A 34kDa protein expressed in vivo by M. a. paratuberculosis exhibits an overall similarity to HtrA, a trypsin like serine protease, of several gram positive and gram negative bacteria. HtrA mutants of several pathogenic bacterial strains have demonstrated attenuation in both in vivo and in vitro models of infection. To investigate the potential role that the 34kDa protein plays in the virulence of M. a. paratuberculosis, the entire open reading frame was cloned into a mycobacterial shuttle vector under the transcriptional control of the M. tuberculosis hsp60 promoter. The construct was introduced into M. smegmatis mc2155, and protein expression detected by western blot using a monoclonal antibody raised against the 34kDa putative serine protease. M. smegmatis mc2155 expressing the recombinant protein exhibited increased survival within ovine ex-vivo alveolar macrophages at 24 hours post infection. Further work has centred on the identification and characterisation of the transcriptional signals responsible for the expression of the 34kDa putative serine protease.
This work was funded by grants from the European Commission and the Scottish Executive Environment and Rural Affairs Department.