Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae

Citation
B. Fleury et al., Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae, J CLIN MICR, 39(8), 2001, pp. 2814-2822
Citations number
35
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
39
Issue
8
Year of publication
2001
Pages
2814 - 2822
Database
ISI
SICI code
0095-1137(200108)39:8<2814:CAAOAS>2.0.ZU;2-S
Abstract
The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two -10 boxes and a possibl e -35 region constituting the potential promoter, and a transcription termi nation site. The gene for the 266-amino-acid protein is preceded by a polyp urine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot a nalysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with spec ific primers for p30 and Southern blot analysis revealed the presence of th e gene in all M. agalactiae strains tested and its absence in the other myc oplasma species. Among 27 strains of M. agalactiae studied, 20 strains belo nging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The o ther seven strains belonging to the rarely isolated serotypes E to H were n egative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not ex press P30. The negative strains carried mutations in both -10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expre ssion in these strains. Analysis of sera from sheep that were experimentall y infected with M. agalactiae revealed that P30 induced a strong and persis tent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.