Year

2011

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

Porcine enzootic pneumoniae is a chronic disease of swine affecting herds worldwide. Mycoplasma hyopneumoniae is the causative pathogen and disease increases susceptibility of the host to secondary pathogens. The cilium adhesin, P97, has been identified as an important adhesin. Heparin binding domains are known to contribute to the adherence potential of P97, but other contributing mechanisms remain unknown. In this study surface plasmon resonance was used to investigate the ability of P97 domains to interact with plasminogen and fibronectin. Plasminogen is a proenzyme, which when activated, is capable of extracellular matrix degradation. A P97 domain containing the R2 region was able to interact with plasminogen (KD = 13 ± 2 nM). Plasminogen binding is often mediated by a C-terminal lysine, when the C-terminal lysine from the R2 region of P97 was deleted, plasminogen binding still occurred, although a decrease in the level of binding was observed. Fibronectin is an extracellular matrix component. An ability to bind fibronectin has been shown to be advantageous for pathogenic bacteria, providing a mechanism for adherence and potentially triggering cytoskeletal rearrangements. The R2 repeat region of P97 bound fibronectin in a physiologically manner with a KD = 18 ± 5 nM, while the R1 + R2 repeat regions bound with a KD of 21 ± 4 nM.

P97 is encoded by mhp183 which is located in a two gene operon with mhp182, encoding P102. Although the function of P102 is unknown, it is expressed during disease and is able to associate with cilia. The genetic linkage between P97 and P102 suggests that P102 has a function related to P97, in the M. hyopneumoniae adherence process. Here we demonstrate that as with P97, P102 is a fibronectin and plasminogen binding protein. P102 undergoes proteolytical processing to generate a surface located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) protein. Surface plasmon resonance was used to show that recombinant P102 (rP102) binds plasminogen in a physiologically relevant manner (KD ~76 nM) with two C-terminal tandem lysine residues contributing to binding. Recombinant proteins mimicking the natural cleavage fragments of P102, P60 (rP60) and P42 (rP42), also bound plasminogen at physiologically significant levels. The ability of plasminogen bound to the surface of M. hyopneumoniae to be biologically functional as the protease plasmin was also demonstrated. In the presence of tissue-specific plasminogen activator (tPA) surface-bound plasminogen was activated and shown to degrade fibrinogen. The presence of plasminogen in porcine ciliated airways was also established, where it is accessible to M. hyopneumoniae during colonisation. rP102 and rP42 were identified as fibronectin binding proteins using surface plasmon resonance (with KDs of 26 nM and 33 nM respectively).

Both P97 (mhp183) and P102 (mhp183) have six paralogs in the M. hyopneumoniae genome. Paralogs are genes which have resulted from a genetic duplication event, and often retain similar but not identical functions. There is a need to increase the understanding of this protein family and the role it plays in M. hyopneumoniae pathogenesis. Thus, we have characterised the proteins encoded by the genes mhp107 and mhp108 which are found in a two gene structure. Mhp107 is a P97 paralog encoded by mhp107. Here we demonstrate that Mhp107 is a surface located multifunctional adhesin. The N-terminal fragment (F1Mhp107) of Mhp107 bound heparin in a dose-dependant, physiologically relevant manner with a KD of 87 ± 9 nM. Surface plasmon resonance was used to show that the C-terminal fragment of Mhp107 (F3Mhp107) binds fibronectin at physiologically relevant concentration (KD = 174 ± 80 nM) and F1Mhp107 binds plasminogen with a KD of 24 ± 2 nM with binding mediated via lysine residues. F3Mhp107 mediated binding to the porcine kidney epithelial-like (PK15) cell line, while F1-F3Mhp107 adhered to swine respiratory cilia.

Likewise we investigated the potential role as an adhesin for the P102 paralog, P116, encoded by mhp108. Proteomic analyses showed P116 is surface located and expressed as a full length protein, but that P116 also undergoes cleavage events with P116 fragments of 17 – 70 kDa observed. Binding studies showed that M. hyopneumoniae binds fibronectin in a dose-dependant and physiologically relevant manner. The Cterminal domain of P116 is a fibronectin binding protein (KD = 24 ± 6 nM). M. hyopneumoniae was able to bind plasminogen in a dose-dependant and physiologically relevant manner. The C-terminal domain was also elucidated as a plasminogen binding protein (KD = 44 ± 5 nM) with a C-terminal lysine residue critical to the plasminogen binding site. P116 fragments are also able to mediate binding to PK15 cells and adhere to swine respiratory cilia. Here we have substantially contributed to understanding the function of the P97/P102 paralog family. Together this data provides strong evidence for a family of multifunctional adhesins which are important M. hyopneumoniae virulence factors.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.