Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Demuth, Donald R.

Author's Keywords

Quorum sensing; A1-2; Aggregatibacter actinomycetemcomitans.

Subject

Quorum sensing (Microbiology); Cellular signal transduction; Bacterial genetics

Abstract

Quorum sensing in the oral pathogen Aggregatibacter actinomycetemcomitans is dependent upon the soluble signaling molecule AI-2, but it is not known how the initial detection of AI-2 is coupled to the downstream regulation of gene expression that confers complex phenotypes such as biofilm formation and iron acquisition. Here we show that expression of a two-component system encoded by qseBC is induced by AI-2 in A. actinomycetemcomitans and that induction of qseBC requires the AI-2 receptors, LsrB and/or RbsB. Inactivation of the sensor kinase gene qseC resulted in a significant reduction of in vitro biofilm growth and in vivo virulence using a murine model of periodontitis. We also show that qseBC regulates the expression of several operons encoding iron acquisition pathways, consistent with previous studies showing that AI-2 regulates iron uptake in A. actinomycetemcomitans. However, some AI-2 regulated iron uptake operons were not controlled by QseBC, suggesting that only a subset of iron acquisition systems are co-regulated by AI-2 and QseBC. Interestingly, the toxin-antitoxin system mqsRA is involved in AI-2 and QseBCdependent regulation of biofilm growth of some strains of Escherichia coli. The genome sequence of A. actinomycetemcomitans was searched and open reading frames Aa00672 and Aa00673 were shown to exhibit sequence similarity to MqsRA and MazEF, two toxin-antitoxin systems encoding mRNA interferases. Aa00673 was shown by RT-PCR to be co-expressed with Aa00672 and the expression of Aa00672-Aa00673 was decreased in A. actinomycetemcomitans strains lacking functional AI-2-receptors or QseC, suggesting that Aa00672-Aa00673 functions downstream from the AI-2 receptors and QseBC in the AI-2 response circuit of A. actinomycetemcomitans. We also demonstrate that Aa00673 encodes a toxin, which causes reversible growth inhibition and may function as an RNase; whereas Aa00672 encodes its cognate antitoxin, negating the effects of the toxin. Thus, our results suggest that the QseBC two-component system is part of the AI-2 regulon and in concert with Aa00673, a putative paralog of MqsR, link the detection of AI-2 to the regulation of genes controlling biofilm formation, iron acquisition, and in vivo virulence of A. actinomycetemcomitans.

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