Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.