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Presence of PPE proteins in Mycobacterium avium subsp. paratuberculosis isolates and their immunogenicity in cattle
Victoria Newton1, Shawn L McKenna, Jeroen De Buck
1Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1.
Abstract:
Johne's disease or paratuberculosis in cattle is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although the stages of infection have been well described, very few virulence factors of MAP have been studied in detail. We aimed to study the localization and immunogenicity of members of the polymorphic PPE protein family which is unique to Mycobacteria and has been linked to virulence in Mycobacterium tuberculosis (Mtb). The presence of PPE proteins in the cell wall was investigated by enzymatic digest of surface exposed proteins of live MAP bacteria and analysis by LC-MS/MS. Polyclonal antisera were generated against a recombinant fragment of one PPE protein and a synthetic peptide of the other to confirm their surface exposure. Sera from naturally infected cows were investigated for the presence of specific antibodies against the recombinant PPE protein. Two PPE proteins, Map3420c and Map1506, were detected by mass spectrometry and confirmed to be surface exposed on live MAP cells by immunohistochemistry. The sera from naturally infected animals contained specific antibodies against recombinantly expressed Map3420c as demonstrated by western blotting. These findings show the in vitro expression of two PPE proteins. Additionally the surface exposure and immunogenicity of PPE proteins of MAP was demonstrated.
Insights
This study investigated PPE proteins in Mycobacterium avium subsp. paratuberculosis (MAP), the cause of Johne's disease in cattle. Two surface-exposed PPE proteins were identified and shown to be immunogenic in infected cattle.
Area of Science:
- Bacteriology
- Immunology
- Veterinary Medicine
Background:
- Johne's disease in cattle is caused by Mycobacterium avium subsp. paratuberculosis (MAP).
- Virulence factors of MAP are not well understood, despite detailed descriptions of infection stages.
- The polymorphic PPE protein family, unique to Mycobacteria, is linked to virulence in Mycobacterium tuberculosis.
Purpose of the Study:
- To investigate the localization and immunogenicity of PPE proteins in MAP.
- To determine if PPE proteins are surface-exposed on live MAP bacteria.
- To assess the immune response to MAP PPE proteins in naturally infected cattle.
Main Methods:
- Enzymatic digestion of surface-exposed proteins on live MAP followed by LC-MS/MS analysis.
- Generation of polyclonal antisera against recombinant PPE protein fragments and synthetic peptides.
- Immunohistochemistry to confirm surface localization and Western blotting to detect specific antibodies in cattle sera.
Main Results:
- Two PPE proteins, Map3420c and Map1506, were detected on the surface of live MAP.
- Immunohistochemistry confirmed the surface exposure of these two PPE proteins.
- Sera from cattle with natural Johne's disease contained antibodies against the recombinant Map3420c protein.
Conclusions:
- Two PPE proteins (Map3420c and Map1506) are expressed on the surface of MAP.
- These surface-exposed PPE proteins are immunogenic in cattle naturally infected with MAP.
- PPE proteins represent potential virulence factors and diagnostic targets for Johne's disease.
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