Streptococccus mutans is a ubiquitous oral bacterium identified as a prominent etiologic agent of human dental caries. Certain strains of S. mutans have also been identified as causative agents of bacterial endocarditis, discovered within atherosclerotic plaque, and demonstrated to invade human coronary epithelial cells. The health care cost associated with dental decay stemming from S. mutans and other cariogenic organisms alone is enormous, over $64 billion annually in the USA. This project addresses membrane biogenesis and protein secretion in S. mutans. Membranes comprised of a semi-permeable lipid bilayer enclose all cells and, by dictating what can and cannot cross by virtue of proteins embedded within them, determine cellular function. S. mutans shares features common to bacterial membrane protein insertion and protein secretion systems common to the most widely studied model organism Escherichia coli, as well as to those of mitochondria and chloroplasts, but it exhibits multiple unique properties as well. For this reason it is gaining in reputation as a model organism to better understand the protein translocation machinery in other streptococci, and Gram-positive bacteria in general. All known virulence properties of S. mutans are a consequence of its membrane protein composition and/or secreted extracellular proteins. Since biological membranes are composed of ~50% proteins by mass, and these represent most known and existing drug targets, a better understanding of the membrane insertion and transport pathways of S. mutans that guide its virulence factors to their necessary locations will facilitate future targeted therapies against this and related pathogens. This projec focuses on two critical co-translational translocation pathways, the signal recognition particle (SRP) pathway found in all living cells, and the YidC insertase pathway found in bacteria/mitochondria/chloroplasts. The presence in Gram-positive bacteria of an additional SRP component called YlxM, and of dual YidC paralogs of differing function, was discovered in S. mutans. Additional components unique to S. mutans appear to exist as well. In this project, a combination of membrane proteomics, directed genetic, biochemical, and biophysical approaches, and complementary in vivo and in vitro analyses will improve the understanding of membrane protein insertion and secretion pathways of S. mutans and will identify specific membrane-localized substrates related to the pivotal virulence properties of competence development and mutacin production since secretion pathway mutants are defective in this regard. These studies are facilitated by the establishment of methodologies to purify transitionally active ribosomes from S. mutans, to assess membrane protein insertion in vivo and in vitro, and to utilize biolayer interferometry, co-immunoprecipitation, and chemical cross-linking to detect and measure interactions of membrane-localized secretion pathway components that are crucial to the transport and insertion of the substrates identified as part of this research.
Streptococcus mutans is an oral bacterium and major global infectious agent that causes human dental caries, as well as bacterial endocarditis and atherosclerosis. Annual health care costs related to dental caries in the US alone exceed 60 billion dollars. Multiple virulence properties of S. mutans stem from the way the organism inserts proteins into its cytoplasmic membrane, but the cellular machinery that transports these proteins is not completely understood. This research will explain how proteins important for disease get into and through the membrane of S. mutans and other related pathogens.
|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|
|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|
|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|
|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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