Date on Master's Thesis/Doctoral Dissertation

5-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Demuth, Donald

Committee Co-Chair (if applicable)

Graham, James E.

Committee Member

Lawrenz, Matthew

Committee Member

Chung, Donghoon

Author's Keywords

aggregatibacter actinomycetemcomitans; type II toxin anti-systems; environmental stress

Abstract

Microbes express many protective mechanisms in response to environmental stress. Toxin/anti-toxin systems encode a biologically active toxin and a labile anti-toxin that inhibits the toxin’s activity. These systems are known to contribute to persister cell and biofilm formation. A. actinomycetemcomitans thrives in the complex oral microbial community and is subjected to continual environmental flux. Little is known regarding the presence and function of TA systems in this organism or their contribution survival in the oral environment. Using BLAST searches and other informatics tools, we identified 11 intact TA systems that are conserved across all seven serotypes of A. actinomycetemcomitans and represent the RelBE, MazEF and HipAB families of TA systems. The A. actinomycetemcomitans TA systems identified selectively responded to various environmental conditions that exist in the oral cavity. Transcription of two putative RelBE-like TA systems, D11S_1194-1195 and D11S_1718-1719, were induced in response to low pH, and were selected for further study. Deletion of D11S_1718-1719 significantly reduced metabolic activity of stationary phase A. actinomycetemcomitans cells during prolonged exposure to acidic conditions. The mutant also exhibited reduced biofilm biomass when cultured under acidic conditions. The D11S_1194 and D11S_1718 toxins inhibited in vitro translation of dihydrofolate reductase (DHFR), and degraded ribosome-associated, but not free mRNA. In contrast, the corresponding antitoxins or equimolar mixtures of toxin and antitoxin had no effect on DHFR production or RNA degradation. Preliminary results comparing the proteomes of acid stressed mutants to the acid stressed wild-type suggest that metabolism proteins are the most affected by these two TA systems. Among these, proteins involved in nucleotide metabolism are largely over-represented in the mutants. Other identified proteins are directly involved in quorum-sensing, iron transport and virulence (e.g. leukotoxin). Results of these studies indicate that the anti-toxin proteins inhibit the activity of the corresponding toxins and suggest that D11S_1194-1195 and D11S_1718-1719 are RelBE-like type II TA systems that are activated under acidic conditions. The toxins of both systems may function to cleave ribosome-associated mRNA to inhibit translation in A. actinomycetemcomitans.

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