Variable protection against experimental broiler necrotic enteritis after immunisation with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant

Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis (NE), a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunisation with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247–370) would provide protection against experimental NE. Immunised animals with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247–370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed challenge

Identification of a key residue for Oligomerisation and pore-formation of Clostridium perfringens NetB

Necrotic enteritis toxin B (NetB) is a β-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis

Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein.

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin-Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore-form of NetB solved to 3.9Å. The heptameric assembly shares structural homology to the Staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerisation and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology

Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids

NetB (necrotic enteritis toxin B) is a recently identified β-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease

The conserved translocase Tim17 prevents mitochondrial DNA loss

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease