Lpp34, a Novel Putative Lipoprotein from Mycobacterium avium subsp. paratuberculosis

• Published in: Journal of veterinary medicine. Series B Vol. 53; no. 1; pp. 34 - 41
• Main Authors: Gioffré, A, Caimi, K, Zumárraga, M.J, Meikle, V, Morsella, C, Bigi, F, Alito, A, Santángelo, M.P, Paolicchi, F, Romano, M.I, Cataldi, A
• Format: Journal Article
• Language: English
• Published: Berlin, Germany Berlin, Germany : Blackwell Verlag GmbH 01-02-2006; Blackwell Verlag GmbH; Blackwell Publishing Ltd
• Subjects: amino acid sequences; Animals; Bacteria; bacterial antigens; bacterial proteins; Bacterial Proteins - classification; Bacterial Proteins - immunology; Bacterial Proteins - isolation & purification; Base Sequence; Blotting, Western - veterinary; Cattle; Cloning, Molecular; DNA, Bacterial - analysis; Escherichia coli; food animals; Gene expression; humoral immunity; lipoproteins; Lipoproteins - classification; Lipoproteins - immunology; Lipoproteins - isolation & purification; membrane proteins; microbial genetics; Molecular Sequence Data; Molecular Weight; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis; Mycobacterium avium subsp. paratuberculosis - classification; Mycobacterium avium subsp. paratuberculosis - immunology; Mycobacterium avium subsp. paratuberculosis - isolation & purification; Mycobacterium tuberculosis; Open Reading Frames; paratuberculosis; polymerase chain reaction; Polymerase Chain Reaction - veterinary; Proteins; Recombinant Proteins - classification; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Rodents; sequence analysis; Sequence Analysis, DNA; Sequence Analysis, Protein; Tuberculosis; Western blotting
• ISSN: 0931-1793; 1863-1959; 1439-0450; 1863-2378
• DOI: 10.1111/j.1439-0450.2006.00905.x

A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.