Identification and characterisation of an exported immunogenic protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
Exported proteins of mycobacteria are available to interact with the immune system at an early stage of infection and are potent inducers of immune responses. Potentially exported proteins of Mycobacterium avium subspecies paratuberculosis were identified using alkaline phosphatase gene fusion technology. A library of partial gene fusions from a New Zealand clinical isolate of M. a. paratuberculosis was constructed in the shuttle vector pJEM11 and expressed in the surrogate hosts E. coli and M. smegmatis. The DNA inserts from a portion of the resulting clones expressing alkaline phosphatase-positive fusion proteins were partially sequenced to identify the proteins. Eleven proteins not previously described for M. a. paratuberculosis were identified as containing signal sequences for export. One of these, a putative lipoprotein named P22 was selected for further study. The full nucleic acid sequence of the p22 gene was determined and the open reading frame was cloned into die mycobacterial expression vector pMIP12. This enabled P22 to be produced as a polyhistidine-tagged protein in M. smegmatis and facilitated purification by chromatography. N-terminal sequencing of the recombinant protein confirmed cleavage of an N-terminal signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. a. paratuberculosis strain 316F using rabbit antibody raised to P22. Investigation of the presence of genes similar to p22 in other mycobacterial species, revealed p22 was present in Mycobacterium avium subspecies avium and similar genes existed in M. intracellularae (88.5% identity) and M. scrofulaceum (87.7% identity). Database searches showed P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in M. leprae and in members of the M. tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit significantly increased interferon-gamma secretion in blood from a group of eight sheep vaccinated with a live, attenuated strain of M. a. paratuberculosis (strain 316F) compared to a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.