Effect of PugNAc on whole-cell hexosaminidase activity
Five-day-old T. forsythia cells were harvested, washed with PBS, and adjusted to a cell density at A600 of 0.05. The hexosaminidase substrates, 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-β-D-glucopyranoside (MUGs) and 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-galactopyranoside (MUGal) were then added to a final concentration of 1.5mM. The inhibitor PugNAc (O-(2-Acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate) was added at the concentrations shown. Reactions were allowed to proceed for 2h before measurement at a fixed excitation wavelength of 355nm and the emission measured at 430±10nm in a fluorimeter. Assays were performed in triplicate, and the readings are presented as a percentage of the cell activity without any PugNAC added; standard error is shown. (Inset) Photograph of end-point assay in the presence of 25μM PugNAc.
(B) Biofilm growth of T. forsythia on glycoprotein coated surfaces. Biofilm growth was assessed for the wild-type strain in the absence (black) or presence of PugNAc (25μM) (grey). Biofilms were set-up in triplicate and wells supplemented with 6 mM sialic acid (sialic), 6mM sialyl-lactose (sia-lac) or in coated wells with 6mM mucin, fetuin and a-sialo fetuin, 2mg/mL human serum and saliva with and without the addition of 25μM PugNAc to the TSB-medium as indicated at the time of inoculation. Glycoproteins were coated on the 96-well plate overnight at 4°C and washed before inoculation with T. forsythia to a final OD600 of 0.05. Data are number of cells after harvesting from wells after 4 days growth with standard deviations. Difference between mean data in this experiment were evaluated by t-test, with p<0.05 being taken as the level of significance (* statistically significant).
(C) Initial adhesion growth was compared as above with the wild-type strain of T. forsythia without and with 25mM PugNAc except that plates were only incubated for 3h before harvesting of bacteria and enumeration.
(D) Initial adhesion of the ΔnanH mutant of T. forsythia in the presence and absence of 25mM PugNAc.