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Proteomic Study on Iron-regulated Outer Membrane Proteins of Two Pasteurella Multocida Strains

Author: ZuoDongDong
Tutor: YuXuPing
School: Zhejiang University
Course: Preventive Veterinary Medicine
Keywords: Pasteurella multocida IROMPs Proteomics SDS-PAGE 2-DE MALDI-TOF
CLC: S852.61
Type: Master's thesis
Year: 2007
Downloads: 5
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Abstract

Pasteurella multocida is a gram-negative bacterium which can cause numerous diseases in a wide range of animal species. It is the causative agent of numerous, economically important diseases, including avian fowl cholera, swine pasteurellosis, bovine haemorrhagic septicaemia, swine atrophic rhinitis, and even human infections caused by animal bites. Fowl cholera, which is generally caused by serotypes A:1, A:3 or A:4, results in significant economic losses to poultry industries worldwide. Regular outbreaks also occur in wild bird populations, resulting high mortality.Out membrane proteins （OMPs） of Pasteurella multocida play an important role in infection and pathogencity, such as invasion, adhesion, iron absorption, toxin secretion and resistance to antibiotics. The expression of various OMPs is influenced by the extracellular environment. Grown in iron-depleted medium, Pasteurella multocida regulates the OMPs’expression to achieve enough iron to live. The present study focused on the alteration of OMPs from Pasteurella multocida C48-19 （serotype A:1） and P1059（serotype A:1） grown in normal and iron depleted brain heart infusion medium （BHI）OMPs of these two stains grown in BHI or BHI broth containing the iron chelator dipyridyl were extracted by SLS method and analyzed by SDS-PAGE. OMPs profiles of these two strains grown in brain heart infusion were heterogeneous, with 39kD and 37kD protein being the major OMP of C48-19 and P1059 respectively. Both strains expressed additional OMPs while grown in brain-heart infusion broth containing the iron chelator dipyridyl. These OMPs were merely expressed in iron deficient medium and were considered to be iron-regulated outer membrane proteins （IROMPs）. The SDS-PAGE patterns showed the existence of 87, 81,79,64,60.5kD IROMPs in both strains. The following western blotting demonstrated that 87,79,60.5kD proteins can be recognized by the convalescent serum from chicken recovered form C48-19’s infection. Furthermore, OMPs of C48-19 grown in chicken allantolic fluid shared similar pattern with those grown in iron depleted media.2-dimentional gel electrophoresis （2-DE） was used to the further study of these IROMPs. We found that there was no significant difference among most of the protein’s expression when C48-19 was grown in BHI or iron-depleted BHI medium. However,7 protein spots changed more than 3 folds in expression level compared with grown in BHI medium, and 9 spots only expressed in iron-depleted medium. Further western-blotting revealed that 2 of these changed proteins can be recognized by the convalescent serum from chicken recovered form C48-19’s infection. Cultivated in iron-depleted medium, P1059 expressed 4 protein spots specifically, with absence of 5 protein spots that expressed in BHI medium.Interested proteins from SDS-PAGE and 2-DE of C48-19 were in-gel Digested for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein C₁02kD, C₈7kD form SDS-PAGE were identified to be hypothetical protein PM0336 and Omp87 of Pm respectively; protein C₄ and C₁6 were identified as the GroEL and hypothetical protein PM1974. PM0336 is highly conserved and is homogeneous with hemoglobin receptor of other bacterium. This protein may be involved in iron absorbtion system when C48-19 was grown in animals.

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