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

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.

Bacteria localise proteins to their surfaces via several mechanisms including their covalent modification with lipids. The mycolata are a major bacterial suborder that contains significant human and veterinary pathogens including Rhodococcus equi; their characteristic envelopes have an outer membrane rich in mycolic acids. Recently O-mycolylation of two Corynebacterium glutamicum porins was identified as a novel protein modification. The main aims of the proposed research are to establish whether O-mycolylation is a mycolata-wide protein localisation mechanism and thus should be considered as a potential drug target. To establish whether protein mycolylation is widespread among the mycolata, lipidated envelope proteins will be selectively extracted from representative species, purified to remove contaminating glycolipids and their modifying acyl groups recovered via saponification and methylation to establish their nature using MALDI-TOF MS. The identity of mycolated proteins will be established by proteomic survey of this protein fraction from R. equi, for which we have a strong candidate for a mycolated virulence factor, and selected representative species that modify proteins in this manner. This data will inform delineation of a mycolylation target sequence motif for predicting substrate proteins. An identified protein mycolyltransferase (MytC) from C. glutamicum and an R. equi orthologue will be cloned and used in the development of a radiometric biochemical assay in which mechanistic aspects will be explored using site-directed mutagenesis of both enzyme and substrate proteins. A non-radioactive biochemical assay suitable for a high throughput screening of compounds will be developed. A mutant in R. equi mytC will be constructed to confirms its function and as a host for in vivo functional characterisation. The virulence of the mutant will also be established using impedance-based monitoring of infections in semi-adherent J774A.1 macrophage-like cells.

This study will take a major step towards defining a novel mechanism of protein localisation which we expect to play a fundamentally important role underpinning host-pathogen interactions in a highly significant group of bacterial pathogens. Consequently, we are confident that there will be translational benefits of the work which will benefit a range of user groups, most obviously in that the pathway we identify is expected to contain components which could be targets for novel drug development. We believe the primary beneficiaries of our work will be the following:

Northumbria University undergraduate and MSc students - our students will benefit from the integration of our research into our teaching (both classroom and project based)

Pharmaceutical industry - it is expected that we will identify targets for novel therapeutic strategies, relevant to all mycolic acid containing bacteria. We will seek academic partners in medicinal chemistry and commercial partners in the pharmaceutical industries to work on drug development programmes which will have profound economic and societal benefits (including improved human health and animal welfare).

Health sector - in addition to targeting the pathway itself, it is likely that this mechanism can be made manipulatable, thereby allowing the development of strains expressing autologous or heterologous antigens to maximise their immunogenicity for vaccine purposes. This will aid vaccine development against various mycolic acid containing pathogens. We will seek partners in the health sector to help us implement this translational research. Significant health and economic benefits can therefore be expected as a long-term consequence of vaccine development.

Biotechnology sector - non-pathogenic mycolic acid containing bacteria are also of major interest for biotechnological applications such as bioremediation and metabolite production. Pathway engineering may be applicable to improving protein surface display in these bacteria, so we expect our work to be of interest to the commercial sector. We will seek partners in the biotechnology industry to help develop this aspect, thereby boosting economic competitiveness of this sector.

General public - Antimicrobial resistance is now established as an area of significant concern for the general public. We will use our public engagement activities to reassure the public that significant research efforts are being directed towards the identification and exploitation of novel targets for antimicrobials i.e. that the antimicrobial pipeline can be refilled.

Post-doctoral training - we expect that this project will provide the appointee with a multi-disciplinary skillset and high impact publication profile which will sustain development of a career in academic and translational research.