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

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 possible -35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-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 analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging 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 other seven strains belonging to the rarely isolated serotypes E to H were negative 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 express P30. The negative strains carried mutations in both -10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.

FIG. 1

Physical map of the p30 locus. The figure shows the physical and genetic map of plasmid pJFFBF1+E3; the p30 gene is represented by a black arrow; the different ORFs are indicated with arrowheads indicating the direction of translation. The putative transcriptional termination site for p30 is indicated by a hairpin. The restriction sites are indicated by the following abbreviations: B, BamHI; S, Sau3AI; P, PstI; H, HindIII; and E, EcoRI. The interrupted line (//) indicates the junction of noncontiguous segments. The EMBL/GenBank accession no. of the DNA sequence is AF327858.

FIG. 2

Presence of p30 in M. agalactiae strains and other mycoplasma species. Detection of p30 in strains of M. agalactiae and other mycoplasma species by amplification of genomic DNA was performed by PCR with primers MagaORF1-L and MagaORF1-R (Table2). The strains are listed in Table 1. The arrows indicate the p30 amplicons. Std, molecular mass standards (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.6 kb).

FIG. 3

Distribution of P30 expression in strains of M. agalactiae and of other mycoplasmas. Immunoblot analysis was carried with 10 μg of total antigen per lane. Total antigen of a selection of strains from M. agalactiae and of type and reference strains of other mycoplasmas was separated by SDS–12% PAGE, transferred onto a nitrocellulose membrane, and probed with the antiserum against P30. Std, molecular mass standards (broad-range marker 7708S; New England Biolabs, Inc., Beverly, Mass.).

FIG. 4

Phylogenetic grouping of 15 strains of M. agalactiae. Sequence similarity (neighbor-joining) tree based on the comparison of the p30 nucleotide sequences from 15 strains of M. agalactiae. The scale represents the genetic distance in percent.

FIG. 5

Immune response to P30 of ewe 1937 artificially infected with type strain PG2. SDS–15% PAGE was performed with approximately 1 μg of P30-His per strip. After blotting onto nitrocellulose, serial serum samples were used at a dilution of 1:200. Abbreviations: C⁻, lamb precolostral serum (negative control); d-23 and d-1, sera collected 23 days and 1 day, respectively, prior to inoculation; dn, serum collected n days after inoculation; C⁺, serum PAL 97 (anti-M. agalactiae) from a naturally infected ewe, diluted 1:600 (positive control); Std, molecular mass standards. Numbers below the panel indicate the titers in international units as determined by ELISA.

FIG. 6

Triton X-114 phase partitioning of M. agalactiae type strain PG2. Mycoplasmas at the end of exponential growth were subjected to Triton X-114 partitioning; 10 μl of fractions and total antigen were separated by SDS–12% PAGE, transferred onto nitrocellulose filter, and probed with monospecific P30 antiserum. We attribute the larger protein band of approximately 60 kDa that is found at low intensity upon reaction with anti-P30 antibodies to formation of a dimeric aggregate of P30, since this band occasionally occurs only with strains that express the protein. Lane1, molecular mass standard (broad-range marker 7708S; 175, 83, 62, 47.5, 32.5, 25, and 16.5 kDa; New England Biolabs); lane 2, total antigen; lane 3, detergent phase; lane 4, aqueous phase.