Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

Abstract

Background: Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.

Results: In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains.

Conclusions: Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.

Figure 1

2DE proteome profiling the three M. hyopneumoniae strains with IEF at pH 4-7. Protein samples (2 mg) from the M. hyopneumoniae strains J (A), 7448 (B), and 7422 (C) were separated by IEF using 17 cm pH 4-7 IPG strips, followed by SDS-PAGE on 12% gels and stained with Coomassie Brilliant Blue G. The approximate molecular weights are shown on the left of the gel and the acid-to-alkaline gradient is from left to right. The rectangle delimited areas (numbered d1-d5) in the gels and panels in (D) show gel regions in which spots corresponding to differentially expressed proteins were identified. Spots corresponding to proteins identified by matching to the previously reported M. hyopneumoniae 7448 proteome maps [16] were named according to the predicted gene products. Spots corresponding to proteins thus far unidentified in proteome maps were numbered.

Figure 2

2DE proteome profiling of the three M. hyopneumoniae strains with IEF at pH 3-10. Protein samples (2 mg) from the three M. hyopneumoniae strains J (A), 7448 (B), and 7422 (C) were separated by IEF using 17 cm pH 3-10 IPG strips, followed by SDS-PAGE on 12% gels and stained with Coomassie brilliant blue G. The approximate molecular weights are shown on the left of the gel and the acid-to-alkaline gradient is from left to right. The rectangle delimited areas (numbered d1-d6) in the gels and panels in (D) show gel regions in which spots corresponding to differentially expressed proteins were identified. Spots corresponding to proteins identified by matching to the previously reported M. hyopneumoniae 7448 proteome maps [16] were named according to the predicted gene products or by the corresponding CDS number. Spots corresponding to proteins thus far unidentified in proteome maps were numbered.

Figure 3

2DE immunoblotting analyses of P97 adhesin. Proteins samples from the M. hyopneumoniae strains J (C), 7448 (B), and 7422 (A) were resolved by 2DE as described in Figures 1 and 2, electroblotted onto PVDF membranes, and probed with the anti-P97 monoclonal antibody F1B6 (1:400 dilution). Anti-mouse IgG alkaline phosphatase-labelled secondary antibody (1:2000 dilution) was used to develop antigen-antibody reactions. The approximate molecular weights are shown on the left of the gel and the acid-to-alkaline gradient is from left to right. Mature P97 (P97), its two main proteolytic products (a and b), and the P97 pathogenic-strain-specific, low-MW proteolytic products (1-4) are indicated.