Cross-talk between the core and pathogen-specific genomes in Escherichia coli via a bacterial anti-activator protein

Mickey, Abigail, Microbiology - School of Medicine, University of Virginia
Nataro, James, Pediatrics, University of Virginia

Bacteria have evolved elegant and complex mechanisms to regulate gene expression and survive in diverse environmental conditions. Pathogenic bacteria are confronted by the compound challenges of both successful competition within their metabolic niche as well as the need to effectively orchestrate virulence gene expression at the right time and place. The regulatory complexities are still being deciphered for a large number of pathogens, and new, highly nuanced systems are being illuminated. This dissertation describes regulatory features of diarrheagenic Escherichia coli heretofore unknown.
Several Gram-negative pathogens colonize and compete in certain niches by acquiring new genes that are characterized by their high AT content (compared with the AT content of E. coli, which is ca. 50%). Although this aberrant AT speaks of exogenous ancestry, the maintenance of such loci permits concerted expression in part by virtue of proteins that bind such AT-rich segments. Diarrheagenic E. coli have thus used the histone-like structuring protein, H-NS, which binds and silences AT-rich genes. Exactly how H-NS is harnessed, and more broadly, how these pathogens both regulate cross-talk between acquired genes and the core E. coli genome as well as control the switch between nonpathogenic and pathogenic lifestyles, are subjects of intense and fascinating investigation.
Enteroaggregative Escherichia coli (EAEC), a common diarrheagenic E. coli pathotype, utilizes a member of the AraC family of transcriptional regulators, AggR, to regulate the expression of virulence genes which are necessary for host colonization and virulence. In addition to controlling/promoting expression of virulence factors, AggR also regulates the expression of its own negative regulator, aar, which encodes a small protein that binds in vitro to the dimerization site of AggR and thus blocks AggR function. Recent transcriptomic profiling of EAEC strain 042 with and without functional aar revealed that core genes outside of the AggR regulon are also regulated by Aar (perhaps via AggR, or independently). One of the non-AggR-regulated genes encoded the global regulator H-NS. Moreover, published data from our research group suggest that Aar directly binds to H-NS and effects H-NS repression.
In this dissertation, I identify the functional significance of both Aar/AggR and Aar/H-NS binding events in the pathogenesis of EAEC. I propose a novel role for Aar that includes acting as an anti-activator of AggR and an anti-repressor of H-NS. My data suggest that during the initial steps of pathogenesis, Aar removes H-NS silencing at AggR-regulated AT-rich genes, thereby allowing unobstructed activation of these genes by AggR. Then, as the concentration of Aar increases, Aar binds to AggR and prevents further gene activation. In addition, I demonstrate that the expression of aar affects bacterial fitness in vivo, and I speculate that this occurs through modulation of aggR and hns expression. In summary, my work extends the characterization of a novel, but common, regulatory protein that regulates both virulence and core metabolic genes, and provides new insights into pathogenic gene regulation.

PHD (Doctor of Philosophy)
Escherichia coli, anti-activator, ANR, AggR, anti-repressor, H-NS
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