Orthologous surface proteins from Mycoplasma hyopneumoniae and Mycoplasma flocculare: in silico comparison and heterologous expression of differential extracellular domains

Abstract

Background: Mycoplasma hyopneumoniae and Mycoplasma flocculare are two closely related mycoplasma species often found in the porcine respiratory tract [1]. However, M. hyopenumoniae is pathogenic, being the causative agent of enzootic pneumonia, while M. flocculare is a commensal bacterium. Enzootic pneumonia is a contagious respiratory disease characterized by chronic cough, growth retardation, low mortality, and a high morbidity. It causes significative economic losses in pig industry worldwide. Some of main interactions between the host and the bacteria are mediated by surface proteins, and a comparison of surface proteins between these two mycoplasmas species can lead to identification of determinants of pathogenicity or commensalism. Methods: In this work the deduced amino acid sequences of M. hyopneumoniae and M. flocculare surface proteins [1,2] were aligned and comparatively analyzed. Comparative analysis was made by sequencing alignments using ClustalW algorithm available in the MEGA 5.05 software package. Orthologous pairs of interest were selected based on the differential presence of amino acid stretches > 5 residues or regions of significantly reduced homology in comparison to flanking sequences. Topology analyses including predictions of extracellular and transmembrane domains were performed using the TopPred, phobius and TMHMM programs. The differential domains of the selected orthologous pairs were amplified by PCR from genomic DNA of the respective species and these amplicons were cloned into the pGEX4T-3 plasmid by in vivo homologous recombination in Escherichia coli KC8 [3]. The recombinant plasmids were expressed in E. coli strains adequate to heterologous expression, and the expressed recombinant polypeptides were purified by affinity chromatography and thrombin cleavage. Results and conclusions: A total of 170 putative surface protein sequences with identified orthologs in both M. hyopneumoniae and M. flocculare (MHP and MF, respectively) were analyzed. From this survey, three pairs of orthologs were selected for functional analysis, based on the presence of differential amino acid stretches in predicted extracellular domains. In the first pair (MHP7448-0556 and MF-00306), the M. flocculare protein presents an exclusive 53 aa-long stretch in its N-terminal extracellular end. In the second pair (MHP7448-0094 and MF-00500) less conserved N-terminal sequences (40.5% identical) are present, in comparison to the rest of the proteins (80% identical). And, finally, the third pair (MHP7448-0612 and MF-00357) showed an 114 aalong stretch with 35% identity between orthologs, flanked by regions with 70%-80% identity. This third pair of orthologs had the differential domains (with 306 bp and 393 bp, respectively) cloned and expressed in E. coli. Star and pLysE strains. GST-tagged recombinant polypeptides of 37 kDa and 40 kDa were expressed and, after purification and cleavage with thrombin, 11.4 kDa and 14.7 kDa polypeptides were recovered for MHP7448-0612 and MF-00357, respectively. The differential domains of the other two pairs of selected orthologs will be also cloned and expressed in E. coli and all the recombinant polypeptides from M. hyopneumoniae and M. flocculare produced will be used for immunization of mice, in order to get evidence of potential differences between the immune responses induced by each ortholog.