Study of an exported protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
Johne's disease is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (M. ptb) from the MAIS complex (M. avium, M. ptb, M. intracellulare and M. scrofulaceum). The lack of specific and sensitive diagnostic tests often leads to M. ptb infected animals being diagnosed with bovine tuberculosis, a member of the MTB complex (M. tb, M. bovis, M. bovis BCG, M africanum, M. microti and M. canetti). Secreted proteins from pathogenic mycobacteria have been found to be important for the development of protective immunity, namely a cell mediated immune response (CMI). The development of reliable differential diagnostic tests will require the use of species-specific secreted protein antigens and the CMI response. Due to the taxonomic distance between the MAIS and MTB complexes our hypothesis was that the M. ptb genome may encode for secreted proteins that are absent from members of the MTB complex. If such proteins can stimulate an immune response they may be suitable for use as antigens in a differential diagnostic test for Johne's disease. To this end, the secreted protein library clone pJEMIl-M ptb281 was examined and its insert found to contain the 5' region of the hypothetical M.ptb281 ORF fused in frame with phoA. The entire ORF was determined using M. avium and M. ptb database sequences then cloned into E. coli and mycobacterial expression systems. These systems incorporate 6x histidine (His6) affinity tags into recombinant proteins allowing them to be semi-purified by Ni-NTA affinity chromatography. Semi-purified recombinant proteins tested positive by western blot analysis to highly specific anti-His6-tag antibodies. Amino acid sequencing to confirm the identity of these recombinant proteins and screening for their ability to stimulate an immune response were prevented by time constraints. Homologs to M. ptb281 were absent from M. tb, M. bovis and M. bovis BCG but present in the MAIS complex, making this protein unsuitable for use as an antigen to differentiate between MAIS complex species in a diagnostic test. M. ptb281 homologs found in the genomes of two members of the Acetomycetes order corresponded to hypothetical proteins predicted by computer software programs trained to identify genes, which may indicate that the hypothetical M. ptb281 ORF may encode a functional protein.