Significance: Vibrio cholerae , responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Colorectal cancer is one of the leading causes of cancer‐related death worldwide. The adenomatous polyposis coli (APC) gene is mutated in hereditary colorectal tumors and in more than 80% of sporadic colorectal tumors. APC mutations impair β‐catenin degradation, leading to its permanent stabilization and increased transcription of cancer‐driving target genes. In colon cancer, impairment of β‐catenin degradation leads to its cytoplasmic accumulation, nuclear translocation, and subsequent activation of tumor cell proliferation. Suppressing β‐catenin signaling in cancer cells therefore appears to be a promising strategy for new anticancer strategies. Recently, we discovered a novel Vibrio cholerae cytotoxin, motility‐associated killing factor A (MakA), that affects both invertebrate and vertebrate hosts. It promotes bacterial survival and proliferation in invertebrate predators but has unknown biological role(s) in mammalian hosts. Here, we report that MakA can cause lethality of tumor cells via induction of apoptosis. Interestingly, MakA exhibited potent cytotoxic activity, in particular against several tested cancer cell lines, while appearing less toxic toward nontransformed cells. MakA bound to the tumor cell surface became internalized into the endolysosomal compartment and induced leakage of endolysosomal membranes, causing cytosolic release of cathepsins and activation of proapoptotic proteins. In addition, MakA altered β‐catenin integrity in colon cancer cells, partly through a caspase‐ and proteasome‐dependent mechanism. Importantly, MakA inhibited β‐catenin‐mediated tumor cell proliferation. Remarkably, intratumor injection of MakA significantly reduced tumor development in a colon cancer murine solid tumor model. These data identify MakA as a novel candidate to be considered in new strategies for development of therapeutic agents against colon cancer.

Bacterial membrane vesicles (BMVs) are spherical extracellular organelles whose cargo is enclosed by a biological membrane. The cargo can be delivered to distant parts of a given habitat in a protected and concentrated manner. This review presents current knowledge about BMVs in the context of bacterial eco-evolutionary dynamics among different environments and hosts. BMVs may play an important role in establishing and stabilizing bacterial communities in such environments; for example, bacterial populations may benefit from BMVs to delay the negative effect of certain evolutionary trade-offs that can result in deleterious phenotypes. BMVs can also perform ecosystem engineering by serving as detergents, mediators in biochemical cycles, components of different biofilms, substrates for cross-feeding, defense systems against different dangers and enzyme-delivery mechanisms that can change substrate availability. BMVs further contribute to bacteria as mediators in different interactions, with either other bacterial species or their hosts. In short, BMVs extend and deliver phenotypic traits that can have ecological and evolutionary value to both their producers and the ecosystem as a whole.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.