A 38-Kilobase Pathogenicity Island Specific forMycobacterium aviumsubsp.paratuberculosisEncodes Cell Surface Proteins Expressed in the Host

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Abstract
We have used representational difference analysis to identify a novelMycobacterium aviumsubsp.paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designatedmptAto -Fand is immediately preceded by two putative Fur boxes. Functional genomics revealed that themptoperon is flanked on one end by afepcluster encoding proteins involved in the uptake of Fe3+and on the other end by asidcluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kbM. aviumsubsp.paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction ofM. aviumsubsp.paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening ofMycobacterium smegmatistransformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes anM. aviumsubsp.paratuberculosispathogenicity island.