Narayanan Ramasubbu

Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.

Enzymatic Detachment of Staphylococcus epidermidis Biofilms

ABSTRACT The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices. S. epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S. epidermidis biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from S. epidermidis cells. Precoating surfaces with the enzyme prevented S. epidermidis biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.

Detachment and Killing of Aggregatibacter actinomycetemcomitans Biofilms by Dispersin B and SDS

The periodontopathogen Aggregatibacter actinomycetemcomitans forms tenacious biofilms on abiotic surfaces in vitro. The objective of the present study was to measure the susceptibility of A. actinomycetemcomitans biofilms to detachment and killing by the anionic surfactant sodium dodecyl sulfate (SDS). We found that biofilms formed by a wild-type strain were resistant to detachment by SDS. In contrast, biofilms formed by an isogenic mutant strain that was deficient in the production of PGA (poly-N-acetyl-glucosamine), a biofilm matrix polysaccharide, were sensitive to detachment by SDS. Pre-treatment of wild-type biofilms with dispersin B, a PGA-degrading enzyme, rendered them sensitive to detachment by SDS and resulted in a > 99% increase in SDS-mediated cell killing. We concluded that PGA protects A. actinomycetemcomitans cells from detachment and killing by SDS. Dispersin B and SDS may be useful agents for treating chronic infections caused by A. actinomycetemcomitans and other PGA-producing bacteria.

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.

Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans.

Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.

Pub: Probing the Interaction of Human Salivary Alpha-Amylase and Amylase Binding Protein A(AbpA) of Streptococcus gordonii

Amylase binding protein A (AbpA) of Streptococcus gordonii serves as a major receptor for human salivary α-amylase (HSAmy), the predominant enzyme in human salivary secretions, to bind to the bacterial cell surface. On enamel surfaces, the binding of AbpA to HSAmy renders S. gordonii act as a scaffold for other oral bacteria to attach to the acquired enamel pellicle leading to complex bacterial communities and invasion of host tissues. While the role of AbpA in adhesion, starch metabolism and biofilm formation in influencing the ecology of the oral biofilms has been established, the structure function relationships of AbpA are yet to be defined. Since distally located aromatic residues of HSAmy are involved in the interaction with AbpA, we hypothesized that AbpA might use separate structural regions to bind to HSAmy. To test this, several deletion mutants of AbpA were constructed and studied to correlate the effect of deletions in binding to HSAmy in the following: 1) their secondary structural features through circular dichroism studies; 2) their ability to alter the capacity of HSAmy to hydrolyze starch and several oligosaccharides after complex formation and 3) their binding to HSAmy using surface plasmon resonance spectroscopy. Our results showed that a) AbpA does not bind at the active site of salivary α-amylase; b) both the N-terminal region (24-56 residues) and a central region (residues 124-165) are required for binding to HSAmy; and c) the C-terminal end residues (166- 195) are not necessary for binding. These results clearly show that AbpA binding to HSAmy encompasses distinct and distal regions of the structure.

Structure of amylase-binding protein A of Streptococcus gordonii: a potential receptor for human salivary α-amylase enzyme.

Amylase‐binding protein A (AbpA) of a number of oral streptococci is essential for the colonization of the dental pellicle. We have determined the solution structure of residues 24–195 of AbpA of Streptococcus gordonii and show a well‐defined core of five helices in the region of 45–115 and 135–145. 13Cα/β chemical shift and heteronuclear 15N‐{1H} NOE data are consistent with this fold and that the remainder of the protein is unstructured. The structure will inform future molecular experiments in defining the mechanism of human salivary α‐amylase binding and biofilm formation by streptococci.

Dispersion Risk Associated With Surgical Debridement Devices

INTRODUCTION Surgical instrumentation is now available to facilitate wound debridement. The 2 primary options involve different energy applications, but both have the potential to spray. The Versajet II (Smith & Nephew, London, UK) utilizes a high-powered water jet to disrupt tissue and remove debris by means of the Venturi effect. The SonicVac (Misonix, Farmingdale, NY) is a direct-contact, low-frequency ultrasound debriding device. It delivers a high-energy ultrasound to a wound surface via a fluid medium, causing bubble cavitation, a physical effect of rapid pressure waves causing bubbles to form and implode that releases mechanical energy. OBJECTIVE This study is designed to assess spray dispersion under ideal and challenging conditions. MATERIALS AND METHODS The 2 aforementioned instruments were tested in a laboratory situation. Bacteria (Escherichia coli [ATCC#54288] or Staphylococcus epidermidis [RP62A]) were seeded onto separate pieces of beef steak. Culture plates were set up in a predesignated position around the specimen; the specimen was then treated for 60 seconds at a power setting of 7 and 70% irrigation (ultrasound device) or 10 (waterjet device). After 60 seconds of debridement, about 4 mm to 5 mm of muscle tissue had been removed by the ultrasound device and 2 mm to 3 mm by the waterjet. In the bony specimen, the bone was more exposed after the treatment. The ultrasound device polished but did not remove the bone. RESULTS Both instruments performed well with minimal dispersion in the ideal setting. In beef steak with bone and grizzle, the waterjet created a lawn of bacterial spray in the plate in front of the surgeon. The ultrasound had a small number of contaminants in the same conditions. CONCLUSIONS Both instruments can be used safely in the proper conditions, but the surgeon needs to be aware of the limitations and risks of spray dispersion.

Abstract 26. Biofilm Management: Attack the Matrix with Ultrasound

CONCLUSION: Despite their similarities in clinical and radiographic presentation, pediatric JE phalangeal fractures are a distinct entity from SH2 fractures. Presenting with significantly more radiographic angulation and clinical instability, JE fractures more frequently required operative fixation compared to SH2 fractures. This distinction is important when determining the treatment strategy employed (operative fixation versus nonoperative management) as well as potential length and degree of immobilization/stabilization for nonoperative management to increase the success of treatment.