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Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis

Chris Dupont¹, Keith Thompson¹, Cord Heuer¹, Brigitte Gicquel² and Alan Murray¹

¹Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, Private Bag 11 222, New Zealand ²Unite de Genetique Mycobacterienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

Correspondence Chris Dupont cdupont{at}sciborg.uwaterloo.ca

Received May 19, 2005
Accepted July 12, 2005

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75 % identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from 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.

Abbreviations: IFN-, interferon-gamma; MAP, Mycobacterium avium subsp. paratuberculosis; PPD, purified protein derivative.

The GenBank/EMBL/DDBJ accession number for the sequence of p22 in Mycobacterium avium subsp. paratuberculosis is AY956313.

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