Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum. Key Points γδ T cells expand and develop cytolytic potential in response to malaria cultures. γδ T cells inhibit parasite growth in a contact- and granzyme-dependent manner. Granulysin delivers granzyme B into RBCs to kill late-stage parasites.

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-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.

Hemolysin β-pore-forming toxins (βPFTs) are key virulence factors of Clostridium perfringens , associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium βPFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 – as does the natural ANTXR2 ligand collagen type VI – NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin βPFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses.

Hemolysin β-pore-forming toxins (βPFTs) are key virulence factors of Clostridium perfringens , associated with severe diseases in humans and animals. Yet, the mechanisms by which Clostridium βPFTs recognize and engage specific target cells remain poorly understood. Here, we identify the cellular receptor for C. perfringens necrotizing enteritis toxin F (NetF), a recently discovered toxin implicated in severe enteritis in dogs and foals. We show that NetF binds to the same receptor as anthrax toxin, namely ANTXR2. Using cryo-electron microscopy, we determined the structure of the oligomeric NetF pre-pore as well as the transmembrane pore, both alone and in complex with the extracellular domain of ANTXR2. Unlike anthrax toxin, which binds to the apical MIDAS motif of ANTXR2 – as does the natural ANTXR2 ligand collagen type VI – NetF engages the receptor laterally, spanning both the von Willebrand A and the Ig-like domains. This interaction positions the toxin near the membrane, facilitating contact with membrane lipids and promoting transmembrane pore formation. Our findings uncover key principles of hemolysin βPFT-receptor recognition and advance our understanding of how pathogenic bacteria use these toxins to breach host defenses.

Several clostridial species can cause severe diseases in humans and animals. Among their most frequent virulence factors are hemolysin β-pore-forming toxins (β-PFTs). These protein toxins are secreted as soluble monomers that oligomerize on host cell membranes to form membrane-inserted β-barrel pores. Recent research on bacterial β-PFTs showed that many, including clostridial PFTs, target their host cells via specific membrane protein receptors. To date, 14 β-PFTs and related homologs have been identified in different Clostridium species, most of which remain uncharacterized. Here we report 11 additional putative β-PFT homologs identified across six Clostridium species through database analyses. We reviewed knowledge from genomic, in vitro and in vivo studies on clostridial β-PFTs, with emphasis on toxin- host cell recognition and the roles in the pathogenesis of diseases.