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Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae
Jody Wilton1, Cheryl Jenkins, Stuart J Cordwell
1NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden, NSW 2570, Australia.
Abstract:
Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO(2) chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide (90)VSELpSFR(96) (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1(P216), F2(P216), F3(P216)) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1(P216), F2(P216) and F3(P216) adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.
Insights
Mycoplasma hyopneumoniae uses Mhp493 to adhere to swine cilia, causing respiratory disease. This protein fragment, P85, binds heparin and is recognized by antibodies during infection, offering potential therapeutic targets.
Area of Science:
- Microbiology
- Veterinary Science
- Molecular Biology
Background:
- Mycoplasma hyopneumoniae causes swine respiratory disease by damaging cilia.
- Heparin binding to M. hyopneumoniae inhibits its adherence to cilia.
Purpose of the Study:
- Investigate the role of Mhp493 (P216) in M. hyopneumoniae adherence to swine cilia.
- Characterize the heparin-binding properties and potential immunogenicity of Mhp493 fragments.
Main Methods:
- Protein cleavage analysis and identification of heparin-binding domains.
- Mass spectrometry (MALDI-MS/MS) for phosphoserine residue identification.
- Binding assays using recombinant proteins and cell monolayers.
Main Results:
- Mhp493 is cleaved into P120 and P85, which bind heparin.
- A phosphoserine residue at position 94 in P85 is identified.
- Mhp493 fragments bind porcine cilia and are recognized by infection-elicited antibodies.
Conclusions:
- Mhp493 is a novel cilium adhesin contributing to M. hyopneumoniae surface architecture.
- Mhp493 fragments P120 and P85 are key for heparin binding and cilia interaction.
- This protein represents a potential target for controlling swine respiratory disease.
