This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Staphylococcus aureus is a Gram-positive pathogen that causes a wide variety of diseases in humans and animals. It is a leading cause of hospital acquired infections, costing the United States more than 4.5 billion dollars annually. The expression of most S. aureus virulence factors is determined by the growth phase.The exponential phase of growth is characterized by the production of cell associated adhesion factors (e.g., fibronectin binding protein) while the post-exponential phase of growth is distinguished by the production of secreted virulence factors (e.g., alpha-toxin). Concomitant with the entry into the post-exponential phase of growth is an increase in tricarboxylic acid (TCA) cycle activity. Aconitase is a TCA cycle enzyme that converts citrate to isocitrate. Eukaryotic organisms have mitochondrial and cytoplasmic aconitase activity. The cytoplasmic aconitase activity is caused by the iron-responsive protein-1 (IRP-1), an mRNA-binding protein that posttranscriptionally regulates the synthesis of iron-regulated proteins. Thus, cytosolic aconitase is a bifunctional protein. Recently, it has been demonstrated that aconitase from Bacillus subtilis and Escherichia coli bind to sequence specific-elements in mRNAs in an iron-dependent fashion. These observations established bacterial aconitase, like eukaryotic cytosolic aconitase/IRP-1, as a bifunctional protein. Inactivation of the sole S. aureus aconitase gene (acnA/citB), significantly decreases virulence factor synthesis, alters host-pathogen interaction, and enhances stationary phase survival. The affects of aconitase inactivation can be the result of a metabolic block in the TCA cycle, the loss of regulatory function, or a combination of the two. We propose to determine which affects of aconitase inactivation are due to a metabolic defect and which are due to the loss of regulatory function. Additionally, we will determine if aconitase will bind to the cognate mRNAs of genes identified as potentially regulated by aconitase. [Lay Summary] Bacteria are single celled organisms that 'eat' and divide; therefore, everything bacteria do is linked to eating and dividing, including causing disease in humans. A fundamental understanding of how bacteria regulate pathogenesis in response to nutrient limitation is critical to developing new treatments designed to kill bacteria. The work contained within this proposal is a first step in designing new strategies to combat bacterial infections.
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