title: Molecular characterisation of the Mycoplasma cynos haemagglutinin HapA.
abstract: Mycoplasma (M.) cynos is a proven pathogen of dogs causing respiratory infections including pneumonia. We examined 19 M. cynos strains isolated from different organs of dogs in Austria, Denmark and Israel. All strains agglutinated mammalian and chicken erythrocytes. Using erythrocytes of chickens or dogs as specific ligands we isolated an approximately 65 kDa protein from cell-free supernatants of 3 M. cynos strains, which showed an apparent capacity for haemagglutination. The N-terminal sequence of a 25 kDa fragment of this protein was identified as NNEMTPKVTVEAKSMELLLSVEK. The identical amino acid sequence is encoded by the gene MCYN_0308 in the genome of M. cynos C142. This gene belongs to a family of some 20 genes which encode putative lipoproteins with proline-rich regions (PRR) in the first third of their molecules. We termed the 65 kDa haemagglutinin HapA and sequenced hapA gene homologues of 16 M. cynos strains. Analyses of hapA gene homologues revealed similar but not identical sequences, some having insertions and/or deletions in the PRR. We produced a recombinant HapA protein (rHapA) and also mouse monoclonal antibodies (mAbs) recognizing HapA. However, enzyme immunoassays using native M. cynos colonies and mAbs 5G2 or 3B7 showed variable expression of HapA in all M. cynos strains. This was further confirmed by Western blot analyses which showed different HapA quantities and also size-variation of HapA among strains. Analyses of cDNA of the expressed hapA genes showed that besides the hapA gene cultures of M. cynos (strains 105, 2002, 2297) can also express other forms of hap genes. In addition, in cloned cultures of strain 2297 altered HapA epitopes for mAbs 5G2 and 3B7 with distinct hapA gene mutations that resulted in altered HapA amino acid sequence were found. Most of the dogs examined had serum antibodies to rHapA. In conclusion, we characterized the M. cynos haemagglutinin HapA protein and encoding gene hapA, a factor involved in cytadherence to host cells and therefore important for M. cynos infection, and showed that expression of HapA is varied in M. cynos by two distinct mechanisms; differential gene expression and nucleic acid substitution within hapA homologues.
authors.affiliation: Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia.
authors.forename: Saša
authors.initials: S
authors.lastname: Kastelic
authors.affiliation: Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia.
authors.forename: Ivanka
authors.initials: I
authors.lastname: Cizelj
authors.affiliation: Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Narat
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.
authors.forename: Nataša
authors.initials: N
authors.lastname: Tozon
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Bacteriology Reference Department, Public Health England, 61 Colindale Avenue, London NW9 5EQ, United Kingdom.
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Chalker
authors.affiliation: Mycoplasma Unit, Division of Avian and Fish Diseases, Kimron Veterinary Institute, Bet Dagan 50250, Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Lysnyansky
authors.affiliation: Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria.
authors.forename: Joachim
authors.initials: J
authors.lastname: Spergser
authors.affiliation: Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia. Electronic address: dusan.bencina@bf.uni-lj.si.
authors.forename: Dušan
authors.initials: D
authors.lastname: Benčina
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics; D004283:Dog Diseases / Q000382:microbiology*; D004285:Dogs; D000939:Epitopes; D004912:Erythrocytes / Q000276:immunology; D015964:Gene Expression Regulation, Bacterial; D006388:Hemagglutinins / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009174:Mycoplasma / Q000166:cytology / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D018076:DNA, Complementary; D000939:Epitopes; D006388:Hemagglutinins; D008074:Lipoproteins; D011994:Recombinant Proteins
keywords: Haemagglutinin; Lipoprotein HapA; Mycoplasma cynos