Data from clinical and basic research have implicated Streptococcus mutans as the primary microbial etiologic agent of dental caries, one of the most common infectious diseases afflicting humans. The virulence of S. mutans resides in three core attributes;its abilities to adhere and form biofilms on tooth surfaces, to produce large quantities of organic acids from a wide range of carbohydrates, and to tolerate environmental stresses, particularly low pH. Because stress tolerance is intertwined with S. mutans virulence, the dissection of the mechanisms that allow these bacteria to thrive in oral biofilms during stressful conditions is central for a complete understanding of the pathogenesis of dental caries. In Gram-positive bacteria, energy-dependent proteases, in particular the ClpXP system, are central for stress tolerance and have been implicated in bacterial pathogenesis. However, a clear picture of the biological role of Clp proteases in pathogenesis has yet to emerge. Our recent work revealed that ClpXP plays an important role in the expression of key virulence attributes of S. mutans, including biofilm formation, cell viability and acid tolerance. In Bacillus subtilis, the global regulator Spx is a substrate of ClpXP, and accumulation of Spx has been correlated to phenotypes of ?clpXP strains. We have demonstrated that inactivation of spx genes (herein designated spxA and spxB) restored many phenotypes observed in the S. mutans ?clpXP strains. Thus, the underlying mechanisms by which ClpXP affects virulence traits in S. mutans are intimately associated with accumulation of Spx. Despite the linkage of Clp proteolysis with bacterial virulence, and the close association of Spx accumulation with phenotypes resulting from clpP and clpX mutations, the effects of global regulation by Spx in pathogenic bacteria remains to be explored. Our working hypothesis is that Clp proteolytic control of the Spx regulator plays a critical role in processes underlying S. mutans pathogenesis. Therefore, this application focuses on the characterization of the SpxAB global regulators, and their role in controlling virulence expression in S. mutans. To prove our hypothesis, three Specific Aims are proposed.
In Aim 1, we will dissect the transcriptional and post-translational mechanisms regulating Spx levels, which will provide additional insights into the molecular and physiologic significance of the interactions between Clp proteases and Spx.
In Aim 2, a thorough in vitro and in vivo characterization of the ?clpXP and ?spx mutant strains will be conducted in order to disclose the significance of Spx regulation in processes underlying pathogenesis. Finally, in Aim 3 whole genomic microarrays will be used to identify the genetic network under Spx control and, when analyzed in conjunction with Aims 1 and 2, to identify new genes that participate in the expression of virulence. Ultimately, the advances from this proposal will help the identification of proteins and signaling pathways that could be used for the development of novel therapeutic agents to prevent dental caries.

Public Health Relevance

Streptococcus mutans is considered the primary causative agent of human dental caries, and is often implicated in cases of endocarditis, a life-threatening inflammation of the heart valves. The objective of this study is to understand the mechanisms used by S. mutans to adhere to the tooth, produce acids, and survive acidic conditions in the oral cavity. Our studies will facilitate the discovery of new products that could be used to reduce or eliminate caries.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE019783-03
Application #
8211850
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2010-02-05
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
3
Fiscal Year
2012
Total Cost
$382,388
Indirect Cost
$134,888
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Baker, J L; Derr, A M; Karuppaiah, K et al. (2014) Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels. J Bacteriol 196:2166-77
Lemos, Jose A; Quivey Jr, Robert G; Koo, Hyun et al. (2013) Streptococcus mutans: a new Gram-positive paradigm? Microbiology 159:436-45
Kajfasz, Jessica K; Mendoza, Jorge E; Gaca, Anthony O et al. (2012) The Spx regulator modulates stress responses and virulence in Enterococcus faecalis. Infect Immun 80:2265-75
Kajfasz, Jessica K; Abranches, Jacqueline; Lemos, Jose A (2011) Transcriptome analysis reveals that ClpXP proteolysis controls key virulence properties of Streptococcus mutans. Microbiology 157:2880-90
Kajfasz, Jessica K; Rivera-Ramos, Isamar; Abranches, Jacqueline et al. (2010) Two Spx proteins modulate stress tolerance, survival, and virulence in Streptococcus mutans. J Bacteriol 192:2546-56