title: Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates.
abstract: Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.
authors: Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors: Lucas
authors: L
authors: Moitinho-Silva
authors: Marcia Y
authors: MY
authors: Kondo
authors: Lilian C G
authors: LC
authors: Oliveira
authors: Debora N
authors: DN
authors: Okamoto
authors: Jéssica A
authors: JA
authors: Paes
authors: Mauricio F M
authors: MF
authors: Machado
authors: Camila L
authors: CL
authors: Veronez
authors: Guacyara
authors: G
authors: Motta
authors: Sheila S
authors: SS
authors: Andrade
authors: Maria A
authors: MA
authors: Juliano
authors: Henrique B
authors: HB
authors: Ferreira
authors: Luiz
authors: L
authors: Juliano
authors: Iuri E
authors: IE
authors: Gouvea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016234:Kallikrein-Kinin System; D007700:Kinetics; D045705:Mycoplasma hyopneumoniae / Q000145:classification / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D010447:Peptide Hydrolases