Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia

Abstract

Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 μM/min ± 1.76 μM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.