Post-translational lipidation of proteins with mycolates in Rhodococcus equi: a novel drug target in the mycolata?

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Novel antimicrobials are desperately needed as both antibiotic resistance increases and new pathogens emerge. The development of novel antimicrobials will be greatly facilitated by the characterisation of as yet unexploited pathways that contribute to the interactions between pathogenic bacteria and their hosts. We propose to study one such pathway, which we expect will prove to be an overlooked but crucial aspect of the interaction between mycolic acid containing bacteria and their human or animal hosts. The mycolic acid containing bacteria are an important group of bacteria which have a common feature of a waxy cell envelope based on characteristic lipids, the mycolic acids. Many very significant pathogens belong to this group of bacteria, including the causative agents of tuberculosis, leprosy and diphtheria in humans and of bovine farcy, bovine tuberculosis, rhodococcal bronchopneumonia in foals and caseous lymphadenitis in sheep and goats. The biosynthesis of the unusual cell envelopes of these bacteria is already established as valid drug target (e.g. in tuberculosis treatments). We hypothesise that there is a pathway in these bacteria by which proteins are localised to the mycolic acid cell envelope by modification of the proteins with mycolic acids, which will provide a lipid anchor holding such proteins onto the waxy cell surface layer. This type of protein modification is thus likely to influence the virulence of mycolic acid containing bacteria as cell envelope proteins are crucial to the interactions between pathogen bacteria and their hosts. We therefore propose to verify that this type of lipid modification is indeed widespread in mycolic acid containing bacteria and to establish the mechanism by which it occurs. We will examine a representative range of mycolic acid containing bacteria to demonstrate that mycolic acid modified proteins are present, using selective extraction and chemical characterisation methods. We will also examine the sites at which the proteins are modified as this should give insights into the underlying mechanism and also allow sequence based prediction of which proteins are likely to be similarly modified. As a model system in which to study the mycolic acid modification pathway in detail, we will use the important equine pathogen Rhodococcus equi (a global cause of rhodococcal bronchopneumonia in foals) as we have extensive experience of methods for studying the cell envelope biology of this bacterium. We have already identified a candidate enzyme that is likely to be the catalyst for mycolic acid modification of proteins. Genetic modifications of the gene encoding this enzyme will allow us to create mutant strains of R. equi that we predict will be attenuated in their ability to cause disease, which we will verify using novel tissue culture method for assessing bacterial virulence in vitro. This will provide evidence for the importance of this pathway that should be relevant to other mycolic acid containing pathogens. Finally, we will characterise biochemically the protein mycolic acid modification machinery in R. equi and thus gain mechanistic insights that will allow us to determine whether this pathway is a suitable target for the development of novel antimicrobial therapies. As part of this work we will also devise assays suitable for adaptation in high throughput screens that can be used for the discovery of novel antimicrobials targeting this pathway. In summary, we expect this project to identify a new pathway influencing host-pathogen interactions in an important group of bacteria and to demonstrate that this pathway represents a target suitable for the development of novel antimicrobials.