Dissecting biofilm formation and metabolite sensing in diabetic urine
Urinary tract infections (UTIs) are frequent, have a high degree of recurrence and are difficult to treat. Patients with diabetes have an elevated risk of developing UTI. Diabetics also excrete higher amounts of certain amino acids, including serine, in their urine, as well as 2-3 times more pyruvate than non-diabetics. Uropathogenic Escherichia coli (UPEC), the primary causative agent of community-acquired and catheter-associated urinary tract infections (UTIs) form extracellular and intracellular biofilms during infection. A self-produced extracellular matrix that is virtually impenetrable to antibiotics encases biofilm-associated bacteria. Combined with the steady rise in antibiotic-resistant UPEC strains, biofilm formation presents a serious health problem, especially in the case of diabetic individuals. We have recently identified the presence of two signal transduction networks, YehUT and YpdAB that respond to serine and pyruvate changes in UPEC and have determined that the genes activated by YehUT and YpdAB in response to serine and pyruvate, are significantly induced during acute bladder infection. Hypothesis: Biofilm organization, which contributes to the resilience of pathogenic biofilms, is controlled by environmental stimuli such as altered nutritional cues in the urine. We hypothesize that UPEC senses and responds to altered nutritional cues in the diabetic urine to form biofilms with different composition that enhance pathogenesis in diabetics. Aims/Experimental methods: We will combine classical genetics approaches with the technology of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and a custom-built biofilm reactor that will enable us to scrutinize biofilm formation by 3 UPEC strains during growth in diabetic and non-diabetic urine. Our goal is to identify biofilm factors and pathways that are distinctly expressed or repressed in diabetic urine and could contribute to enhanced pathogenesis in diabetics (Aim 1). Aim 2 will elucidate the molecular mechanisms underlying the activation of two bacterial pyruvate sensors in response to diabetic urine and will test the fitness potential of deletion mutants devoid of these sensors in a diabetic murine model of acute and chronic UTI.Combined with the well-established murine models of UTI, the impressive mass spectrometry facilities and other resources at Vanderbilt University, as well as the inter-disciplinary expertise of our collaborators, we are confident that we will elucidate UPEC biofilm signatures and regulatory pathways that are critical in enhancing pathogenesis in the diabetic population. Our long-term goal is to leverage the outcomes of these studies to develop better UTI therapies in diabetic and non-diabetic hosts.

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