Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum

Abstract

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gaUlisepticum protein band of Mr approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 pg of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. galisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

“…All pMGA genes with determined sequences (pMGA1.1p MGA1.9) have highly conserved leader peptide sequence and encode proline-rich regions (PRR) at the N-terminus with the cysteine (C 1 ) at the ¢rst position [6,8,10]. Our study was focused on the pMGA1.1 and pMGA1.2 genes because of the importance of their products as adhesins and immunodominant antigens [3,4,12]. The presence of the large pMGA gene family in M. gallisepticum required a selection of pMGA1.1/pMGA1.2 speci¢c PCR primers (PMGA1.F/PMGA1.R), which directed the synthesis of the 1.3-kb PCR fragment (PMGA1FR).…”

“…The origin of M. gallisepticum strains (S6 208 , S6 JMB , S6 B , A5969 HK , PG31, 6/85, G11, IHB1, ULB921 and ULB931) and their classi¢cation on the basis of reactivity with 12 monoclonal antibodies (mAbs) was described elsewhere [12,13]. Other reference strains used were S6 PFM [4], TS100, a temperature sensitive mutant of the S6 strain [20] and A5969 LG , a low passage culture from a chicken trachea. Our recent M. gallisepticum isolates PPT 2 from a chicken trachea, ULB971 from a pheasant trachea and ULB992 from the infraorbital sinus of a peacock were also examined.…”

“…The mAb 66 that reacts with pMGA1.1 and mAbs 71 and 86 that react also with pMGA1.2 were described elsewhere [4,8,15]. The mAb A3 reacts with pMGA1.1 and pMGA1.2 [14,16].…”

“…However, im-munoa¤nity-puri¢ed 67-kDa protein, obtained from the PG31 strain using mAb K1, reacted with mAbs 71, 86 and A3, indicating that mAb K1 recognizes a pMGA1.2 protein (data not shown). Antibodies against the synthetic peptide C 1 TTPTPSPAPNPNPPSNG+C, which was coupled to keyhole limpet hemocyanin and represented the N-terminal part of pMGA1.1 [4,17], were raised in a rabbit and designated pAb D1. The rabbit was immunized intramuscularly with 1 mg synthetic peptide in Freund's complete adjuvant and boosted ¢ve times with 1 mg peptide in Freund's incomplete adjuvant in 1^3-week intervals.…”

“…Immunoblotting with the PhastSystem (Pharmacia-LKB, Uppsala, Sweden) was used to determine regions of the pMGA molecule recognized by mAbs [12]. The whole length puri¢ed pMGA1.1 protein, its fragments, cleaved either with protease V-8 (Glu-C) or trypsin, or a truncated recombinant pMGA (residues from 37 to about 300) were used for the analysis [4,5]. Mapping of the epitopes for mAbs was conducted with the SPOTS system (Genosys, Cambridge, UK).…”