REGULATIONS OF IRON ACQUISITATION BY S AUREUS

Illinois State University, Normal IL

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Abstract

DESCRIPTION (Adapted from the Applicant's Abstract): Staphylococcus aureus is a major cause of human diseases, ranging from basic skin abscesses to complicated life-threatening diseases such as toxic shock syndrome, pneumonia, septicemia, and infective endocarditis. The acquisition of broad antibiotic resistance in this bacterium, especially the methicillin and vancomycin-resistant S. aureus strains, poses a major threat to public health. The virulence factors involved in the pathogenicity of S. aureus have been extensively studied, but the molecular mechanisms governing S. aureus invasion, colonization, and survival within the host environment remains largely unknown. Various stress conditions within the host are proposed to simulate the expression of virulence genes in S. aureus. Iron, which has been shown to be an important virulence determinant and required for many vital cellular processes, is not readily available for bacterial cells in the animal host. To cope with an iron-deficient host environment, the bacteria have evolved several mechanisms of iron acquisition and its regulation. However, these pathways are poorly understood in S. aureus. The PI has recently cloned and partially characterized a S. aureus chromosomal gene, fur, that shows homology to the ferric-uptake regulatory genes of other bacteria. The fur gene codes for a protein that binds to a specific DNA sequence in the promoter region of at least two genes, sirA and fhu. The long-term goal of this project is to investigate how Fur regulates iron acquisition in S. aureus and its role in the regulation of the expression of virulence genes. The specific objectives of this study are 1) cloning and molecular analysis of fur, 2) construction of a mutation in fur and determination of its effect on pathogenicity, 3) molecular analysis of Fur, and 4) characterization of the Fur regulated iron uptake operon, fhu of S. aureus. To accomplish this first objective, the PI has cloned fur which will be overexpressed and the protein purified to conduct gel-shift and footprint assays. Northern blots will be done to determine the mechanism of fur regulation. To accomplish the second objective, Fur- mutants will be generated by site-directed mutagenesis and the effect of the mutation on pathogenicity will be determined using a rat model of endocarditis. The Fur- mutant and the parent will be used to identify Fur-regulated genes in an in vitro cycle selection procedure or alternatively by 2-D gel electrophoresis to accomplish the third objective. The fourth objective will be accomplished by sequencing the fhu operon, followed by the construction of a mutation in each reading frame to determine their roles in the pathogenicity and uptake of iron. The results of this study may identify novel targets that interfere with iron uptake and its regulation and may lead to alternative therapies for staphylococcal infections.