A Pipeline for Structural Characterization of Protein Targets from Staphylococcus aureus and Acinetobacter baumannii
Healthcare-associated infections cause significant morbidity and mortality in the United States. The bacteria Staphylococcus aureus and Acinetobacter baumannii are both considered serious threats by the CDC due to multi-drug resistance, making any infection they cause difficult to remedy. These pathogens often infect immunosuppressed hospital patients which can result in pneumonia or bloodstream infections. Subsequently, it is vital that these organisms are studied to discover alternative treatments for resistant infections. By structurally analyzing proteins from these bacteria that may have a role in survival mechanisms, it is possible to establish important regions for protein function. To select our protein targets, we used multiple bioinformatic tools to analyze protein characteristics that could influence expression and crystallization. Initial recombinant protein expression trials identified two promising candidates for crystallization, HemA from S. aureus and HutC from A. baumannii. We have mutated several residues near the N-terminus of HemA, an enzyme involved in the heme biosynthesis pathway, to improve its stability. Additionally, we have optimized expression and purification of the transcriptional regulator HutC. Examining these two proteins in broad crystallization screens will reveal favorable conditions for crystal formation. Ultimately the information gathered from these structures will inform functional studies that may uncover downstream effects that these proteins have within the cell . Such knowledge could give us a better understanding of their function and may reveal new targets for the next generation of antimicrobials.