Rhodococcus equi is a soil-borne facultative bacterial pathogen which causes a severe pyogranulomatous pneumonia in young foals. Despite the availability of antibiotics, mortality rates are high and there is currently no vaccine licensed to prevent disease caused by R. equi. The R. equi bacterium grows rapidly on simple organic acids which are abundant in the faeces of grazing animals. The major route of R. equi infection is by inhalation of contaminated faeces particles into the lungs. Here the bacteria enter alveolar macrophages and proliferate, evading normal macrophage killing mechanisms by processes currently unknown. The ability of R. equi to survive in this environment is linked to the possession of a virluence plasmid encoding a family of virulence-associated proteins (VapA-VapH). These proteins are related by sequence and are unique to R. equi. VapA has been found to be highly immunogenic in foals and is a defining member of this protein family. Amino acid sequence analysis of the proteins predicts a common overall structure connected to a disordered N-terminal region of variable length. In VapA this region constitutes 30% of the protein while in VapD it is only 13%.
Studies to investigate the structural properties of the virulence-associated proteins will be presented. VapA, VapG and VapD were overexpressed and purified for crystallisation trials and NMR studies. Preliminary crystals were obtained for VapD. Solution studies using SEC-MALS revealed that VapA forms dimers while VapD and VapG are monomeric. An N-terminally truncated form of VapA loses some of its capacity to form dimers, suggesting a role for the N-terminus in dimer formation. These studies provide evidence that the variable N-terminal region confers different properties to the proteins which may relate to different functions in vivo.