Streptococcus mutans is recognized as the major etiologic agent of dental caries in humans. Its pathogenicity is enhanced by a number of virulence factors including an adhesin (P1), which promotes colonization in the absence of dietary sucrose and the ability of this species to withstand the effects of low pH caused by the homofermentation of sugars. The present application focuses on each of these virulence traits. The gene encoding the P1 adhesin, spaP, has been cloned and sequenced and the gene product is well characterized. Nothing is known, however, of environmental or genetic regulatory mechanisms controlling its expression. Environmental regulation will be studied by growing a heterodiploid strain containing a chromosomal spaP::lacZ reporter gene fusion construct in a chemostat under varying growth conditions. Both adherent and non-adherent cells will be assayed for gene expression by measuring beta-galactosidase activity. Genetic regulatory loci will be interrupted by using transposon Tn 917 or random chromosomal fragments and screened for loss of adhesin expression. Regulatory genes will be characterized after marker rescue or cloning into E. coli. Also, putative enzymes and transport proteins that aid in the folding and localization of P1 will be isolated by affinity chromatography methods and responsible genes characterized as above. The second thrust of this proposal involves the Ffh-dependent protein translocation system, only recently discovered in S. mutans. An acid-sensitive mutant, known to be interrupted in the ylxM-ffh (sat) operon and unable to assemble optimal amounts of membrane-bound H+/ATPase, will be studied by two-dimensional gel electrophoresis. Major proteins differentially expressed between mutant and wild-type membranes will be recovered, subjected to N-terminal amino acid sequence analysis and appropriate degenerate oligonucleotides synthesized for use in screening the investigator's genomic libraries for responsible genes. By this approach, the investigator hopes to determine the key proteins transported into membranes by the Ffh-pathway. The yeast two-protein hybrid technique will be employed to analyze the binding of S. mutans Ffh-containing fusion proteins to those containing S. mutans ATP-ase subunits or other relevant gene products in vivo in Saccharomyces cerevisiae. Such reactions will be confirmed by affinity chromatography on Ffh- GST columns. Finally, environmental regulation will be studied by growing a heterodiploid strain containing a chromosomal ylxM promoter::lacZ reporter gene fusion construct in a chemostat under varying growth conditions including pH, osmolarity, and nutrient sources.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE008007-14
Application #
2896996
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1986-03-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Florida
Department
Dentistry
Type
Schools of Dentistry
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Binepal, Gursonika; Gill, Kamal; Crowley, Paula et al. (2016) Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance. J Bacteriol 198:1087-100
Crowley, P J; Brady, L J (2016) Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production. Mol Oral Microbiol 31:59-77
Tang, Wenxing; Bhatt, Avni; Smith, Adam N et al. (2016) Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy. J Biomol NMR 64:153-64
Sullan, Ruby May A; Li, James K; Crowley, Paula J et al. (2015) Binding forces of Streptococcus mutans P1 adhesin. ACS Nano 9:1448-60
Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J et al. (2015) Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface. J Biol Chem 290:9002-19
Lewis, N E; Brady, L J (2015) Breaking the bacterial protein targeting and translocation model: oral organisms as a case in point. Mol Oral Microbiol 30:186-97
Williams, Matthew L; Crowley, Paula J; Hasona, Adnan et al. (2014) YlxM is a newly identified accessory protein that influences the function of signal recognition particle pathway components in Streptococcus mutans. J Bacteriol 196:2043-52
Liao, Sumei; Klein, Marlise I; Heim, Kyle P et al. (2014) Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteriol 196:2355-66
Heim, Kyle P; Crowley, Paula J; Long, Joanna R et al. (2014) An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1. Proc Natl Acad Sci U S A 111:15746-51
Heim, Kyle P; Crowley, Paula J; Brady, L Jeannine (2013) An intramolecular interaction involving the N terminus of a streptococcal adhesin affects its conformation and adhesive function. J Biol Chem 288:13762-74

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