Biofilm formation and virulence expression by Streptococcus mutans are altered when grown in dual-species model

BMC Microbiology RBeiseoarfcihlamrticlfeormation and virulence expression by Streptococcus mutans are altered when grown in dual-species model Zezhang T Wen 0 David Yates 1 Sang-Joon Ahn 1 Robert A Burne 1 0 Department of Oral and Craniofacial Biology and Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center , New Orleans, LA 70119 , USA 1 Department of Oral Biology, College of Dentistry, University of Florida , Box 100424, Gainesville, FL 32610 , USA Background: Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis. Results: As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Cocultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes 1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis. Conclusions: These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans. - Background Oral biofilms are compositionally and structurally complex bacterial communities. To date, more than 750 different species or phylotypes of bacteria have been identified in mature dental plaque [1]. Microbial cell-cell interactions in the oral flora and their impact on bacterial adherence and biofilm formation are beginning to be appreciated [1-4]. Cross-feeding or metabolic cooperation is well-documented among certain bacterial species in the oral flora. Veillonellae can utilize the lactic acid produced by streptococci and Porphyromonas gingivalis benefits from succinate produced by T. denticola. Similarly, isobutyrate secreted by P. ginivalis stimulates the growth of T. denticola [2,3]. Adhesin-ligand mediated physical interactions such as those between Streptococcus gordonii and P. gingivalis may be important for secondary colonizers like P. gingivalis to establish and persist in the oral cavity [5]. A recent study has also provided evidence that a mutualistic effect in biofilm formation between Actinomyces naeslundii and Streptococcus oralis is facilitated by autoinducer-2 (AI-2) [6]. Intra- and inter-species interactions are believed to play a crucial role in community dynamics, contributing to the formation of plaque and, ultimately, the development of polymicrobial diseases, including caries and periodontitis [2,5]. Therefore, a better understanding of cell-cell interactions between oral pathogens and commensal bacteria, and the impact of these interactions on expression of virulence factors and pathogenicity, could lead to development of novel preventive and therapeutic strategies against dental caries and periodontitis. As the principal etiological agent of human dental caries, Streptococcus mutans has developed multiple mechanisms to colonize the tooth surface and, under certain conditions, to become a numerically significant species in cariogenic biofilms [7]. The multi-functional adhesin SpaP, also called P1 and PAc1, is considered the primary factor mediating early attachment of S. mutans to tooth enamel in the absence of sucrose [8]. S. mutans also produces at least three glucosyltransferases (GtfB, -C & -D), which polymerize the glucosyl moiety from sucrose and starch carbohydrates into 1,3- and 1,6-linked glucans [7,9]. Binding to glucans by glucan binding proteins (GbpA, -B, -C and -D) and by the Gtfs facilitates bacterial adherence to tooth surfaces, inter-bacterial adhesion and accumulation of biofilms [9,10]. GtfBC&D and GbpABC&D, together with the adhesive extracellular glucans, constitute the sucrose-dependent pathway for S. mutans to establish on the tooth surface and are of central importance in plaque formation and development of caries [7,9-14]. Multiple regulatory networks that integrate external signals, including the cell density-dependent Com system and other two-component regulatory systems, including CiaHR, LiaSR and VicRK, with CiaH, LiaS and VicK being the sensor kinases and CiaR, LiaR and VicR the response regulators of two-component system, are required for biofilm formation [15-21]. S. mutans also possesses a LuxS-mediated signaling pathway that affects biofilm formation and bacteriocin production [18,22,23]. LuxS is the enzyme that catalyzes the reactions leading to the production of the AI-2 signal molecule [24]. In addition, a number of other gene products, such as BrpA (a cell surface-associated biofilm regulatory protein), have also been shown to play critical roles in environmental stress responses and biofilm development by S. mutans [25,26]. While much effort has been devoted to understanding the molecular mechanisms of adherence, biofilm development and virulence gene expression by S. mutans in pure cultures, there are large gaps in our knowledge of how this cariogenic bacterium behaves in response to inter-generic interactions with bacteria commonly found in the supragingival plaque. In this study, we developed a dual-species in vitro model to examine the impact of co-cultivation of S. mutans with S. oralis or S. sanguinis, two primary colonizers and members of the normal flora, or with Lactobacillus casei, a bacterium frequently isolated from carious sites, on biofilm formation by these bacteria and expression of known virulence factors of S. mutans. Data presented here suggest that growth in dual-species impacts surface biomass accumulation by some of the bacterial species analyzed, as compared to the respective monospecies biofilms and that the expression of known virulence factors by S. mutans can be differentially modulated by growth with other bacteria commonly found in dental plaque. Such int (...truncated)