Hounsome, Jonathan David Arthur (2012) Comparative outer membrane proteomic analyses of bovine and ovine isolates of Mannheimia haemolytica and Mannheimia glucosida. PhD thesis, University of Glasgow.Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.
In the present study, recombinant proteins representing the transmembrane domain of M. haemolytica outer membrane protein A (OmpA) from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were over-expressed, purified and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. An examination of the binding specificities of these antibodies to M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognised OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognised OmpA2-type proteins but not OmpA1 type proteins. This demonstrated that OmpA1 and OmpA2 are surface-exposed and could potentially bind to different receptors in cattle and sheep. The outer membrane subproteomes of seven M. haemolytica isolates and one M. glucosida isolate were also characterised and compared after growth in complex growth medium in order to identify OMPs with putative roles in host-specificity and virulence. First, a simple bioinformatic workflow (E-Komon et al., 2011b) was used to confidently predict 93 unique OMPs encoded among the genomes of a bovine serotype A1 M. haemolytica isolate and two serotype A2 isolates (one bovine and one ovine). Secondly, a combination of gel-based and gel-free proteomic approaches employing MALDI-TOF-TOF and LC-ESI-QqTOF mass spectrometry identified 55 unique OMPs among the outer membrane fractions of seven M. haemolytica isolates and one M. glucosida isolate (of which 50 were predicted by the bioinformatic approach). A role in host-specific adaptation could not be established for any of the identified OMPs, however, the study represents the most comprehensive analysis of M. haemolytica and M. glucosida outer membrane subproteomes to date. In order to identify putative virulence-associated OMPs, the outer membrane subproteomes of the same representative isolates were also characterised after in vitro growth under conditions that were designed to mimic the in vivo host respiratory tract microenvironment. These conditions included growth in iron-restricted medium, serum-supplemented tissue culture media and growth on solid-surface agar (in the absence or presence of Congo red). This approach allowed the identification of 13 additional OMPs that were not identified after growth in complex medium alone.
|Item Type:||Thesis (PhD)|
|Additional Information:||Chapter two has been published as follows: Hounsome, J. D. A., Baillie, S., Noofeli, M., Riboldi-Tunnicliffe, A., Burchmore, R. J. S., Isaacs, N. W. & Davies, R. L. (2011). Outer membrane protein A (OmpA) of bovine and ovine isolates of Mannheimia haemolytica is surface-exposed and contains host-species specific epitopes. Infection and Immunity 79, 4332-4341.|
|Keywords:||Mannheimia haemolytica, proteomics, bioinformatics, protein, purification, crystallisation, OmpA, outer membrane, outer membrane protein, OMP|
|Subjects:||Q Science > QR Microbiology|
|Colleges/Schools:||College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation|
|Supervisor's Name:||Davies, Dr. Robert|
|Date of Award:||2012|
|Embargo Date:||1 February 2015|
|Depositing User:||Mr Jonathan Hounsome|
|Copyright:||Copyright of this thesis is held by the author.|
|Date Deposited:||06 Feb 2012|
|Last Modified:||10 Dec 2012 14:04|
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