Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae.
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.
Wilton
Jody
J
NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden, NSW 2570, Australia.
Jenkins
Cheryl
C
Cordwell
Stuart J
SJ
Falconer
Linda
L
Minion
F Chris
FC
Oneal
David C
DC
Djordjevic
Michael A
MA
Connolly
Angela
A
Barchia
Idris
I
Walker
Mark J
MJ
Djordjevic
Steven P
SP
D018829:Adhesins, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000378:metabolism; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006493:Heparin / Q000378:metabolism*; D008969:Molecular Sequence Data; D045705:Mycoplasma hyopneumoniae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013552:Swine
D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D011994:Recombinant Proteins; D006493:Heparin