Characterisation of the bacterial proteins YjeE, YeaZ and YgjD and evaluation as a potential novel antimicrobial target
Lead Research Organisation:
University of Dundee
Department Name: Molecular Medicine
Abstract
Bacteria have a number of proteins that are not found in any other form of life, and some of these have been shown to be essential for bacteria to survive. This project aims to explore the way a set of three essential bacterial proteins interact with each other and to try to understand the critical roles that they play within the bacterium. Armed with this knowledge we would then like to discover small molecules that disrupt these processes, which might be ultimately developed as new drugs to treat bacterial infections.
Antibiotic resistance in bacteria is increasing, and with it the number of hard-to-treat infections also increases. The network of proteins we will investigate is not attacked by the antibiotic agents we currently use, and so may be a potential target for new antibiotic agents that may be effective when the bacteria are resistant to out current antibiotics.
Antibiotic resistance in bacteria is increasing, and with it the number of hard-to-treat infections also increases. The network of proteins we will investigate is not attacked by the antibiotic agents we currently use, and so may be a potential target for new antibiotic agents that may be effective when the bacteria are resistant to out current antibiotics.
Technical Summary
The conserved bacterial proteins YjeE, YeaZ and YgjD form an interacting network, and have been shown to be generally essential for bacterial viability. YjeE and YeaZ are found only in eubacteria, whilst the YgjD protein has orthologues in all other branches of life. The essential nature of these proteins, coupled with the fact that two of them are found only in bacteria, makes the pathway a potential target for antimicrobial chemotherapy. The aims of this project are to explore the interactions between the proteins and to investigate possible agents to disrupt the functions of one or more components of this pathway.
Objectives
A two-stranded research project is proposed. Firstly to explore the interactions between the proteins YjeE, YeaZ and YgjD to identify preferential partnerships under different conditions; to investigate potential protease and DNA binding activities; and to determine the structure of one or more of the different partner complexes. The second stage of the project will involve the screening of a compound library for agents that impair the function of one or more proteins in the network.
Methodology
Plasmids will be constructed carrying the genes for the YjeE, YeaZ and YgjD proteins labelled with split yellow fluorescent protein, which will fluoresce when two labelled proteins interact. The individual components will be labelled in pairs to establish preferentially pairing under a variety of culture conditions (including pH, osmolarity, oxygen tension, and different chemical sources).
Electrophoretic mobility shift assays will be used to investigate the DNA binding of YeaZ and YgjD to DNA, either in isolation or in partnership.
The stability YjeE/YeaZ and YgjD/YeaZ complexes will be probed, for example to determine the effects of nucleotides, metal ions or pH, and of adding the missing third protein from the network. Stability will be assessed by gel filtration and proteins analysed by SDS PAGE. Crystallisation trials will be undertaken to elucidating a high resolution structure for one or more of these complexes.
An assay will be developed to permit screening of a compound library of drug-like molecules. Potential assays for development include a reporter gene format, split-YFP labelling, or the measurement of specific protein activity.
Scientific and Medical Opportunities
Exploration of the YgjD/YjeE/YeaZ network will increase our understanding of the functions of these proteins in Escherichia coli. This will hopefully lead to the discovery of potential new antimicrobial agents acting at a target site not affected by current classes of antibiotic.
Objectives
A two-stranded research project is proposed. Firstly to explore the interactions between the proteins YjeE, YeaZ and YgjD to identify preferential partnerships under different conditions; to investigate potential protease and DNA binding activities; and to determine the structure of one or more of the different partner complexes. The second stage of the project will involve the screening of a compound library for agents that impair the function of one or more proteins in the network.
Methodology
Plasmids will be constructed carrying the genes for the YjeE, YeaZ and YgjD proteins labelled with split yellow fluorescent protein, which will fluoresce when two labelled proteins interact. The individual components will be labelled in pairs to establish preferentially pairing under a variety of culture conditions (including pH, osmolarity, oxygen tension, and different chemical sources).
Electrophoretic mobility shift assays will be used to investigate the DNA binding of YeaZ and YgjD to DNA, either in isolation or in partnership.
The stability YjeE/YeaZ and YgjD/YeaZ complexes will be probed, for example to determine the effects of nucleotides, metal ions or pH, and of adding the missing third protein from the network. Stability will be assessed by gel filtration and proteins analysed by SDS PAGE. Crystallisation trials will be undertaken to elucidating a high resolution structure for one or more of these complexes.
An assay will be developed to permit screening of a compound library of drug-like molecules. Potential assays for development include a reporter gene format, split-YFP labelling, or the measurement of specific protein activity.
Scientific and Medical Opportunities
Exploration of the YgjD/YjeE/YeaZ network will increase our understanding of the functions of these proteins in Escherichia coli. This will hopefully lead to the discovery of potential new antimicrobial agents acting at a target site not affected by current classes of antibiotic.
