Analysis of the AgrA-mediated transcription regulation in Listeria monocytogenes

About This Grant

Abstract. Listeria monocytogenes is a low G+C Gram-positive bacterium that can cause severe disease in immunocompromised individuals, pregnant women, newborns, and the elderly. Pathogenic strains frequently use two-component regulatory systems to control the timing and level of virulence gene expression. In L. monocytogenes, a quorum-sensing-like two-component system, AgrCA, is involved in virulence, biofilm development, and adaptation to environment. It governs expression of dozens of listerial genes. The agr locus is highly expressed in laboratory media and during infection, and AgrA is, apparently, one of the most abundant transcriptional regulators in listerial cells. The functions of most AgrA-regulated genes and the mechanisms by which AgrA affects virulence and biofilm development remain unknown. Importantly, it is not even known which genes are directly regulated by AgrA and under which conditions the regulation occurs. We propose to determine the entire set of genes that are able to interact with AgrA directly either in vitro or in vivo. Using a novel approach, in vitro DNA affinity purification coupled with sequencing, we are able to visualize protein-binding sites on a genome-wide basis at near-single-nucleotide resolution and rank the sites according to their relative strengths. We now achieve similar, near-single-nucleotide resolution in ChIP- Seq experiments, which will be used to define sites that interact with AgrA in vivo. The precise information on the locations and relative strengths of AgrA-binding sites will provide the foundation for future research on the detailed mechanisms by which AgrA regulates individual genes. We have shown that the L. monocytogenes agr locus is the strongest target of regulation by CodY, a global transcriptional regulator of metabolism and virulence in most low G+C Gram-positive bacteria. CodY- mediated regulation of the agr locus is positive and direct. The agr locus is also subject to a very strong AgrA- mediated positive autoregulation, and requires binding of another transcriptional regulator, MouR, for its expression. The requirement for three direct positive regulators to achieve expression of a single locus above the low basal level is very unusual. The mechanisms of this triple regulation have not yet been determined. To begin unraveling this complex regulation, we will determine the modes of interaction of AgrA, CodY, and MouR with their binding sites and establish whether CodY and/or MouR are required for AgrA binding and activity at the agr locus and other AgrA-regulated genes. DNase I footprinting, high-resolution ChIP-Seq experiments, and transcription assays with purified proteins will be used as in vitro and in vivo approaches. L. monocytogenes is one of the deadliest foodborne pathogens in the United States. By revealing the mechanisms of transcriptional regulation of the agr locus and identifying the full complement of direct AgrA targets, we will be able to provide novel insight into how L. monocytogenes senses its environment and uses that information to modulate its physiology and the level of virulence.