Resumo

Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes respiratory epithelial cells causing porcine enzootic pneumonia. Cell adhesion and other processes important for infection depend on membrane proteins, most of which are transported by the Sec-dependent pathway. Type I signal-peptidase (SPase I) is a membrane protease that acts in the release of translocated proteins through Sec-dependent pathway. Therefore, SPase I is critical for protein export, which may play important roles in pathogen-host relationships. The aim of this work is the functional characterization of M. hyopneumoniae SPase I (MhSPase I), in order to analyze its relevance for bacterial cell viability and pathogenicity. For that, in vivo functional activity of MhSPase I was initially assessed by complementation of temperature sensitive SPase I mutant Escherichia coli IT89.The MhSPase I coding DNA sequence, sipS, was cloned into the expression vector pEX50 and E. coli IT89 was transformed with pEX50:sipS clone or with the parental plasmid. Growth of E. coli IT89 transformed with pEX50:sipS or pEX50 at non-permissive (42°C) temperature was monitored by measuring OD540 for 72 h. Expression of recombinant MhSPase I (rMhSPase I) in E. coli IT89 transformed with pEX50:sipS was confirmed by immunoblot using polyclonal antibodies anti-rMhSPase I. In the performed assays, rMhSPase I was able to partially complement SPase I activity of E. coli IT89 in the non-permissive temperature, allowing the bacterial to survive. In vitro assays will now be performed to demonstrate rMhSPase I activity on M. hyopneumoniae preprotein. For that, M. hyopneumoniae putative substrates for SPase I activity were identified in silico using the signal-peptide prediction software SignalP 4.1, PrediSi, Phobius and Signal-Blast. Among the eight identified targets, the P216 adhesin was selected and the coding DNA sequence of its amino-terminal portion containing the putative signal-peptide was cloned and expressed in E. coli BL21 Codon Plus RP. The recombinant amino-terminal portion of P216 will be used to assess rMhSPase I activity in vitro and its demonstration will be taken as evidence of its contribution for pathogenicity in vivo. Subsequently, new in vitro cleavage assays will be performed using others recombinant preproteins, in order to confirm the enzymatic activity of rMhSPase I.