Doctor of Philosophy
School of Biological Sciences
Bogema, Daniel Ross, Genomic and proteomic analyses of Mycoplasma hyopneumoniae, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2012. http://ro.uow.edu.au/theses/3485
Mycoplasma hyopneumoniae is the aetiological agent of mycoplasmal enzootic pneumonia (MEP), a chronic respiratory disease affecting pigs that inflicts significant economic damage on the swine industry. M. hyopneumoniae causes MEP by colonizing the ciliated respiratory epithelia, causing extensive damage and disrupting mucociliary clearance. A group of highly cleaved adhesins, known as the P97/P102 family, have been identified as important in attachment of M. hyopneumoniae to respiratory cilia, as well as to biomolecules, such as fibronectin, plasminogen and glycosaminoglycans, which may be important to the pathogen’s colonisation and survival. While several P97/P102 family members have been characterized as adhesins, some of the more highly expressed members are yet to be examined. This study examined the posttranslational processing and binding capacity of two P97/P102 family members found to be highly expressed in broth culture, Mhp683 (P135) and Mhp684 (P146). Additionally, P97/P102 family members and the immunogenic lipoprotein P65 were compared at the gene and protein level in two variants of M. hyopneumoniae strain 232 that display high and low levels of adherence to porcine cilia.
Examination of post-translational processing in Mhp683 and Mhp684 revealed that both pre-proteins were cleaved into three fragments. Mhp683 fragments were identified at very similar masses and designated P45683, P48683 and P50683. N-terminal sequencing of the three Mhp683 fragments revealed (1) no signal peptide cleavage and (2) that cleavage occurred at the motif TTKF ↓ QE. This cleavage motif was found to be predictive of post-translational cleavage in P216 (Mhp493) and similar motifs are found in identified cleavage regions of several P97/P102 family members. All three Mhp683 fragments were found on the surface of M. hyopneumoniae and cleavage was consistent in multiple field isolates. Recombinant fragments (F1683 – F5683) spanning the entire Mhp683 sequence were shown to bind heparin in a dose-dependant and saturable manner that was inhibited by unlabelled heparin and other sulphated glycosaminoglycans, suggesting that sulphate groups are important in heparin binding. Denatured F4683 protein, representing a C-terminal portion of Mhp683, was also found to bind heparin, indicating that a linear heparin-binding motif is present in this fragment. Mhp683 expressed regions were found to bind cilia and antisera generatedfrom these recombinants were observed to significantly inhibit binding of M. hyopneumoniae cells to porcine cilia.
Mhp684 (P146) fragments were identified at three different masses in twodimensional gels and immunoblots and designated P40P146, P50P146 and P85P146. All three cleavage fragments were found on the surface of M. hyopneumoniae and immunoblots revealed that efficiency of proteolytic cleavage varied between field isolates. Three recombinant Mhp684 fragments (F1P146 – F3P146) were generated and two of these (N-terminal F1P146 and C-terminal F3P146) bound heparin in a dosedependant and saturable manner that was inhibited by unlabelled heparin and fucoidan, again suggesting sulphate groups are important in this interaction. However, mucin and chondroitin sulphate B did not inhibit this binding, suggesting that the position of sulphate groups is important. Versions of recombinant F3P146 from M. Hyopneumoniae strains J and 232, containing a C-terminal lysine or arginine respectively, bound porcine plasminogen in dot-blots and surface plasmon resonance experiments; an interaction that was significantly reduced by removal of the C-terminal residue, indicating that this residue is critical to plasminogen binding by P146.
Analysis of the gene and protein differences of P97/P102 family members in high and low adherence variants of strain 232 revealed only small differences in these variants. Immunoblots using antisera to P97/P102 family members revealed differences in the processing of one these proteins (Mhp107), but no significant differences in their expression were observed. Examination of the nucleotide sequences of the P97/P102 family members revealed no difference between high and low adherent variants; however, small differences between both of these variants and the published 232 genome sequence were identified. While the immunogenic lipoprotein P65 had previously been shown to be down-regulated in the low adherence variant, we found no evidence of this, as expression levels of P65 in the high and low adherence variants were not significantly different. Finally, despite the presence of a poly-adenine repeat near the promoter region of P65, we found no evidence that P65 underwent phase variation in both the high and low adherence variants and various laboratory strains and field isolates of M. hyopneumoniae.