Date on Master's Thesis/Doctoral Dissertation

12-2015

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)

Alard, Pascal

Committee Member

Scott, David

Committee Member

Graham, James

Committee Member

Lawrenz, Matthew

Author's Keywords

two-component systems; catecholamines; anaerobic metabolism; toxin/anti-toxin systems; biofilms

Abstract

In order for a pathogen to successfully colonize the host, it must be able to acquire essential nutrients and regulate gene expression to respond to environmental fluctuations. One mechanism bacteria have evolved in order to detect these fluctuations and respond is the two component signal transduction systems (TCS). Aggregatibacter actinomycetemcomitans, a dental commensal associated with localized aggressive periodontitis, contains a TCS designated QseBC that was previously shown to regulate expression of several iron acquisition genes, biofilm formation and virulence, but the activating signal was unknown []. QseBC is a conserved TCS in the Enterobacteriaceae and Pasteurellaceae, but the activating signal for QseC varies for each species [125]. The QseC in Escherichia coli responds to catecholamines whereas the QseC homolog (FirS) in Haemophilus influenzae responds to ferrous iron. Here we show that the activating signal for the A. actinomycetemcomitans QseC is a combination of iron and catecholamines (CAT-Fe). The sensing of the CAT-Fe complex leads to a significant stimulation of biofilm and planktonic growth, as well as induction of genes involved in anaerobic metabolism, electron transport and oxidative stress while down-regulating expression of genes involved in iron acquisition and fatty acid and LPS biosynthesis. Using a combination of RNA analysis and metabolomics, we identified an increased flux through the TCA cycle to pyruvate and then to anaerobic respiration and metabolism with exposure to CAT-Fe. Cells exposed to Cat-Fe also expressed higher levels of catalase and were more resistant than untreated cells to oxidative stress. Finally, QseB also appears to play a role in regulating the general stress response in A. actinomycetemcomitans since a ΔqseB mutant exhibits increased transcription of several stress response genes, including the Lon protease. Ectopic expression of lon resulted in an increase in transcription of ygiW-qseBC. Lon is involved in activating type II toxin/anti-toxin (TA) systems, one of which (D11S_2133-2134) was shown to be highly up-regulated during iron starvation. Further, it was found that ectopic expression of the putative anti-toxin D11S_2134 resulted in a significant increase in transcription of qseBC. Based on this data, it is likely that D11S_2133-2134 regulates expression of qseBC allowing QseBC and D11S_2133-2134 to tightly control iron acquisition and regulate adaptation to the host.

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