Jeffrey B. Kaplan

Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite

Adherence of Actinobacillus actinomycetemcomitans to hard-tissue surfaces was evaluated by comparing a phenotypically stable, well-maintained clinical isolate (strain CU1000) to Streptococcus gordonii G9B, an extensively studied oral-colonizing bacterium. Standard innocula of radiolabelled bacteria were added to saliva-coated hydroxyapatite (SHA) and the ratio of bound to unbound cells counted. Several other clinical isolates as well as laboratory strain Y4 were studied. In other experiments, cell detachment from SHA was compared in static and shaking vessels to calculate controlled desorption of cells over time. A sonic-displacement assay was used to measure avidity of binding to HA and SHA. To better define the attachment properties of CU1000, bacteria were treated with a variety of agents including detergents, salts and enzymes before or after incubation with SHA. Results indicated that CU1000 bound better than G9B (a minimum of 10-fold greater; p < or = 0.05) and did not desorb from SHA, while G9B desorbed to equilibrium in 4 h. Furthermore, Langmuir isotherm calculations indicated that, unlike G9B, CU1000 did not follow second-order adsorption kinetics and thus did not achieve saturation. In addition, of the agents tested only periodate reduced attachment and resulted in detachment of CU1000 from surfaces. These experiments suggest that clinical isolates of A. actinomycetemcomitans possess unique binding properties that promote adsorption to and impede desorption from SHA. The characteristics described for the actinobacillus in this study have been previously underestimated, appear to be mediated by glycoconjugates, and may resemble attachment described for several biofilm-forming, non-oral pathogens.

Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci

Extracellular DNA is an adhesive component of staphylococcal biofilms. The aim of this study was to evaluate the antibiofilm activity of recombinant human DNase I (rhDNase) against Staphylococcus aureus and Staphylococcus epidermidis. Using a 96-well microtiter plate crystal-violet binding assay, we found that biofilm formation by S. aureus was efficiently inhibited by rhDNase at 1–4u2009μgu2009l−1, and preformed S. aureus biofilms were efficiently detached in 2u2009min by rhDNase at 1u2009mgu2009l−1. Pretreatment of S. aureus biofilms for 10u2009min with 10u2009mgu2009l−1 rhDNase increased their sensitivity to biocide killing by 4–5 log units. rhDNase at 10u2009mgu2009l−1 significantly inhibited biofilm formation by S. epidermidis in medium supplemented with sub-MICs of antibiotics. We also found that rhDNase significantly increased the survival of S. aureus-infected Caenorhabditis elegans nematodes treated with tobramycin compared with nematodes treated with tobramycin alone. We concluded that rhDNase exhibits potent antibiofilm and antimicrobial-sensitizing activities against S. aureus and S. epidermidis at clinically achievable concentrations. rhDNase, either alone or in combination with antimicrobial agents, may have applications in treating or preventing staphylococcal biofilm-related infections.

A second Aggregatibacter actinomycetemcomitans autotransporter adhesin exhibits specificity for buccal epithelial cells in humans and Old World primates

ABSTRACT Previous work showed that the Aggregatibacter actinomycetemcomitans adhesin Aae demonstrated species specificity and tissue tropism to buccal epithelial cells (BECs) derived from humans and Old World primates, but a second, lower-affinity adhesin was noted. This study was designed to determine if Omp100 (also known as ApiA), a surface-expressed A. actinomycetemcomitans adhesin, is that second adhesin and if so to investigate its tissue tropism and species specificity. A targeted mutagenesis protocol was used to construct an isogenic apiA mutant and an aae apiA double mutant with wild-type A. actinomycetemcomitans. In addition, Escherichia coli strain DH5α was used to express apiA to further assess binding parameters. Results indicated that the apiA mutant strain showed significantly less binding to BECs than its parent strain (P ≤ 0.05). Further, binding mediated by ApiA was specific to BECs from humans and Old World primates, as seen in both wild-type A. actinomycetemcomitans and E. coli expressing ApiA (P ≤ 0.05). Pretreatment of wild-type A. actinomycetemcomitans cells with anti-ApiA antiserum reduced binding in a dose-dependent manner. The aae apiA double mutant completely abrogated A. actinomycetemcomitans binding to both human and Old World primate BECs. Taken together, these studies indicate that ApiA and Aae, in concert, modulate binding of A. actinomycetemcomitans to human BECs. Since the BEC is a prominent reservoir for A. actinomycetemcomitans, identification of this second adhesin could lead to important therapeutic strategies.

Role of active-site residues of dispersin B, a biofilm-releasing β-hexosaminidase from a periodontal pathogen, in substrate hydrolysis

Dispersinu2003B (DspB), a familyu200320 β‐hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves β(1,6)‐linked N‐acetylglucosamine polymer. In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and nonconserved residues in the vicinity of the active site. The active sites of DspB and other familyu200320 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsiteu2003−1. These residues were mutated using site‐directed mutagenesis and characterized for their enzyme activity. Our results show that a highly conserved acid pair in β‐hexosaminidases D183 and E184, and E332 play a critical role in the hydrolysis of the substrates. pH activity profile analysis showed a shift to a higher pH (6.8) in the optimal activity for the E184Q mutant, suggesting that this residue might act as the acid/base catalyst. The reduction in kcat observed for Y187A and Y278A mutants suggests that the Y187 residue (unique to DspB) located on a loop might play a role in substrate specificity and be a part of subsiteu2003+1, whereas the hydrogen‐bond interaction between Y278A and the N‐acetyl group might help to stabilize the transition state. Mutation of W237 and W330 residues abolished hydrolytic activity completely suggesting that alteration at these positions might collapse the binding pocket for the N‐acetyl group. Mutation of the conserved R27 residue (to R27A or R27K) also caused significant reduction in kcat suggesting that R27 might be involved in stabilization of the transition state. From these results, we conclude that in DspB, and possibly in other structurally similar familyu200320 hydrolases, some residues at the active site assist in orienting the N‐acetyl group to participate in the substrate‐assisted mechanism, whereas other residues such as R27 and E332 assist in holding the terminal N‐acetylglucosamine during the hydrolysis.

Genome Sequence of Kingella kingae Septic Arthritis Isolate PYKK081

Kingella kingae is a human oral bacterium that can cause infections of the skeletal system in children. The bacterium is also a cardiovascular pathogen causing infective endocarditis in children and adults. We report herein the draft genome sequence of septic arthritis K. kingae strain PYKK081.