Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

Platelet Endothelial Cell Adhesion Molecule 1 (CD31) Is Essential for Clostridium perfringens Beta-Toxin Mediated Cytotoxicity in Human Endothelial and Monocytic Cells.

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (ß-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals.

CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens ß-Toxin.

Clostridium perfringens ß-toxin (CPB) is a highly active ß-pore-forming toxin (ß-PFT) and the essential virulence factor for fatal, necro-hemorrhagic enteritis in animals and humans. The molecular mechanisms involved in CPB's action on its target, the endothelium of small intestinal vessels, are poorly understood. Here, we identify platelet endothelial cell adhesion molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression corresponds with the cell-type specificity of CPB, and it is essential for toxicity in cultured cells and mice. Ectopic CD31 expression renders resistant cells and liposomes susceptible to CPB-induced membrane damage. Moreover, the extracellular Ig6 domain of mouse, human, and porcine CD31 is essential for the interaction with CPB. Hence, our results explain the cell-type specificity of CPB in vitro and in the natural disease caused by C. perfringens type C.

Clostridium perfringens type C necrotic enteritis in pigs: diagnosis, pathogenesis, and prevention.

Clostridium perfringens type C causes severe and lethal necrotic enteritis (NE) in newborn piglets. NE is diagnosed through a combination of pathology and bacteriologic investigations. The hallmark lesion of NE is deep, segmental mucosal necrosis with marked hemorrhage of the small intestine. C. perfringens can be isolated from intestinal samples in acute cases but it is more challenging to identify pathogenic strains in subacute-to-chronic cases. Toxinotyping or genotyping is required to differentiate C. perfringens type C from commensal type A strains. Recent research has extended our knowledge about the pathogenesis of the disease, although important aspects remain to be determined. The pathogenesis involves rapid overgrowth of C. perfringens type C in the small intestine, inhibition of beta-toxin (CPB) degradation by trypsin inhibitors in the colostrum of sows, and most likely initial damage to the small intestinal epithelial barrier. CPB itself acts primarily on vascular endothelial cells in the mucosa and can also inhibit platelet function. Prevention of the disease is achieved by immunization of pregnant sows with C. perfringens type C toxoid vaccines, combined with proper sanitation on farms. For the implementation of prevention strategies, it is important to differentiate between disease-free and pathogen-free status of a herd. The latter is more challenging to maintain, given that C. perfringens type C can persist for a long time in the environment and in the intestinal tract of adult animals and thus can be distributed via clinically and bacteriologically inapparent carrier animals.

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module.

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.

Effect of Clostridium perfringens ß-Toxin on Platelets.

Clostridium perfringensß-toxin (CPB) is the major virulence factor of C.perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but without obvious thrombosis of affected vessels, suggesting altered hemostasis in the early phase of the disease. The objective of the present study was to investigate the effect of CPB on platelets, with respect to primary hemostasis. Our results demonstrate that CPB binds to porcine and human platelets and forms oligomers resulting in a time- and dose-dependent cell death. Platelets showed rapid ultrastructural changes, significantly decreased aggregation and could no longer be activated by thrombin. This indicates that CPB affects the physiological function of platelets and counteracts primary hemostasis. Our results add platelets to the list of target cells of CPB and extend the current hypothesis of its role in the pathogenesis of C. perfringens type C enteritis.

Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR.

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.