydrates, including 3' and 6'sialyllactose, but differences in moderately binding ligands, including blood groups A and B. An N-acetylglucosamine-binding lectin function which binds to PNAG identified on the A. baumannii cell surface may contribute to biofilm structure and PNAG surface presentation on A. baumannii. Overall, these data indicated differences in PNAG presentation and accessibility for interactions on Gram-positive and Gram-negative cell surfaces which may play an important role in biofilm-mediated pathogenesis.

Experimental study for evaluation of the efficacy of a biofilm-embedded bacteria-based vaccine against Staphylococcus chromogenes-associated mastitis in sheep.

Although coagulase-negative staphylococci are the primary aetiological agents of subclinical mastitis in ewes, there is little information regarding vaccination against that infection. The objective of this study was to evaluate the efficacy of a vaccine against staphylococcal mastitis in ewes under experimental conditions. The antigen in the vaccine is based on a bacterin of Staphylococcus aureus strain, expressing the exopolysaccharide poly-N-acetylglucosamine (PNAG), which is involved in biofilm formation by these bacteria. Ewes in groups A (n = 17) or B (n = 6) were given an initial vaccination 5 weeks before expected lambing, followed by a repeat administration 21 days later. Ewes in groups C (n = 8) or D (n = 6) were unvaccinated controls. Ewes in group A (n = 17) or C (n = 8) were challenged with a biofilm-forming S. chromogenes; animals in subgroups A1 or C1 were challenged on the 10th and those in A2 or C2 on the 50th day after lambing. Ewes in groups B or D were uninoculated controls. Clinical examinations of ewes, ultrasonographic examinations of udder, milk yield measurements, blood sampling for detection of anti-PNAG specific antibodies and milk sample collection for bacteriological and cytological examinations were performed up to 52nd day post-challenge. Finally, biopsies were performed for mammary tissue collection for histopathological examination. Among group A ewes, 29% developed systemic signs and 59% signs in the inoculated gland; the respective figures for group C were 50% and 100% (P =  0.040 for mammary signs). The median total clinical score was 2.0 for A and 5.5 for C ewes (P =  0.025). For A, but not for C, clinical scores decreased progressively during the study (P =  0.018 and P =  0.47, respectively). The duration of mastitis was shorter in A (4 days) than in C (17.5 days) ewes (P =  0.022). Bacterial counts were lower in milk samples from A than from C ewes, for samples collected from the inoculated and the uninoculated (P <  0.01) mammary glands of these ewes. Somatic cell counts in samples from inoculated and uninoculated mammary glands of A ewes were higher than in samples of C ewes (P <  0.02). There were differences for gray-scale evaluations during ultrasonographic examination and for milk yield measurements between groups (P <  0.01). Median bacterial counts in tissue samples from A ewes (0 cfu g-1) were lower than in ones from C (6.5 cfu g-1) ewes (P =  0.041). The median score for histopathological findings in tissue samples from inoculated glands of A was lower than that for C ewes: 1 versus 2 (P =  0.014). It is concluded that mastitis was less severe in vaccinated animals, as indicated by a wide array of measures.

σB Inhibits Poly-N-Acetylglucosamine Exopolysaccharide Synthesis and Biofilm Formation in Staphylococcus aureus.

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.IMPORTANCE Due to its multifaceted lifestyle, Staphylococcus aureus needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named σB (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of σB in the regulation of the biofilm lifestyle in S. aureus Our study demonstrated that σB is not necessary for exopolysaccharide-dependent biofilms and, even more, that S. aureus produces stronger biofilms in the absence of σB The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.

Production of poly-ß-1,6-N-acetylglucosamine by MatAB is required for hyphal aggregation and hydrophilic surface adhesion by Streptomyces.

Streptomycetes are multicellular filamentous microorganisms, and major producers of industrial enzymes and bioactive compounds such as antibiotics and anticancer drugs. The mycelial lifestyle plays an important role in the productivity during industrial fermentations. The hyphae of liquid-grown streptomycetes can self-aggregate into pellets, which hampers their industrial exploitation. Here we show that the Mat complex, which is required for pellet formation, catalyzes the synthesis of extracellular poly-ß-1,6-N-acetylglucosamine (PNAG) in the model organisms Streptomyces coelicolor and Streptomyces lividans. Extracellular accumulation of PNAG allows Streptomyces to attach to hydrophilic surfaces, while attachment to hydrophobic surfaces requires a cellulase-degradable extracellular polymer (EPS) produced by CslA. Over-expression of matAB was sufficient to restore pellet formation to cslA null mutants of S. lividans. The two EPS systems together increase the robustness of mycelial pellets. These new insights allow better control of liquid-culture morphology of streptomycetes, which may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.

A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus.

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization.

Detection of the biofilm component polysaccharide intercellular adhesin in Staphylococcus aureus infected cow udders.

Biofilms are communities of microorganisms embedded in a self-produced extracellular matrix made up of polymeric substances. They reduce the effects of antibiotics and allow the microorganisms to evade the innate immune system. This can lead to persistent or recurrent infections. In dairy cow herds, mastitis is a serious problem. The present study aimed to investigate the occurrence of biofilms in the udders of dairy cows infected with Staphylococcus (S.) aureus, because biofilms may affect the response to treatment of bovine mastitis. Immunofluorescence staining of polysaccharide intercellular adhesin (PIA), a component of S. aureus biofilms, was carried out based on swabs taken from different areas of S. aureus infected udders. We were able to demonstrate the presence of PIA in S. aureus infected bovine udders. However, the applied method is invasive and therefore only really suitable for scientific research and not for clinical diagnosis.

The protein BpsB is a poly-ß-1,6-N-acetyl-D-glucosamine deacetylase required for biofilm formation in Bordetella bronchiseptica.

Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni(2+) and Co(2+). The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms.

Enzymatic modifications of exopolysaccharides enhance bacterial persistence.

Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes.

Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.

Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-ß(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection.