Department of Oral Biology
1395 Center Drive, DG-32
Gainesville, FL 32610-0424
- Ph.D., University of California, Berkeley, Physiology, 1981
- B.S., University of California, Berkeley, Biology, 1974
Project 1: Innate immunity in the oral cavity in the context of oral epithelial cells and how specific commensals help protect epithelial cells from invasion by the periodontal pathogen, Porphyromonas gingivalis.
In health, gingival epithelial cells are in homeostasis with the commensal oral microbiota. Pathogenesis begins with a breach in the balance of normal host-microbiome interactions. We hypothesize that interactions between gingival epithelial cells and commensal streptococci help to protect gingival cells from adherence and/or invasion by P. gingivalis. To test this hypothesis, we are exploring interactions of telomerase immortalized gingival keratinocytes (TIGKs) with strains of commensal streptococcal species highly prevalent in the gingival sulcus (S. gordonii, S. mitis, S. intermedius) to alter invasion and/or adherence of TIGKs by P. gingivalis.
To date, we find that S. mitis and S. intermedius enhance the innate immunity of TIGKs in the context of decreased adherence and invasion of P. gingivalis, whereas S. gordonii has comparably little or no effects. Enhanced innate immunity is not due to TIGK cell death, apoptosis or to changes in integrin binding sites. Evidence is consistent with increases in expression of membrane glycoproteins, in glycoprotein sialylation and in cross-linking via cell derived lectins, factors that may function to attenuate adherence of P. gingivalis. We have also established protocols for analyses of P. gingivalis-glycoprotein interactions. Of 20 chemokines and cytokines examined, only TNF-alpha, GM-CSF, IL6, and IL8 are altered (up-regulated) differentially by both S. intermedius and S. mitis when compared to S. gordonii, suggesting one or more of these cyto/chemokines may have an autocrine effect to enhance protection against P. gingivalis.
We are interested in delineating streptococcal-host cell interactions and host cell signaling responsible for enhanced innate immunity. Preliminary data suggests components within the cell-free supernatant of S. intermedius is sufficient to mediate enhanced innate immunity. Future efforts are to employ proteomic and transcriptomic techniques combined with pathways analyses.
Project 2: In vivo and in vitro assessment of the innate immune functions of specific salivary components in protection against the cariogenic bacterium, Streptococcal mutans.
Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with S. mutans, a pathogen also linked to endocarditis and atheromatous plaques. We generated knockout mice with targeted gene deletions of specific constituents of saliva (i.e., Muc19, Muc10 and amylase). Knockout mice were used in in vivo caries experiments to test the influence of each salivary molecule in protecting teeth against induction of carious lesion by S. mutans.
Gel-forming mucins are a major constituent of saliva, and in mice Muc19 is the dominant salivary gel-forming mucin. We find deletion of Muc19 greatly increases the incidence and severity of carious lesions. In vitro studies of S. mutans and saliva ± Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. Surprisingly, amylase also has a marked effect in protection against caries whereas Muc10 (a small soluble mucin in saliva) has very modest effects. Determination of the influence of host determinants in caries development may ultimately provide important targets for therapeutic intervention in the treatment of patients at high risk for caries.
Project 3: Examine probiotic strategies of beneficial oral commensal streptococci to prevent dental caries and examine the role of specific genetic determinants in caries protection.
This is a multi-investigator project in which our lab’s component is focused on the analysis, in vivo, of a panel of commensal isolates from healthy humans. We are using our established mouse model of dental caries to ascertain whether isolates with select competitive phenotypes against S. mutans, in vitro, inhibit the establishment and persistence of this pathogen as well as the induction and progression of dental caries. Competitive phenotypes of interest include streptococci that utilize arginine to moderate extracellular pH, generate H2O2, or produce antagonistic proteases or bacteriocins. Further efforts will test, in vivo, the role of genes responsible for competitiveness in vitro. Elucidation of mechanisms for probiotic effects of beneficial oral streptococci will assist in development of new therapies to help prevent dental caries.
Culp DJ, Robinson B, Cash MN, Bhattacharyya I, Stewart C, Cuadra-Saenz G. Salivary mucin 19 glycoproteins: innate immune functions in Streptococcus mutans-induced caries in mice and evidence for expression in human saliva. J Biol Chem. 2015 Jan 30;290(5):2993-3008. PubMed PMID: 25512380
Moffatt-Jauregui CE, Robinson B, de Moya AV, Brockman RD, Roman AV, Cash MN, Culp DJ, Lamont RJ. Establishment and characterization of a telomerase immortalized human gingival epithelial cell line. J Periodontal Res. 2013 Dec;48(6):713-21. PubMed PMID: 23441958
Das B, Cash MN, Robinson B, Kuhns CS, Latchney LR, Fallon MA, Elliott RW, Hand AR, Culp DJ. The sld genetic defect: two intronic CA repeats promote insertion of the subsequent intron and mRNA decay. J Biol Chem. 2013 May 24;288(21):14742-55. PubMed PMID: 23580649
Culp DJ, Zhang Z, Evans RL. Role of calcium and PKC in salivary mucous cell exocrine secretion. J Dent Res. 2011 Dec;90(12):1469-76. PMID: 21933938
Culp DJ, Robinson B, Parkkila S, Pan PW, Cash MN, Truong HN, Hussey TW, Gullett SL. Oral colonization by Streptococcus mutans and caries development is reduced upon deletion of carbonic anhydrase VI expression in saliva. Biochim Biophys Acta. 2011 Dec;1812(12):1567-76. PubMed PMID: 21945428
Das B, Cash MN, Hand AR, Shivazad A, Grieshaber SS, Robinson B, Culp DJ. Tissue distribution of murine Muc19/smgc gene products. J Histochem Cytochem. 2010 Feb;58(2):141-56. PubMed PMID: 19826070
Das B, Cash MN, Hand AR, Shivazad A, Culp DJ. Expression of Muc19/Smgc gene products during murine sublingual gland development: cytodifferentiation and maturation of salivary mucous cells. J Histochem Cytochem. 2009 Apr;57(4):383-96. PubMed PMID: 19110483