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Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF?QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683-F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683-F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family. [ABSTRACT FROM AUTHOR]

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcuspneumoniae n-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN[supK252 + 255A], SEN[supK344A], SEN[supK334A], SEN[supK344E], SEN[supK435L, and SEN[supΔ434-435]) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN[supK344E], as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN[supK3444E] had lost a-enolase activity, which was also reduced in SEN[supK334A] and SEN[supΔ434-435]. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN[supK252 + 255A], SEN[supK435L], and SEN[supΔ434-435]. The lysine residues at positions 252, 255,434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures. [ABSTRACT FROM AUTHOR]