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      Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans

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          Abstract

          Streptococcal glucosyltransferases (Gtf) synthesize α-glucan exopolymers which contribute to biofilm matrix. Streptococcus oralis interacts with the opportunistic pathogen Candida albicans to form hypervirulent biofilms. S. oralis 34 has a single gtf gene ( gtfR). However, the role of gtfR in single and mixed species biofilms with C. albicans has never been examined. A gtfR deletion mutant, purified GtfR, and recombinant GtfR glucan-binding domain were tested in single and mixed biofilms on different substrata in vitro. A mouse oral infection model was also used. We found that in single species biofilms growing with sucrose on abiotic surfaces S. oralis gtfR increased biofilm matrix, but not bacterial biomass. In biofilms with C. albicans, S. oralis encoding gtfR showed increased bacterial biomass on all surfaces. C. albicans had a positive effect on α-glucan synthesis, and α-glucans increased C. albicans accretion on abiotic surfaces. In single and mixed infection of mice receiving sucrose S. oralis gtfR enhanced mucosal burdens. However, sucrose had a negative impact on C. albicans burdens and reduced S. oralis burdens in co-infected mice. Our data provide new insights on the GtfR-mediated interactions between the two organisms and the influence of biofilm substratum and the mucosal environment on these interactions.

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          Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.

          Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.
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            Bacterial coaggregation: an integral process in the development of multi-species biofilms.

            Coaggregation is a process by which genetically distinct bacteria become attached to one another via specific molecules. Cumulative evidence suggests that such adhesion influences the development of complex multi-species biofilms. Once thought to occur exclusively between dental plaque bacteria, there are increasing reports of coaggregation between bacteria from other biofilm communities in several diverse habitats. A general role for coaggregation in the formation of multi-species biofilms is discussed.
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              Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms.

              Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.
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                Author and article information

                Contributors
                mmv4@buffalo.edu
                adongari@uchc.edu
                Journal
                ISME J
                ISME J
                The ISME Journal
                Nature Publishing Group UK (London )
                1751-7362
                1751-7370
                10 February 2020
                10 February 2020
                May 2020
                : 14
                : 5
                : 1207-1222
                Affiliations
                [1 ]ISNI 0000000419370394, GRID grid.208078.5, Department of Oral Health and Diagnostic Sciences, , University of Connecticut School of Dental Medicine, ; Farmington, CT USA
                [2 ]ISNI 0000 0001 0723 2494, GRID grid.411087.b, Department of Prosthodontics and Periodontology, Piracicaba Dental School, , University of Campinas (UNICAMP), ; Piracicaba, São Paulo Brazil
                [3 ]ISNI 0000 0004 1936 9887, GRID grid.273335.3, Department of Oral Biology, School of Dental Medicine, , University at Buffalo, ; Buffalo, NY USA
                [4 ]ISNI 0000000419370394, GRID grid.208078.5, Departments of Medicine, Pediatrics and Molecular Biology and Biophysics, , University of Connecticut Health Center, ; Farmington, CT USA
                [5 ]ISNI 0000 0001 0860 4915, GRID grid.63054.34, Microbial Analysis, Resources, and Services Core, , University of Connecticut, ; Storrs, CT USA
                Article
                608
                10.1038/s41396-020-0608-4
                7174356
                32042100
                7cf9f71a-3d46-419b-9b40-ae08dd53b19e
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 21 October 2019
                : 8 January 2020
                : 30 January 2020
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000072, U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR);
                Award ID: RO1 DE013986
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000060, U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID);
                Award ID: RO1AI029735
                Award ID: R21AI126146
                Award Recipient :
                Funded by: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
                Categories
                Article
                Custom metadata
                © International Society for Microbial Ecology 2020

                Microbiology & Virology
                microbiology,biofilms
                Microbiology & Virology
                microbiology, biofilms

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