SummaryMycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A 271), two decapeptide repeats (designated R2271), and a second set of six pentapeptide repeats (designated R1B271). To determine their function, recombinant proteins containing R1A271 (F1271) and R2271-R1B271 (F2271) were constructed and used in in vitro binding assays. F2271, but not F1271, bound heparin (KD = 8.1 Ϯ 0.4 nM), fibronectin (KD = 174 Ϯ 13 nM) and porcine cilia. Pre-incubation of F2271 with 100 mM heparin blocked cilium binding bỹ 69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules.

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K D 24 ؎ 6 nM). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K D 44 ؎ 5 nM). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

SummaryMycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K D~76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K Ds of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.

ABSTRACTMycoplasma hyopneumoniaecauses 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 P85P146that 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 sequence672ATEF↓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-F3P146that mimic P50P146, P40P146, and P85P146were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146generated fromM. hyopneumoniaestrain 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 ofM. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by whichM. hyopneumoniaeregulates protein topography.IMPORTANCEVaccines used to controlMycoplasma hyopneumoniaeinfection 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 ofM. 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 ofM. hyopneumoniae.