Year

2010

Degree Name

Doctor of Philosophy

Department

University of Wollongong. School of Biological Sciences

Abstract

M. hyopneumoniae is the causative agent of porcine enzootic pneumonia (PEP) and inflicts a severe economic burden on swine production. Colonisation involves adherence of M. hyopneumoniae to the porcine respiratory epithelia in a process believed to be mediated by surface proteins capable of binding target cells. This thesis involved the identification and characterisation of several immunoreactive M. hyopneumoniae antigens in an effort to increase the current understanding of the surface topography of this organism.

The first antigen identified in this research was the now well-characterised cilium adhesin of M. hyopneumoniae, P97. P97 was found in earlier research to contain two regions of reiterated repeats (RR1 and RR2). The repeats were found in this research to differ in number amongst strains of M. hyopneumoniae from different geographical localities and did not appear present in related porcine mycoplasmas M. hyorhinis or M. flocculare. Sequences like the proline rich tandem repeats of 5 and 10 amino acids found in RR1 and RR2 respectively are typically involved in protein-protein interactions. Proline rich repeats are usually highly immunogenic (Williamson, 1994) providing the recombinant 35 kDa antigen (encompassing the repeats) produced in this work with vaccine potential.

The second antigen cloned and characterised in this research was identified as a fragment of M. hyopneumoniae pyruvate dehydrogenase E1α (PdhA). Its intracellular location was indicated in trypsination studies of whole M. hyopneumoniae but its identification in the insoluble phase as well as the aqueous phase of triton extractions raises the question of a dual function; a phenomenon reported for PdhA in M. pneumoniae (Layh-Scmitt et al., 2000) and for NrdF in M. hyopneumonia (Fagan et al., 1996).

The final antigen characterised in this research was identified as a 216 kDa adhesin-like protein of M. hyopneumoniae (P216). Four recombinant P216 antigens, covering 75% of the molecule, were cloned, expressed, purified and used to raise antisera in rabbits. P216 was found to be rapidly processed to cleavage fragments P120 and P85, both located on the surface of M. hyopneumoniae. A putative transmembrane domain was identified in P120 providing it with a mechanism for cell surface attachment. It has been hypothesized that P85 maintains its association with the cell surface through a KEKE motif and the two regions of glutamine repeats identified in its’ sequence. Heparin binding of all three recombinant P216 fragments suggests that P120 contains at least two heparin-binding domains and P85 at least one, but further studies are required to define the exact location of these sites.

This research provided a foundation for continued research on P97 and P216, which remain the only two cilium adhesins of M. hyopneumoniae identified to date. Defining the colonisation mechanism of M. hyopneumoniae has implications for the treatment and prevention of disease, therefore research in this area provides an improved understanding of pathogenesis and potential vaccine development.

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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.