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      The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

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          Abstract

          Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

          Author Summary

          Virulent biofilms formed on surfaces are associated with many human infections. The disease dental caries, expressed as cavities, is a prime example of the consequences arising from interactions between bacteria and sugars on tooth-surfaces. When Streptococcus mutans metabolize sugars, they produce a glue-like polymer termed glucan, helping them to adhere firmly to teeth. Glucan is also formed on bacterial surfaces in the mouth, and will accumulate and enmesh additional microorganisms creating the gelatinous formation known as dental plaque-biofilm. We found unique islets of bacteria within these biofilms, particularly close to the tooth-surface, providing safe havens in which bacteria thrive and produce acids that erode teeth. One intriguing mystery is why acids accumulate on the tooth-surface when there is an abundance of neutral-pH saliva surrounding the teeth. We found that bacterial-islets are particularly protected by glucan, which retards neutralization. We noticed that, within biofilms, the interiors of these islets are acidic, where only acid-tolerant bacteria can prosper, ensuring continued localized acid production. Our study demonstrates that construction of biofilms mediated by glucans forms complex 3D architectures, creating a variety of acidic-microenvironments that are essential for virulence expression. These results may aid in the development of enhanced methods to modulate biofilm formation.

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          Most cited references63

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          Quorum sensing: cell-to-cell communication in bacteria.

          Bacteria communicate with one another using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules termed autoinducers . This process, termed quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Most quorum-sensing-controlled processes are unproductive when undertaken by an individual bacterium acting alone but become beneficial when carried out simultaneously by a large number of cells. Thus, quorum sensing confuses the distinction between prokaryotes and eukaryotes because it enables bacteria to act as multicellular organisms. This review focuses on the architectures of bacterial chemical communication networks; how chemical information is integrated, processed, and transduced to control gene expression; how intra- and interspecies cell-cell communication is accomplished; and the intriguing possibility of prokaryote-eukaryote cross-communication.
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            Physiological heterogeneity in biofilms.

            Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.
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              Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

              We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2012
                April 2012
                5 April 2012
                : 8
                : 4
                : e1002623
                Affiliations
                [1 ]Center for Oral Biology, University of Rochester Medical Center, Rochester, New York, United States of America
                [2 ]State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China
                [3 ]Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States of America
                [4 ]Department of General Medicine, Glostrup Hospital, Glostrup, Denmark
                [5 ]Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
                Carnegie Mellon University, United States of America
                Author notes

                Conceived and designed the experiments: JX HK. Performed the experiments: JX MIK CMD BL MLF. Analyzed the data: JX MIK HK CMD BL MLF AH JRYIII. Contributed reagents/materials/analysis tools: HK JRYIII AH. Wrote the paper: JX MIK MLF HK. Developed DUOSTAT and updated COMSTAT: AH.

                Article
                PPATHOGENS-D-11-01814
                10.1371/journal.ppat.1002623
                3320608
                22496649
                86ed80fe-70a9-454f-8a0b-bb4526b5446f
                Xiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 17 August 2011
                : 22 February 2012
                Page count
                Pages: 16
                Categories
                Research Article
                Biology
                Microbiology
                Bacterial Pathogens
                Bacteriology
                Medicine
                Infectious Diseases
                Bacterial Diseases
                Oral Medicine

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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