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

Abstract

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library ofMycobacterium aviumsubsp.paratuberculosisand expressed inMycobacterium 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 inM. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates ofM. aviumsubsp.paratuberculosisstrain 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 inMycobacterium aviumsubsp.aviumand similar genes existed inMycobacterium intracellulareandMycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found inMycobacterium lepraeand in members of theMycobacterium tuberculosiscomplex. 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 ofM. aviumsubsp.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.