Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms.
The production of extracellular poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-D-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Gießen, Germany.
Anke
A
Gökçen
Andreas
A
Vilcinskas
Jochen
J
Wiesner
D018441:Biofilms; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013212:Staphylococcus epidermidis / Q000737:chemistry / Q000378:metabolism*
D011134:Polysaccharides; D006026:Glycoside Hydrolases
Staphylococcus epidermidis; biofilm; exopolysaccharide; high-pH anion-exchange chromatography; poly-β-1,6-N-acetyl-d-glucosamine