Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium

ORIGINAL RESEARCH published: 20 December 2017 doi: 10.3389/fcimb.2017.00524 Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium Zezhang T. Wen 1, 2, 3*, Sumei Liao 1 , Jacob P. Bitoun 1 , Arpan De 2 , Ashton Jorgensen 2 , Shihai Feng 4 , Xiaoming Xu 2 , Patrick S. G. Chain 4 , Page W. Caufield 5 , Hyun Koo 6 and Yihong Li 5 1 Center of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center, New Orleans, LA, United States, Department of Comprehensive Dentistry and Biomaterials, Louisiana State University Health Sciences Center, New Orleans, LA, United States, 3 Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, United States, 4 Genome Science Group, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, United States, 5 Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, United States, 6 Biofilm Research Labs, Levy Center for Oral Health, Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, United States 2 Edited by: Justin Merritt, Oregon Health and Science University, United States Reviewed by: Ping Xu, Virginia Commonwealth University, United States Jens Kreth, Oregon Health and Science University, United States *Correspondence: Zezhang T. Wen Received: 09 August 2017 Accepted: 11 December 2017 Published: 20 December 2017 Citation: Wen ZT, Liao S, Bitoun JP, De A, Jorgensen A, Feng S, Xu X, Chain PSG, Caufield PW, Koo H and Li Y (2017) Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium. Front. Cell. Infect. Microbiol. 7:524. doi: 10.3389/fcimb.2017.00524 Like Streptococcus mutans, lactobacilli are commonly isolated from carious sites, although their exact role in caries development remains unclear. This study used mixed-species models to analyze biofilm formation by major groups of oral lactobacilli, including L. casei, L. fermentum, L. rhamnosus, L. salivarius ssp. salivarius, and L. gasseri. The results showed that lactobacilli did not form good biofilms when grown alone, although differences existed between different species. When grown together with S. mutans, biofilm formation by L. gasseri and L. rhamnosus was increased by 2-log (P < 0.001), while biofilms by L. fermentum reduced by >1-log (P < 0.001). L. casei enhanced biofilm formation by ∼2-log when grown with S. mutans wild-type, but no such effects were observed with S. mutans deficient of glucosyltransferase GtfB and adhesin P1. Both S. mutans and L. casei in dual-species enhanced resistance to acid killing with increases of survival rate by >1-log (P < 0.001), but drastically reduced the survival rates following exposure to hydrogen peroxide (P < 0.001), as compared to the respective mono-species cultures. When analyzed by RNA-seq, more than 134 genes were identified in S. mutans in dual-species with L. casei as either up- or down-regulated when compared to those grown alone. The up-regulated genes include those for superoxide dismutase, NADH oxidase, and members of the mutanobactin biosynthesis cluster. Among the down-regulated genes were those for GtfB and alternative sigma factor SigX. These results further suggest that interactions between S. mutans and oral lactobacilli are species-specific and may have significant impact on cariogenic potential of the community. Keywords: S. mutans, oral lactobacilli, mixed-species biofilms, dental caries, RNA-seq Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 December 2017 | Volume 7 | Article 524 Wen et al. S. mutans in Dual-Species with L. casei INTRODUCTION using in vitro mixed-species models for their abilities to form biofilms with and without the presence of S. mutans. An in vitro continuous biofilm model, transcriptional profiling via RNA-seq and metabolite analysis via HPLC were then used to further investigate the interactions between S. mutans and L. casei and identify the factors that mediate these interspecies interactions. Results demonstrated that interactions between S. mutans and the major oral lactobacilli are species-specific and may have an impact on the pathogenicity of the community. The oral microbiome, represented by dental plaque, harbors diverse and abundant microbial communities consisting of over 700 different species or phylotypes (Jenkinson, 2011). Both intra- and inter-species interactions in the oral flora have been well documented, although the underlying mechanisms remain unclear (Kuramitsu et al., 2007). The oral cavity is featured with fluctuating and often unpredictable conditions, such as nutrient source and availability and pH. Both microbemicrobe and microbe-environment interactions can profoundly influence the composition and relative proportions of major groups in the dynamic communities, leading to dysbiosis and consequently, development of oral diseases such as dental caries and periodontitis (Kuramitsu et al., 2007; Jenkinson, 2011; Burne et al., 2012; Hajishengallis et al., 2017). Cariogenic plaque, for instance, is characterized by dramatic increases in the proportion of acidogenic and aciduric species, which include mutans streptococci and lactobacilli (Jenkinson, 2011). As a major causative agent of dental caries, S. mutans possesses multiple mechanisms to colonize and persist on the tooth surface, and under certain conditions to become numerically significant, causing carious lesions (Hamada and Slade, 1980; Bowen et al., 1991; Bowen and Koo, 2011; Burne et al., 2011). Multi-functional adhesin P1 (also Antigen I/II, SpaP, or PAc) functions as the primary factor mediating early attachment to the tooth surface via interaction with salivary agglutiningp340 (Crowley et al., 1993). S. mutans also produces at least three glucosyltransferases (GtfB, -C, and -D) that polymerize the glucosyl moiety from sucrose, generating adhesive glucans (Bowen and Koo, 2011). The Gtfs and their glucan products, along with the glucan-binding proteins (Gbps), constitute the sucrose-dependent pathway central in plaque formation and caries development (Banas et al., 2007; Gregoire et al., 2011). In addition, multiple two-component signal transduction systems, molecular chaperones, and biofilm regulatory protein BrpA are shown to play an important role in S. mutans biofilm formation (Burne et al., 2011). As the first microorganisms implicated in human dental caries (Owen, 1949), lactobacilli are frequently identified at carious sites, esp. in patients with advanced caries, with L. casei, L. fermentum, L. gasseri, L. salivarius, and L. rhamnosus among the most prevalent groups (Caufield et al., 2007; Badet and Thebaud, 2008; Gross et al., 2012). Lactobacilli can utilize various kinds of sugars, generating lactic acid and other (...truncated)