Award details

Structure-function studies of the virulence associated proteins of Rhodococcus equi

Reference	BB/J007900/1
Principal Investigator / Supervisor	Professor Anthony Wilkinson
Co-Investigators / Co-Supervisors	Dr Jean Whittingham
Institution	University of York
Department	Chemistry
Funding type	Research
Value (£)	378,655
Status	Completed
Type	Research Grant
Start date	01/04/2012
End date	31/03/2015
Duration	36 months

Abstract

R. equi is a pulmonary pathogen in foals and in immuno-compromised humans. It is an intracellular pathogen whose capacity to persist and multiply in macrophages is key to disease development. Isolates of the bacteria from foals with R. equi pneumonia harbour an ~80 kbp plasmid that is critical for virulence. This plasmid contains a pathogenicity island that encodes a set of related virulence associated proteins (Vaps). The vap genes are induced by changes in temperature, pH and oxidative stress - conditions encountered upon entry into the macrophage. The vap genes encode signal sequences indicating that the proteins are exported from the cell. Amongst the Vap proteins, VapA plays a dominant role in R.equi immunology and infection. It is a surface-expressed protein that is highly immunogenic in R. equi infected animals. It has been shown to be essential for survival in the macrophage and for pathogenicity. The objective of this work is to understand the role of VapA in R. equi biology by addressing the questions of (i) how VapA is associated with the surface of R. equi which has a complex mycolic acid-containing cell wall and (ii) how VapA contributes to virulence. Firstly we propose to determine by modern analytical mass spectrometry methods whether VapA is lipid-modified. This has been suggested by earlier research even though VapA lacks recognised post-translational modification signature sequences. We propose to determine the structure of VapA and/or one or more of the Vap homologues using X-ray crystallography. The Vap proteins have no sequence similarity to proteins of known structure and their structure may reveal a novel fold. In contrast, they share sequence similarity to one another such that the structure of one member of the set will give insight into the group as a whole. Finally, VapA is proposed to sit at the centre of an interaction network featuring VapD, VapG, VapI and Orf10 and we plan to characterise in vitro the interactions of these proteins.

Summary

The bacterium Rhodococcus equi (R. equi) infects a range of grazing animals and poses a threat to animal health especially to that of horses. In foals aged 3-5 months, R. equi infection causes a lung disease, which if untreated, can lead to death. The bacteria, which are transmitted through the faeces, are inhaled into the lungs in the form of aerosolised dusts. In common with type of bacterium which causes tuberculosis in humans, R. equi cells are surrounded by a complex protective cell wall rich in compounds called mycolic acids, moreover these bacteria share the ability to survive within macrophages, a type of cell of the human immune system. Macrophages normally sweep up and destroy infecting bacteria by first engulfing them and then exposing them to lethal chemical stresses. Many pathogenic bacteria evade macrophage engulfment altogether, while others such as R. equi have developed survival strategies that prevent their chemical destruction once inside the macrophage. Survival in this 'lion's den' is a critical first step in establishing infection and is essential for the later development of virulence. In this study, we are concerned with virulence-associated proteins (Vaps) of R. equi and with one particular member of this set of proteins, VapA. Serum taken from foals with R. equi pneumonia, are rich in antibodies that react with VapA consistent with this protein being exposed on the surface of the bacterium where it is 'visible' to the host. It therefore has potential as a vaccine against R. equi infection. VapA has also been shown to be indispensible for R. equi virulence making it a potential target for the design of new drugs to cure R. equi infections. Interestingly, VapA production by the bacteria is turned on by elevated temperature, increased acidity and by oxidative stress. These are the conditions that the bacteria encounter upon entry into the macrophage. It has therefore been proposed that VapA plays an important role in enabling R. equi to survive and then persist in this hostile environment inside the macrophage. In view of its important role in R. equi virulence, we wish to extend the understanding of VapA to include its molecular structure and function. A first question concerns how VapA is exposed on the surface of the bacterium. In many other bacterial species, cell surface proteins possess a tagging sequence that leads to the attachment of a hydrocarbon chain which facilities tethering of the protein to the cell membrane. VapA does possess the requisite tagging sequence suggesting either that VapA is not tagged or that it is tagged by a novel mechanism. We propose to extract and isolate VapA from R. equi cells and determine whether it is tagged by analysing the protein by state of the art techniques which measure molecular mass. A further goal is to determine the three dimensional structure of VapA using X-ray diffraction methods. Sometimes it is possible to model the structure of a protein if its sequence has sufficient similarity to the sequence of another protein of known structure. This is not possible for VapA. The only proteins with sequence similarity to VapA are a set of nine other Vap proteins from R. equi. For structure determination we will prepare crystals of VapA, and/or its Vap relatives and use these crystals to collect X-ray scattering data. The structure of the protein once determined will be scrutinised for its functional implications for cell surface localisation and virulence. Finally there is evidence to suggest that the Vap proteins interact with one another and this may be important for virulence. We propose to purify the individual proteins and in mixing experiments explore the strength of these interactions and the composition of the complexes that are formed.

Impact Summary

The research to be carried out here has beneficiaries outside the academic constituencies listed above. Anyone with an interest in horses, be it commercial, medical or recreational, is a potential beneficiary since a better understanding of R. equi biology will improve our capacity to develop strategies to counter its threat to animal health and the associated economic burden that it presents. This would include horse-breeders, horse owners and horse trainers whose livelihoods depend, directly or indirectly, on the welfare of young animals. Associated with this is the horse-racing industry which is economically important to this country as well as forming an important element in its culture. The threat posed by R. equi to the prosperity of this industry prompted the Horse-Racing Betting Levy board to part fund the sequencing of this organism's genome. Research on R. equi is motivated by animal welfare and the results of research in this field are clearly of primary interest to Veterinary Practitioners. VapA is the single most important protein to emerge from studies of R. equi pathogenesis. It is the major immunogen and it is essential for virulence. A vaccine against R. equi infection is being actively sought and VapA has been considered as a potential vaccine candidate. The results of the work described here have the potential to inform the design and production of such a vaccine. Similarly, there is a demand for cheaper alternatives to antibiotic therapy to cure R. equi infections and again the structure of VapA has the potential to define the direction of drug discovery strategies.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Animal Health, Immunology, Microbiology, Structural Biology
Research Priority	Animal Health
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search