Dynamics of Streptococcus mutans Transcriptome in Response to Starch and Sucrose during Biofilm Development

et al. (2010) Dynamics of Streptococcus mutans Transcriptome in Response to Starch and Sucrose during Biofilm Development. PLoS ONE 5(10): e13478. doi:10.1371/journal.pone.0013478 Dynamics of Streptococcus mutans Transcriptome in Response to Starch and Sucrose during Biofilm Development Marlise I. Klein 0 Lena DeBaz 0 Senyo Agidi 0 Herbert Lee 0 Gary Xie 0 Amy H.-M. Lin 0 Bruce R. Hamaker 0 Jose A. Lemos 0 Hyun Koo 0 Frank R. DeLeo, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America 0 1 Center for Oral Biology and Eastman Department of Dentistry, University of Rochester Medical Center , Rochester , New York, United States of America, 2 Department of Microbiology and Immunology, University of Rochester Medical Center , Rochester , New York, United States of America, 3 Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America, 4 Department of Food Science, Whistler Center for Carbohydrate Research, Purdue University , West Lafayette, Indiana , United States of America The combination of sucrose and starch in the presence of surface-adsorbed salivary a-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. mutanstranscriptome 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+stbiofilm, 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+stbiofilms. 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. - Funding: This research was partially supported by grants from the National Institutes of Health (NIH Y1-DE-6006-02) and the University of California Office of President-UCOP 2009 Lab Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Dental caries continues to be the single most common biofilmdependent oral infectious disease worldwide [1]. Dental caries result from the interaction of specific bacterial and salivary constituents with dietary carbohydrates in biofilms tightly adherent on the tooth surface [1,2]. Streptococcus mutans, a member of the oral microbial community, plays a key role in modulating the transition from non-pathogenic form to highly cariogenic biofilms [3], although additional organisms may be also associated with this ubiquitous disease (as reviewed in [4]). This bacterium is able to thrive and compete in the complex biofilm microbiome, and contribute to the pathogenesis of dental caries because it: (i) effectively utilizes dietary sucrose to rapidly synthesize exopolysaccharides (EPS) through glucosyltransferases and a fructosyltransferase that adsorb to surfaces, (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is highly acidogenic and aciduric [2,5]. Sucrose and starch, the main dietary carbohydrates in modern societies, are potentially more cariogenic in combination than is either alone, both in animals and humans [68]. Host salivary aamylases digest starches to maltose, maltodextrins and other oligosaccharides and some of these starch hydrolysates can be incorporated during glucan synthesis by glucosyltransferases (Gtfs) via acceptor reactions [9,10]. More importantly, these host- and bacterial-derived enzymes adsorb to the pellicle in active form [10]. In the presence of sucrose and starch, surface adsorbed-GtfB and -amylase acting in concert increase the synthesis of structurally distinct glucans, which provide enhanced bacterial binding (including S. mutans) to apatitic surfaces [10]. S. mutans has multiple sugar transport systems involved in the uptake of starch hydrolysates (e.g. maltose and maltotriose) [1113], which can be further metabolized into acids [14]. Furthermore, we have collected evidence that the interaction of sucrose and starch through surface-adsorbed salivary a-amylase and Gtf enzymes (particularly GtfB) modulates in situ the development of biofilms that are both structurally and metabolically distinctive [15,16]. These interactions promote formation of biofilms with elevated amounts of EPS and increased acidogenicity [15] by up-regulating the expression of gtfB and msm operon genes [15,16]. The gtfB gene (and its GtfB product) is a recognized virulence factor associated with the pathogenesis of dental caries in rodents and in humans [17,18]. Therefore, analysis of the dynamics of transcriptomic responses of S. mutans to starch and sucrose during the biofilm formation process would enhance our understanding of the molecular mechanisms involved with the biochemical and structural changes, and increased pathogenicity observed previously [68,15,16]. In this study, we used a whole genomic profiling approach to further characterize how S. mutans responds to this unique hostpathogen-diet interaction at distinct time points over the course of biofilm formation on a saliva-coated hydroxyapatite surface. In addition, we developed a new software to analyze the microarray data, focusing on how specific transcriptome changes may be associated with enhanced biofilms accumulation, survival and virulence of (...truncated)