Genetic and functional analysis of the novel toxin NetB from clostridium perfringens chicken necrotic enteritis isolates

The overall objective of this thesis was to develop a structural and functional understanding of NetB toxin from chicken necrotic enteritis isolate of Clostridium perfringens. The thesis contains a literature review, three published papers presenting the research outcomes of this project, and a general discussion. The impetus for this research project was the discovering of NetB toxin and its implication for the development of vaccines that could be used to prevent necrotic enteritis in commercial poultry flocks. The aims of this PhD project were (i) to determine the genetic variation of the netB gene (Chapter 2); (ii) to localize the netB gene in the necrotic enteritis-causing type A C. perfringens isolate EHE-NE18 (Chapter 3); and (iii) to identify the functional domains of the NetB protein (Chapter 4). To investigate netB genetic variation, the netB genes from 23 necrotic enteritis-causing C. perfringens strains were sequenced and analyzed, which indicated that the netB gene was highly conserved. The majority (17/23) were completely identical in the netB coding region at the nucleotide level, only one variant, NetBA168T, was identified in six strains at the amino acid level. Biological analysis of NetBA168T suggested that it was as active as wild type NetB toxin. To answer the question whether the netB gene was carried on a conjugative plasmid, conjugation experiments and subsequent plasmid sequencing analysis were carried out, which revealed that the netB gene was located on a conjugative plasmid pJIR3535 independent from a tetracycline conjugative plasmid pJIR3537, and a third plasmid, pJIR3844, carrying a β2-toxin-encoding gene. To determine the functional domains of NetB toxin, site-directed and random mutagenesis analysis of netB gene were carried out, and several critical residues of NetB were identified. Biological analysis of these mutants demonstrated that some of these derivatives had almost completely lost haemolytic activity, including R230Q, W287R and S254L. Mapping them onto the crystal structure of the NetB monomer indicated that residue R230 and W287 were located in the potential binding domain, and S254 was in the putative oligomerization region. All of these results have been published and the papers are presented as Chapter 2, 3 and 4. In conclusion, the research described in this thesis determined that the netB gene was highly conserved and located on a conjugative plasmid. It revealed that three closely related independent conjugative The overall objective of this thesis was to develop a structural and functional understanding of NetB toxin from chicken necrotic enteritis isolate of Clostridium perfringens. The thesis contains a literature review, three published papers presenting the research outcomes of this project, and a general discussion. The impetus for this research project was the discovering of NetB toxin and its implication for the development of vaccines that could be used to prevent necrotic enteritis in commercial poultry flocks. The aims of this PhD project were (i) to determine the genetic variation of the netB gene (Chapter 2); (ii) to localize the netB gene in the necrotic enteritis-causing type A C. perfringens isolate EHE-NE18 (Chapter 3); and (iii) to identify the functional domains of the NetB protein (Chapter 4). To investigate netB genetic variation, the netB genes from 23 necrotic enteritis-causing C. perfringens strains were sequenced and analyzed, which indicated that the netB gene was highly conserved. The majority (17/23) were completely identical in the netB coding region at the nucleotide level, only one variant, NetBA168T, was identified in six strains at the amino acid level. Biological analysis of NetBA168T suggested that it was as active as wild type NetB toxin. To answer the question whether the netB gene was carried on a conjugative plasmid, conjugation experiments and subsequent plasmid sequencing analysis were carried out, which revealed that the netB gene was located on a conjugative plasmid pJIR3535 independent from a tetracycline conjugative plasmid pJIR3537, and a third plasmid, pJIR3844, carrying a β2-toxin-encoding gene. To determine the functional domains of NetB toxin, site-directed and random mutagenesis analysis of netB gene were carried out, and several critical residues of NetB were identified. Biological analysis of these mutants demonstrated that some of these derivatives had almost completely lost haemolytic activity, including R230Q, W287R and S254L. Mapping them onto the crystal structure of the NetB monomer indicated that residue R230 and W287 were located in the potential binding domain, and S254 was in the putative oligomerization region. All of these results have been published and the papers are presented as Chapter 2, 3 and 4. In conclusion, the research described in this thesis determined that the netB gene was highly conserved and located on a conjugative plasmid. It revealed that three closely related independent conjugative plasmids were carried in one necrotic enteritis strain. Also it generated several mutants of netB, and their derivatives were demonstrated to be critical for NetB function. The findings of this thesis therefore provided an increased understanding of the structure and function of NetB toxin, which is significant for control strategies for chicken necrotic enteritis, especially for development of new vaccines.