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Tricarboxylic Acid Cycle Mediated Regulation of Staphylococcal Biofilm Formation

Somerville, Greg
University of Nebraska - Lincoln
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Staphylococcus aureus and Staphylococcus epidermidis are the leading causes of hospital-associated infections in the United States. Frequently, these hospital-associated infections involve the formation of a biofilm on an indwelling medical device. A biofilm is a complex aggregation of bacteria encapsulated by an adhesive exopolysaccharide matrix. This exopolysaccharide matrix provides structural stability to the biofilm, enhanced adhesion to surfaces, and protection from host defenses and antibiotics.

Our lab recently demonstrated that the staphylococcal exopolysaccharide matrix is synthesized when the tricarboxylic acid cycle is repressed. Of importance, tricarboxylic acid cycle activity is regulated by the availability of nutrients, oxygen, and iron. The linkage of tricarboxylic acid cycle activity and exopolysaccharide synthesis and the susceptibility of the tricarboxylic acid cycle to environmental inactivation led us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in tricarboxylic acid cycle activity. Thus, the central hypothesis of this application is that the tricarboxylic acid cycle acts as a novel signal transduction pathway to translate external stimuli/conditions into intracellular signals (e.g., NADH, ATP, aplha-ketoglutarate) that can stimulate or repress the activity of regulatory proteins.

The goal of this proposal is to identify tricarboxylic acid cycle associated metabolites that regulate exopolysaccharide synthesis. The identification of small molecules that regulate biofilm formation will provide a starting point for the design of novel compounds to prevent biofilm formation.

The long-term goal of this research is to determine how staphylococci perceive external environmental conditions to regulate adaptation to the host environment and to develop therapeutic strategies to disrupt bacterial adaptation to the host.

Potential Impact: Approximately 50,000 to 120,000 persons develop a hospital-acquired bacteremia annually due to vascular catheterization; staphylococci account for nearly 50% of these infections. The pathogenesis of these biomaterial-related infections is directly linked to the ability of staphylococci to produce of PIA and form a biofilm. Identification of TCA cycle associated metabolites that regulate PIA synthesis is the first step in designing drugs to interfere with this synthesis.

Due to the prevalence of staphylococcal biofilms in indwelling medical device associated infections, development of therapies to treat or prevent these infections would enhance the health of Nebraska patients and reduce the associated health care costs. If this research can be extrapolated to industrially important bacteria, then this work could represent a major economic boost to Nebraska industries (e.g., ethanol production facilities) and provide a stimulus for the development of new industries within Nebraska.

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Non-Technical Summary: Bacterial growth occurs in one of two states: free-floating or attached to a surface (biofilm state). Biofilms are a complex aggregation of bacteria encapsulated in an adhesive exopolysaccharide (an excreted sugar polymer) matrix. These biofilms can be beneficial as in the case of ?microbial fuel cells? or deleterious, such as biofouling on the hulls of naval vessels; contamination of ethanol fuel production fermentors; biocorrosion of metals in the medical and food industries; dental caries; and bacterial infections associated with indwelling medical devices (e.g., catheters and artificial heart valves). The economic impact of biofilms in the United States has been estimated to be between several billion dollars to greater than 100 billion dollars annually due to equipment damage, product contamination, energy losses, and increased health care costs. Although bacterial biofilms negatively affect many major industries in Nebraska and the USA, our primary goal is in enhancing the health and well-being of Nebraskans by understanding and controlling staphylococcal biofilm-associated infections on indwelling medical devices. In support of this goal, we will identify the metabolic signals that mediate biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis. The identification of small molecules that regulate biofilm formation will provide a starting point for the design of novel compounds to prevent biofilm formation and biofilm associated infections.

Approach: PIA synthesis is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. To determine the metabolic changes that correspond with increased PIA synthesis, we will grow Staphylococcus aureus and S. epidermidis in conditions known to enhance PIA synthesis (e.g., in with 4% ethanol, low-oxygen, or low-iron), harvest the bacteria, isolate the cytosolic fractions, and analyze the metabolic status using NMR metabolomics (metabolomics is the study of the chemical patterns generated by cellular processes, like PIA synthesis.) In addition, we will determine the amount of cell-associated PIA produced as previously described. We have developed the differential NMR metabolomics technology as a systems biology tool. The approach enables us to follow functional changes by monitoring perturbations in the bacterial metabolome, where changes in the relative concentration of metabolites occur as a direct result of a modification in protein activity. Basically, NMR spectrum obtained from similar metabolomic data will cluster together in a 2D plot (2D scores plot). Therefore, an important component of the technique is the simultaneous preparation of reference cultures (untreated) and test cultures (e.g., 4% ethanol.) Test systems are bacterial cultures grown under conditions that perturb the natural state of the bacteria, such as mutations, addition of a drug, or changes in growth media or temperature. Briefly, ten reference and ten test cultures are prepared and grown to a uniform optical density, the bacteria are harvested by centrifugation, lysed, and suspended in a phosphate buffer. The cell debris is removed by centrifugation and the cell-free lysate is transferred to an NMR tube. A standard 1D 1H NMR spectrum is collected for each sample on a Bruker 500 MHz NMR spectrometer equipped with a cryoprobe, sample changer and software to automate the data collection. The NMR spectrum provides a "snap-shot" of the state of the bacteria, where each metabolite is represented by peaks in the NMR spectra and intensity is proportional to concentration. Major variations in the NMR spectra are analyzed using principal component analysis (PCA), a well-established statistical tool that transforms a multidimensional data set (NMR spectra) into a single-point in PC-space. The relative location of an NMR spectrum depends on the number and intensity of peaks, where spectra collected from cell-free lysates with similar metabolite composition will have a similar peak pattern and will therefore cluster together in a 2D scores plot. The metabolites associated with peaks from the spectra of cell-free lysates that cluster together will be correlated with the PIA data to determine if a correlation exists. To determine if a correlation is valid, we will incubate bacteria with an excess of the metabolite and examine the effect on PIA accumulation.

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Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens