The making of a Superbug. Understanding the mechanisms of high level antibiotic resistance in Methicillin Resistant Staphylococcus aureus (MRSA)

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

Background Information:
Antibiotic resistance is a global challenge to modern human healthcare. Methicillin Resistant Staphylococcus aureus (MRSA) is endemic in many countries with huge associated human and financial costs. If we are to effectively develop new control regimes these must be based on knowledge of the pathogen and how it interacts with the host. The molecular basis of MRSA is via the acquisition of a novel Penicillin Binding Protein (MecA) that shows a low affinity for the range of beta-lactam antibiotics. MecA can substitute for the native PBPs and allow bacterial cell wall synthesis in the presence of drug. Extraordinarily we understand very little as to how MecA fulfills its function and can act with the rest of the cell wall synthesis machinery. The supervisory team have discovered that the presence of MecA in S. aureus does not itself lead to high level AMR but stresses the cells. This paradox that is resolved by the bacteria acquiring mutations in the genes encoding RNA polymerase with a concomitant massive increase in AMR. RNA polymerase has been isolated from the parental and high level AMR strains. This has revealed alterations to overall RNA polymerase properties. This background data has established a firm framework for the proposed project bringing together expertise in molecular microbiology and host:pathogen interaction with protein biochemistry and structural biology to address a problem of great societal importance.

Experimental Approach:
1. How does MecA work?
MecA itself leads to metabolic stress and only gives high level resistance in rpo backgrounds. Molecular interactions of MecA with other proteins will be determined by bacterial 2 hybrid analysis, fluorescence microscopy and protein analysis. A combination of biochemical (HPLC) and biophysical (AFM force curves) will elucidate the effect of AMR on cell wall properties.
2. What is the role of RNA polymerase in high level AMR?
We have shown, using RNA-seq that the rpo mutations lead to changes in expression of a set of genes involved in metabolism. Our hypothesis is that AMR is controlled by a sensing mechanism. We will purify RNA polymerase and determine the effect of the mutations on signal transduction, using a combination of protein chemistry and transcriptional assays.
3. What are the fitness costs and the role of the host environment?
For the first time, we have a defined set of strains only carrying the mecA and rpo mutations and thus can be directly compared in models of disease with and without antibiotic intervention to determine the cost of AMR in vivo. Importantly we have defined that high-level AMR is linked to the ability to grow anaerobically. However, the effect of antibiotics has never been routinely tested under this in vivo relevant condition. The role of anaerobiosis drug efficacy will be tested.

Training:
The project will provide an exciting and interdisciplinary training spanning from molecular genetics, protein chemistry, advanced microscopy approaches, biophysics through to in vivo analysis. The studentship will be set within a wider initiative in Sheffield (http://www.floreyinstitute.com) that brings together researchers from the basic science to clinical application to address the challenge of AMR.

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