There is growing evidence that certain facets of heart disease may be influenced by infections arising from the oral cavity. Streptococcus mutans, a major etiologic agent of dental caries, is also a leading causative agent of infective endocarditis (IE) and has been implicated in the development and progression of atherosclerosis. We have recently demonstrated that certain S. mutans strains invade and persist in the cytoplasm of Human Coronary Artery Endothelial Cells (HCAEC). Invasion of HCAEC was associated with an ability to avidly bind to collagen and laminin, and invasive strains were significantly more virulet than non-invasive strains in the Galleria mellonella model for systemic infection. A collagen-binding protein (Cnm) was found only in invasive strains, and inactivation of the cnm gene significantly impaired collagen and laminin binding capacity, abolished HCAEC invasion, and attenuated virulence in G. mellonella. Characterization of a gene encoding a putative glycosyltransferase (CsbB) co-transcribed with cnm suggested that CsbB is involved in Cnm glycosylation. Preliminary data using a rabbit endocarditis model suggested that expression of Cnm enables infection of the underlying endothelium by S. mutans whereas a cnm mutant was trapped in the heart valve vegetation. Collectively, our data suggest that the ability to invade an persist in the host cytoplasm is an important, previously unrecognized, virulence trait of S. mutans. Our working hypothesis is that S. mutans strains expressing Cnm have an enhanced capacity to colonize human tissues, and that this ability is a major virulence trait in systemic infections. The major goals of this application are to conduct the functional analysis of Cnm and to characterize the role of Cnm in host-bacteria interactions. To achieve these goals, we propose three well-integrated Specific Aims.
In Aim 1 (Molecular characterization of the cnm locus), we will (i) conduct the transcriptional characterization of the cnm operon, (ii) employ mutagenesis approaches to continue the characterization of the Cnm and CsbB, and (iii) identify the essential machinery for intracellular invasion using an heterologous host system.
In Aim 2 (Functional analysis of Cnm), we will (i) use recombinant variant forms of Cnm to identify the functional domains of Cnm, and (ii) determine whether Cnm is glycosylated and, if so, how Cnm is modified.
In Aim 3 (Streptococcus mutans-host interactions), we will (i) use in vitro models to assess Cnm-platelet interactions, (ii) use an ex vivo adhesion model to examine the ability of invasive and non-invasive strains to colonize heart tissues, and (iii) use a rabbit endocarditis model to establish the contribution of Cnm to the pathogenesis of IE and to test the efficacy of active immunization with Cnm to prevent S. mutans infections. This study has strong potential to be readily translated into clinical settings as Cnm could serve as a biomarker for detection of hypervirulent strains in patients in need to receive prophylactic treatment, or as a potential target for the development of effective strategies for prevention and treatment of S. mutans extra-oral infections.

Public Health Relevance

Streptococcus mutans is a major etiological agent associated with caries and a causative agent of infective endocarditis. Herein, we propose to investigate the contribution of a collagen binding protein (Cnm), a newly discovered virulence property of S. mutans, to heart diseases. This work will facilitate the discovery of new strategies that could be used to prevent or treat heart diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE022559-05
Application #
9024352
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2013-05-17
Project End
2018-03-31
Budget Start
2016-04-01
Budget End
2017-03-31
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Florida
Department
Dentistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Araújo Alves, Lívia; Ganguly, Tridib; Mattos-Graner, Renata O et al. (2018) CovR and VicRKX Regulate Transcription of the Collagen Binding Protein Cnm of Streptococcus mutans. J Bacteriol 200:
Avilés-Reyes, A; Freires, I A; Kajfasz, J K et al. (2018) Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans. Mol Oral Microbiol 33:257-269
Avilés-Reyes, Alejandro; Freires, Irlan Almeida; Besingi, Richard et al. (2018) Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins. Sci Rep 8:4705
Abranches, J; Zeng, L; Kajfasz, J K et al. (2018) Biology of Oral Streptococci. Microbiol Spectr 6:
Avilés-Reyes, A; Miller, J H; Lemos, J A et al. (2017) Collagen-binding proteins of Streptococcus mutans and related streptococci. Mol Oral Microbiol 32:89-106
Alves, Livia A; Harth-Chu, Erika N; Palma, Thais H et al. (2017) The two-component system VicRK regulates functions associated with Streptococcus mutans resistance to complement immunity. Mol Oral Microbiol 32:419-431
Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd et al. (2017) Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence. Virulence 8:18-29
Dodo, Cindy Goes; Meirelles, Luiz; Aviles-Reyes, Alejandro et al. (2017) Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis. Braz Dent J 28:428-434
Freires, Irlan Almeida; Lemos, José A; Abranches, Jacqueline (2017) A New Perspective of an Old Villain: Revisiting Biomarkers of Caries Development. EBioMedicine 25:14-15
Papadimitriou, Konstantinos; Alegría, Ángel; Bron, Peter A et al. (2016) Stress Physiology of Lactic Acid Bacteria. Microbiol Mol Biol Rev 80:837-90

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