Structure-based Functional Studies of Resuscitation Promoting Factors (Rpfs) of Pathogenic Bacteria

The bacteria that cause tuberculosis are able to lie dormant in the lungs for decades and only start to actively divide again when an infected patient is weakened by age or disease. We are studying proteins that are a crucial part of the reactivation of the dormant infection. We have recently proposed that these proteins work through digestion of some component of its own cell wall. The current project will identify exactly what this component is and work out how it turns on the growth of the bacteria. This is potentially useful as if tuberculosis can be prevented from going dormant in a healthy individual the infection can be totally cleared by drugs and the immune system. Using the results of recent genomic sequencing projects we have identified that similar proteins are found in another group of bacteria that include the so called ?superbug?, multidrug-resistant Staphylococcus aureus, which is responsible for a large number of serious hospital acquired infections. The life cycle of these bacteria are different and we want to study what role these proteins have in controlling the growth of this serious infection, which again may provide a novel means of treatment.
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Resuscitation promoting factors (Rpfs) are found in high G+C bacteria including the human pathogens M.tuberculosis and M. leprae. Rpfs promote the growth of dormant cultures of bacteria. We have recently shown that the conserved core of these proteins resembles lysozyme including the potentially catalytic glutamate residue. Building on our recent NMR structure and the crystal structure that we expect shortly, we wish to identfiy the biochemical mechanism of action of these proteins. We will isolate and identify the substrates of the Rpfs, using HPLC and mass spectrometry and test whether the products released are second messengers that stimulate bacterial growth. This will be coupled to site directed mutagenesis of the active site and in silico binding studies to understand the biochemical mechanism of action. We will then go on to identify the receptor whereby the extracellular activity is transmitted to growth of the cell. We will also investigate a homologous but little studied group of proteins in the Firmicutes, including Staphylococcus aureus the major source of hospital caught infection in the UK.