Lpp34, a novel putative lipoprotein from Mycobacterium avium subsp. paratuberculosis.

ANDREA, GIOFFRÉ; KARINA, CAIMI; MARTÍN J., ZUMÁRRAGA; VIRGINIA, MEIKLE; CLAUDIA, MORSELLA; FABIANA, BIGI; ALICIA, ALITO; MARÍA P., SANTÁNGELO; FERNANDO, PAOLICCHI; MARÍA I., ROMANO; ANGEL, CATALDI

JOURNAL OF VETERINARY MEDICINE SERIES B

Año: 2006 vol. 53 p. 34 - 41

0931-1793

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.