Role of glucosyltransferase B in the interactions of Candida albicans with 1 Streptococcus mutans and experimental pellicle formed on hydroxyapatite 2 surface 3

23 Candida albicans and mutans streptococci are frequently detected in dental plaque- 24 biofilms from toddlers afflicted with early-childhood caries. Glucosyltransferases (Gtfs) secreted 25 by Streptococcus mutans bind to saliva-coated apatitic (sHA) and to bacterial surfaces, 26 synthesizing exopolymers in situ, which promote cell clustering-adherence to the tooth enamel. 27 We investigated the potential role Gtfs may play in mediating the interactions between C. 28 albicans -SC5314 and S. mutans -UA159, both with each other and with the sHA surface. GtfB 29 adhered effectively to the C. albicans yeast-cell surface in an enzymatically active form as 30 determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the 31 yeast-cell surface were more susceptible to dextranase than those synthesized in solution or on 32 sHA and bacterial surfaces ( P <0.05), indicating elevated content of α 1,6-linked glucose. 33 Fluorescence imaging revealed that larger numbers of S. mutans cells bound to the glucans 34 present on the C. albicans surface, as compared to yeast-cells without surface-glucans (uncoated). 35 The glucans formed in situ also enhanced C. albicans interactions with sHA as determined by a 36 novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast- 37 cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans 38 incubated alone or mixed with uncoated C. albicans , P <0.05). These data reveal a novel cross- 39 kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast 40 surface. The surface-bound GtfB promote the formation of a glucan-rich matrix in situ and may 41 enhance the accumulation of S. mutans on the tooth-enamel surface, thereby modulating the 42 development of virulent biofilms. of 155 [ 14 C]glucose from labeled sucrose into glucans using scintillation counting (51). All assays were 156 done in quadruplicate in at least three different experiments. Results from our preliminary 157 experiments show that GtfB binds the most effectively to C. albicans surfaces followed by GtfC. continuous exopolysaccharides production over time. Our results show that the adhesive 504 bacteria-yeast interaction may be more specific than just physical entrapment associated with de novo glucan synthesis. We propose that the in situ glucans formed on the yeast surface by GtfB 506 act as binding sites for S. mutans forming a bacterial-fungal-EPS complex attached to the sHA 507 surface. These results indicate physical interactions which are fundamentally different from those 508 observed between C. albicans adhesins and streptococcal surface proteins, and salivary protein in 509 the pellicle. GtfB may represent an additional class of bacterial-derived products other than 510 quorum sensing molecules (3, 46), that could directly influence biofilm formation by both of 511 these species (from two distinct kingdoms) by modulating: (i) initial interaction between S. mutans and C. albicans as well as between the yeast cell and sHA surface, and (ii) establishment 513 of the extracellular matrix (possibly in conjunction with EPS, such as β 1,3-glucan, produced by C. albicans ). In addition, it is equally possible that C. albicans could also adsorb GtfB from 515 saliva, produce glucans and then bind to S. mutans attached to sHA.