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Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase.

ABSTRACT: The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expressed in Escherichia coli and purified. Its amylase-binding activity was verified in an amylase ligand binding assay, and its cross-reactivity was verified with an anti-AbpB antibody. Both recombinant His-AbpB and partially purified native AbpB displayed dipeptidase activity and degraded human type VI collagen and fibrinogen, but not salivary amylase. Salivary amylase precipitates not only AbpA and AbpB but also glucosyltransferase G (Gtf-G) from S. gordonii supernatants. Since Streptococcus mutans also releases Gtf enzymes that could also be involved in multispecies plaque interactions, the effect of S. gordonii AbpB on S. mutans Gtf-B activity was also tested. Salivary amylase and/or His-AbpB caused a 1.4- to 2-fold increase of S. mutans Gtf-B sucrase activity and a 3- to 6-fold increase in transferase activity. An enzyme-linked immunosorbent assay verified the interaction of His-AbpB and amylase with Gtf-B. In summary, AbpB demonstrates proteolytic activity and interacts with and modulates Gtf activity. These activities may help explain the crucial role AbpB appears to play in S. gordonii oral colonization.

SUBMITTER: Chaudhuri B

PROVIDER: S-EPMC2546850 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase.

Chaudhuri Biswendu B Paju Susanna S Haase Elaine M EM Vickerman M Margaret MM Tanzer Jason M JM Scannapieco Frank A FA

Infection and immunity 20080804 10

The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expresse ...[more]

PMID: 18678669

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Project description:Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that amylase binding to S. gordonii modulates expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect differentially expressed genes in S. gordonii strain CH1 in response to the binding of purified human salivary amylase as compared to exposure to heat-denatured amylase. Selected genes found to be differentially expressed were validated by qRT-PCR. Five genes from the fatty acid synthesis (FAS) cluster were highly (10-35 fold) up-regulated in amylase treated S. gordonii CH1 cells compared to the denatured-amylase treated cells. An abpA-deficient strain of S. gordonii exposed to amylase did not show a similar response in FAS gene expression as observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 associated with increased expression of FAS genes leading to changes in fatty acid synthesis were noted, as evidenced by increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase exposed abpA-deficient strain, suggesting for the role of AbpA in amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotype adjustment that is potentially advantageous for bacterial survival in the oral environment. In order to identify amylase-regulated genes, S. gordonii CH1 was grown statically in 40 ml CDM at 37°C in a candle jar to mid-log phase corresponding to an optical density at 600 nm of 0.5 to 0.6.The mid-log phase bacterial culture was divided into two aliquots of equal volume. Bacterial cells from all aliquots were pelleted by centrifugation at 6,000 x g in a Sorvall RC6 centrifuge at 20°C, and washed once with simulated salivary buffer preconditioned to 37°C. Simulated salivary buffer containing 0.4 mg/ml purified, non-glycosylated salivary amylase (native amylase) and preconditioned to 37°C was added to the cells of the fist aliquot; to the cells of the second aliquot simulated salivary buffer containing 0.4 mg/ml of the same salivary amylase denatured by heating to 100°C {Heinen, 1976} and cooled to 37°C was added, as a negative control. Each aliquot, amylase treated and control, was incubated statically for 15 min at 37°C in a candle jar. Total RNA was immediately isolated by the hot acid phenol method as described previously {Vickerman, 2007}, followed by treatment with TurboDNase (Applied Biosystems/Ambion, Austin, TX) according to manufacturer’s protocol. Remaining contaminants were removed using the RNeasy minikit column (Qiagen, Valencia, CA) with the cleanup protocol. Total RNA was quantified using the Nanodrop 2000 spectrophotometer and RNA integrity determined by agarose gel electrophoresis. Total RNA was used immediately for cDNA synthesis. The Cy dye-labeled cDNA from the amylase-treated aliquot of the culture was mixed with Cy dye-labeled cDNA from the denatured amylase-treated control aliquot, and used to probe the S. gordonii microarray slides. Each amylase-exposure experiment was repeated from four biological replicates. To confirm microarray results the cDNA from each strain was labeled with the opposite Cy dye and hybridized to similar arrays for the flip-dye comparison. Overall design 4 samples were analyzed. The quality controls were 4 biological replicates and dye-swap technical replicates for each biological replicate.

Dataset Information

Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase.

Publications

Amylase-binding protein B of Streptococcus gordonii is an extracellular dipeptidyl-peptidase.

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