A combined, chemical-microbiology approach to understand the function of novel antibiotic resistance determinant in Gram-negative bacteria.
Dr Khondaker Rahman, Institute of Pharmaceutical Science, King's College London
Dr Charlotte Hind, Public Health England
Dr Mark Sutton, Public Health England
Bacteria are equipped with a wide range of resistance mechanisms to help them deal with the effects of antibiotics. Some of these mechanisms are well characterised, notably those associated with horizontal transmission (acquired resistance) between bacteria, the basis for intrinsic resistance to antibacterial agents is less well understood. This is largely because this type of resistance may be multifactorial with a number of different factors (e.g. limiting access to the cell, increased efflux, increased expression / modification of targets, binding to decoy targets) contributing to the overall resistance of the cell. Some of these mechanisms may be restricted to a limited number of strains, whilst others are widely distributed across different species.
An ongoing collaboration between King’s College London and Public Health England has recently identified a class of regulator proteins that may play an important role in mediating intrinsic resistance, via direct binding to particular types of antibiotic, in the important Gram-negative pathogen Klebsiella pneumoniae. We have now identified two unrelated chemical entities, but with a shared molecular target (DNA-gyrase), for which resistance appears to be mediated by this regulator. This raises the possibility that this protein is either a multidrug binding domain, capable of binding and responding to a wide range of antibiotics or is able to functionally mimic the gyrase-binding pocket. Both of these possibilities are intriguing, and we plan to use a combination of homology modelling and chemical synthesis, coupled with molecular microbiology techniques to provide tools to unpick the mechanism of action of these proteins. This family of proteins is widely distributed across a range of Gram-negative pathogens and with, some modifications to predicted domain structure, in Gram-positive pathogens also. They are also found in a number of bacteria clustered with polyketide synthase or non-ribosomal peptide synthases, where they may act as immunity proteins with, as yet, unidentified compounds. This may represent a pool of resistance determinants that impact on intrinsic susceptibility to existing and novel antibiotics. It may also represent a series of affinity ligands to be used in vitro or in vivo, that could be used to identify and characterise novel antimicrobial compounds from a variety of sources.
We expect the student to gain a working knowledge of a range of skills working at the interface of medicinal chemistry and microbiology, but specific skills will be supported by senior staff within the respective supervisory teams . The project would involve -
- Use of structural prediction and homology modelling to understand the substrate specificity of the Klebsiella regulator protein based on docking studies with current known substrates, other gyrase inhibitors, existing antibiotics and novel ligands.
- Cloning and expression of the Klebsiella regulator proteins in E. coli to allow further characterisation and to assess the impact on antibiotic resistance. This work will measure whether expression of the regulators affects susceptibility directly but also develop reporter constructs which will be activated on binding of potential substrates by the regulator. This will help define the molecular basis for compound recognition and the events leading to promoter de-repression and/or activation.
- Sesign and synthesise derivatives or analogues of known ligands to probe the “functional” structure activity relationship between regulator-binding and gyrase inhibition.
- Use a combination of susceptibility measurement in recombinant systems and synthesised substrate analogues to compare the specificity of regulator proteins from other species with that from Klebsiella.
- Establish protocols for HPLC and NMR analysis of molecules trapped by regulator proteins, using known ligands to provide proof-of-concept. Validate using analogues designed above with known affinity for the regulator protein.
The project is likely to generate a significant amount of data on the structure-function activity of the compound series in vitro and in vivo. Key analysis will relate to the assessment of antimicrobial properties (minimum inhibitory/bacteriocidal concentrations, time-kill etc) for different modified compounds against both clinical strains of pathogens and strains generated to look at specific efflux pumps
Public Health England pay an enhancement to the student stipend, contributing £2000 extra each year.
Interested applicants are encouraged to contact Dr K Miraz Rahman (email@example.com) in advance of the deadline.
Closing date is 19th January. Please ensure that you read the Guidelines before submitting an application. Your application and supporting documents should be sent in a single email to LIDo.Admissions@ucl.ac.uk
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