Identification of two Mycobacterium avium subspecies paratuberculosis geneproducts differentially recognised by sera from rabbits immunised with live mycobacteria but not heat-killed mycobacteria

The investigation of environmentally regulated proteins has led to a better understanding of host-pathogen interactions and identified novel vaccine c andidate antigens for several bacterial pathogens. In an effort to identify such proteins in Mycobacterium avium subsp. paratuberculosis (M. paratuber culosis), a genomic expression library was differentially screened with ser a from rabbits that had been immunised with live M. paratuberculosis (alpha -live) as well as sera from rabbits immunised with heat-killed M. paratube rculosis (alpha -killed). These experiments identified seven recombinant pl aques that were uniquely recognised by the alpha -live sera. Sequence data showed that five of these clones overlapped with each other and contained a common open-reading frame encoding a 25-kDa protein, termed Csp1. The 25-k Da antigen shows weak similarity to a secreted Corynebacterium glutamicum p rotein. The remaining two clones overlapped with each other and contained t wo partial open-reading frames, both encoding proteins with strong homology to polyketide synthase from various species of mycobacteria. Antisera were produced against a peptide of the polyketide synthase gene product designa ted Pks7. Csp1-specific antibodies were affinity purified from the alpha -l ive sera. These purified antibodies demonstrated that Csp1 was present with in infected macrophages. Collectively, these data identify novel M. paratub erculosis antigens that may be important in pathogenesis.