Comparative proteomic analyses of outer membranes and outer membrane vesicles of Pasteurella multocida isolates recovered from different host species

Sulaiman, Nurshahira (2017) Comparative proteomic analyses of outer membranes and outer membrane vesicles of Pasteurella multocida isolates recovered from different host species. PhD thesis, University of Glasgow.

Abstract

Pasteurella multocida is a Gram-negative bacterium that resides in the upper respiratory tract of domesticated animals. It is responsible for pneumonia in cattle, sheep and pigs, atrophic rhinitis in pigs, fowl cholera in poultry and haemorrhagic septicaemia of cattle and buffaloes. Different strains of P. multocida are associated with disease in different host species but little is known about the molecular basis of virulence and host-specificity in this pathogen. The outer membrane of P. multocida is at the interface between pathogen and host and it is highly likely that outer membrane proteins (OMPs) and lipopolysaccharide (LPS) are involved in disease pathogenesis and host-specificity. Outer membrane vesicles (OMVs) are derived from the outer membrane and there is increasing evidence of their involvement in disease pathogenesis. Hence, the present study aimed to compare the composition of OMPs and OMVs derived from different strains of P. multocida using proteomic approaches.
Proteomic characterisation was carried out on the outer membranes of eight P. multocida isolates using gel-based and gel-free methods. A combination of gel-based and gel-free proteomic approaches identified a total of 67 OMPs associated with outer membrane biogenesis and integrity (11 proteins), transport and receptor activities (19 proteins), adherence (9 proteins), enzymatic activity (6 proteins) and unknown functions (22 proteins). Of these, 44 proteins were identified by both methods, 21 proteins were identified only by the gel-based method and two proteins were identified only by the gel-free approach. The study also characterised OMV production from the same P. multocida isolates and assessed the optimal OMV protein concentrations at different stage of growth. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that OMVs were enriched with OMPs and LPS. The OmpA protein was used as an outer membrane marker protein and its presence in OMVs was demonstrated by Western-blotting and immunogold-labelling.
Proteomic analysis of the OMVs derived from the same eight P. multocida isolates was carried out by the gel-free approach in three biological replicates. A total of 200 proteins were identified and these included 58 OMPs, 43 periplasmic proteins, eight inner membrane proteins, 85 cytoplasmic proteins and six extracellular proteins. Interestingly, the number of proteins identified in the OMVs varied in different P. multocida isolates. Subsequently, OMPs identified in the outer membrane and OMV fractions were compared and the findings showed that OMPs associated with OM biogenesis and integrity were more likely to be present in the outer membrane, whereas OMPs associated with transport and receptor activities were highly enriched in the OMVs. These results highlight the potentially important role that OMVs of P. multocida play in disease pathogenesis and identified proteins for further evaluation as putative virulence factors and candidate vaccine antigens.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Pasteurella multocida, outer membrane proteins, outer membrane vesicles, proteomics.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Bacteriology
• Supervisor's Name: Davies, Dr. Robert and Burchmore, Dr. Richard
• Date of Award: 2017
• Embargo Date: 19 December 2021
• Depositing User: Miss Nurshahira Sulaiman
• Unique ID: glathesis:2017-8639
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 Jan 2018 14:25
• Last Modified: 04 Jan 2021 07:43
• URI: https://theses.gla.ac.uk/id/eprint/8639