Liskin Swint-Kruse photoLiskin Swint-Kruse (2014-2015)
Associate Professor, Department of Biochemistry and Molecular Biology
University of Kansas Medical Center

The Cra-FruK complex alters regulation of central metabolism of gamma-proteobacteria

By illuminating the functional significance of the Cra-FruK interaction, results will identify new ways to perturb central metabolism in gamma-proteobacteria, which might be exploited to target a select group of enteric bacteria. After years of complacency, studies of bacteria are now recognized as critical: Antibiotic resistance is a growing problem, and we are beginning to appreciate microbiome contributions to healthy and non-healthy host states.

For γ-proteobacteria (including E. coli, Shigella, Enterobacter, Salmonella, Klebsiella, and Yersinia), several key processes are affected by the Catabolite Repressor Activator protein (“Cra”). The normal activity of Cra is to regulate many genes of central metabolism via binding to unique DNA sites. Cra-DNA binding is alleviated by binding to inducer fructose-1-phosphate (F1P). Since F1P is the first metabolite in fructose catabolism, Cra induction provides is one means by which enteric γ-proteobacteria respond to changes in host diet. In addition, deletion experiments show that Cra is necessary for pathogenesis in several human pathogens; bacteria with mis-regulated central metabolism are ineffective pathogens.

The Cra regulon includes the enzyme 1-phosphofructokinase (FruK). The known role of FruK is to convert F1P to the next compound in fructose metabolism, which concomitantly re-establishes DNA binding by Cra. In addition to their relationship through F1P, we recently identified a direct protein-protein interaction between E. coli Cra and FruK. We hypothesize that this direct interaction plays a previously undetected role in regulating bacterial metabolism. This interaction appears to be unique to γ-proteobacteria (other bacteria lack Cra orthologs) and thus might be exploited to target a select group of pathogens. We propose experi-ments to determine the impact of Cra-FruK complex formation on central metabolism.

First, we hypothesize that complex formation alters both Cra-DNA binding and FruK catalysis of F1P. These hypotheses are supported by preliminary data from DNA binding, enzyme catalysis, and size exclusion measurements; the latter were performed and will be completed using the PPG Core Facility. Results from proposed experiments will complete the work needed for our first manuscript, which appears to be required for a successful R01 application. Second, we will identify the interaction interface of Cra and FruK via crystallography experiments performed in the PSL Core Facility. Among other outcomes, this interface could be targeted for drug binding, for which the COBRE-PSF Fragment Library Screen could be used. Long term, proteins with interface mutations will be used in animal/cell models of virulence.