Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography

ABSTRACT Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections....

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Autores principales:	Daniel R. Bogema, Ania T. Deutscher, Lauren K. Woolley, Lisa M. Seymour, Benjamin B. A. Raymond, Jessica L. Tacchi, Matthew P. Padula, Nicholas E. Dixon, F. Chris Minion, Cheryl Jenkins, Mark J. Walker, Steven P. Djordjevic
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/45d56da8c52a49888729027e2bca1d13
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spelling	oai:doaj.org-article:45d56da8c52a49888729027e2bca1d132021-11-15T15:39:08ZCharacterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography10.1128/mBio.00282-112150-7511https://doaj.org/article/45d56da8c52a49888729027e2bca1d132012-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00282-11https://doaj.org/toc/2150-7511ABSTRACT Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146 that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence 672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146 that mimic P50P146, P40P146, and P85P146 were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146 generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. IMPORTANCE Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50P146, P40P146, and P85P146. Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.Daniel R. BogemaAnia T. DeutscherLauren K. WoolleyLisa M. SeymourBenjamin B. A. RaymondJessica L. TacchiMatthew P. PadulaNicholas E. DixonF. Chris MinionCheryl JenkinsMark J. WalkerSteven P. DjordjevicAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 2 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Daniel R. Bogema Ania T. Deutscher Lauren K. Woolley Lisa M. Seymour Benjamin B. A. Raymond Jessica L. Tacchi Matthew P. Padula Nicholas E. Dixon F. Chris Minion Cheryl Jenkins Mark J. Walker Steven P. Djordjevic Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
description	ABSTRACT Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146 that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence 672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146 that mimic P50P146, P40P146, and P85P146 were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146 generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. IMPORTANCE Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50P146, P40P146, and P85P146. Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.
format	article
author	Daniel R. Bogema Ania T. Deutscher Lauren K. Woolley Lisa M. Seymour Benjamin B. A. Raymond Jessica L. Tacchi Matthew P. Padula Nicholas E. Dixon F. Chris Minion Cheryl Jenkins Mark J. Walker Steven P. Djordjevic
author_facet	Daniel R. Bogema Ania T. Deutscher Lauren K. Woolley Lisa M. Seymour Benjamin B. A. Raymond Jessica L. Tacchi Matthew P. Padula Nicholas E. Dixon F. Chris Minion Cheryl Jenkins Mark J. Walker Steven P. Djordjevic
author_sort	Daniel R. Bogema
title	Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
title_short	Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
title_full	Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
title_fullStr	Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
title_full_unstemmed	Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography
title_sort	characterization of cleavage events in the multifunctional cilium adhesin mhp684 (p146) reveals a mechanism by which <named-content content-type="genus-species">mycoplasma hyopneumoniae</named-content> regulates surface topography
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/45d56da8c52a49888729027e2bca1d13
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Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which <named-content content-type="genus-species">Mycoplasma hyopneumoniae</named-content> Regulates Surface Topography

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