Dataset Information

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary ?-Amylase, and Starch.

ABSTRACT: Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary ?-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA(-) mutant KS1?abpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1?abpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance.

SUBMITTER: Haase EM

PROVIDER: S-EPMC4510181 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch.

Haase Elaine M EM Feng Xianghui X Pan Jiachuan J Miecznikowski Jeffrey C JC Scannapieco Frank A FA

Applied and environmental microbiology 20150529 16

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the r ...[more]

PMID: 26025889

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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.

Project description:The combination of sucrose and starch in the presence of surface-adsorbed salivary ?-amylase and bacterial glucosyltransferases increase the formation of a structurally and metabolically distinctive biofilm by Streptococcus mutans. This host-pathogen-diet interaction may modulate the formation of pathogenic biofilms related to dental caries disease. We conducted a comprehensive study to further investigate the influence of the dietary carbohydrates on S. mutans-transcriptome at distinct stages of biofilm development using whole genomic profiling with a new computational tool (MDV) for data mining. S. mutans UA159 biofilms were formed on amylase-active saliva coated hydroxyapatite discs in the presence of various concentrations of sucrose alone (ranging from 0.25 to 5% w/v) or in combination with starch (0.5 to 1% w/v). Overall, the presence of sucrose and starch (suc+st) influenced the dynamics of S. mutans transcriptome (vs. sucrose alone), which may be associated with gradual digestion of starch by surface-adsorbed amylase. At 21 h of biofilm formation, most of the differentially expressed genes were related to sugar metabolism, such as upregulation of genes involved in maltose/maltotriose uptake and glycogen synthesis. In addition, the groEL/groES chaperones were induced in the suc+st-biofilm, indicating that presence of starch hydrolysates may cause environmental stress. In contrast, at 30 h of biofilm development, multiple genes associated with sugar uptake/transport (e.g. maltose), two-component systems, fermentation/glycolysis and iron transport were differentially expressed in suc+st-biofilms (vs. sucrose-biofilms). Interestingly, lytT (bacteria autolysis) was upregulated, which was correlated with presence of extracellular DNA in the matrix of suc+st-biofilms. Specific genes related to carbohydrate uptake and glycogen metabolism were detected in suc+st-biofilms in more than one time point, indicating an association between presence of starch hydrolysates and intracellular polysaccharide storage. Our data show complex remodeling of S. mutans-transcriptome in response to changing environmental conditions in situ, which could modulate the dynamics of biofilm development and pathogenicity.

Dataset Information

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary ?-Amylase, and Starch.

Publications

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch.

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