Pore-forming toxins (PFT) comprise a large, structurally heterogeneous group of bacterial protein toxins. Nucleated target cells mount complex responses which allow them to survive moderate membrane damage by PFT. Autophagy has recently been implicated in responses to various PFT, but how this process is triggered is not known, and the significance of the phenomenon is not understood. Here, we show that S. aureus α-toxin, Vibrio cholerae cytolysin, streptolysin O and E. coli haemolysin activate two pathways leading to autophagy. The first pathway is triggered via AMP-activated protein kinase (AMPK). AMPK is a major energy sensor which induces autophagy by inhibiting the target of rapamycin complex 1 (TORC1) in response to a drop of the cellular ATP/AMP-ratio, as is also observed in response to membrane perforation. The second pathway is activated by the conserved eIF2α-kinase GCN2, which causes global translational arrest and promotes autophagy in response to starvation. The latter could be accounted for by impaired amino acid transport into target cells. Notably, PKR, an eIF2α-kinase which has been implicated in autophagy induction during viral infection, was also activated upon membrane perforation, and evidence was obtained that phosphorylation of eIF2α is required for the accumulation of autophagosomes in α-toxin-treated cells. Treatment with 3-methyl-adenine inhibited autophagy and disrupted the ability of cells to recover from sublethal attack by S. aureus α-toxin. We propose that PFT induce pro-autophagic signals through membrane perforation–dependent nutrient and energy depletion, and that an important function of autophagy in this context is to maintain metabolic homoeostasis.

c Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small ␤-pore-forming toxin, and termed it phobalysin P (PhlyP), for "photobacterial lysin encoded on a plasmid." PhlyP formed stable oligomers and small membrane pores, causing efflux of K ؉ , with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence.

The small β-pore-forming α-toxin, also termed α-hemolysin or Hla is considered to be an important virulence factor of Staphylococcus aureus. Perforation of the plasma membrane (PM) by Hla leads to uncontrolled flux of ions and water. Already a small number of toxin pores seems to be sufficient to induce complex cellular responses, many of which depend on the efflux of potassium. In this article, we discuss the implications of secondary membrane lesions, for example, by endogenous channels, for Hla-mediated toxicity, for calcium-influx and membrane repair. Activation of purinergic receptors has been proposed to be a major contributor to the lytic effects of various pore forming proteins, but new findings raise doubts that this holds true for Hla. However, the recently discovered cellular pore forming proteins gasdermin D and Mixed lineage kinase domain-like pseudokinase (MLKL) which perforate the PM from the cytosolic side might contribute to both calcium-influx-dependent damage and membrane repair. Activation of endogenous pore forming proteins by Hla above a threshold concentration could explain the apparent dependence of pore characteristics on toxin concentrations. If secondary membrane damage in the aftermath of Hla-attack contributes significantly to overall PM permeability, it might be an interesting target for new therapeutic approaches.

Background:The constitutive reverter of eIF2␣ phosphorylation (CReP/PPP1r15B) targets the catalytic subunit of protein phosphatase 1 (PP1c) to p-eIF2␣ to promote translation initiation. Results: CReP associates with membranes and regulates membrane traffic in a PP1c-independent manner. Conclusion: CReP is co-opted by the traffic machinery. Significance: The data reveal a novel link between the molecular machineries regulating translation and traffic.

Membrane repair emerges as an innate defense protecting target cells against bacterial pore-forming toxins. Here, we report the first paradigm of Ca2+-dependent repair following attack by a small β-pore-forming toxin, namely, plasmid-encoded phobalysin of Photobacterium damselae subsp. damselae. In striking contrast, Vibrio cholerae cytolysin, the closest ortholog of phobalysin, subverted repair. Mutational analysis uncovered a role of channel width in toxicity and repair. Thus, the replacement of serine at phobalysin´s presumed channel narrow point with the bulkier tryptophan, the corresponding residue in Vibrio cholerae cytolysin (W318), modulated Ca2+ influx, lysosomal exocytosis, and membrane repair. And yet, replacing tryptophan (W318) with serine in Vibrio cholerae cytolysin enhanced toxicity. The data reveal divergent strategies evolved by two related small β-pore-forming toxins to manipulate target cells: phobalysin leads to fulminant perturbation of ion concentrations, closely followed by Ca2+ influx-dependent membrane repair. In contrast, V. cholerae cytolysin causes insidious perturbations and escapes control by the cellular wounded membrane repair-like response.

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not known how α-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all α-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of α-toxin, leading to significant increases in intracellular [Ca(2+)]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for α-toxin-dependent xenophagic targeting of S. aureus, whereas a role for α-toxin attack on the plasma membrane was confirmed. The catalytic site of ADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for α-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of α-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of α-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus α-toxin action.

Autophagy is a catabolic process of paramount importance for cellular homeostasis during starvation. Generally, autophagy and translation are inversely regulated. Many kinds of stress lead to attenuation of translation via phosphorylation of eukaryotic translation initiation factor alpha (eIF2α). This response is conserved from yeast to man and can be either protective or detrimental depending on strength and duration of stress, and additional factors. During starvation or viral infection, phosphorylation of eIF2α is required for induction of autophagy. As exemplified here by α-hemolysin, a small pore-forming toxin (PFT) of Staphylococcus aureus and (S)-3-oxo-C12-homoserine lactone [(S)-3-oxo-C12-HSL], a quorum-sensing hormone of Pseudomonas aeruginosa, bacterial exoproducts may also impact translation and autophagy. Thereby, PFT and (S)-3-oxo-C12-HSL act differentially. Damage of the plasma membrane by PFT causes efflux of potassium, which leads to amino acid starvation and energy loss. This triggers amino acid-sensitive eIF2α-kinase GCN2, as well as energy sensor AMPK, and deactivates mTORC1. The output of this response, that is, transient metabolic reprogramming is an essential part of a defense program which enables cells to survive attack by a pore-forming agent. Thus, nutrient/energy sensors serve as sentinels of plasma membrane integrity. In contrast to PFT, (S)-3-oxo-C12-HSL does not cause acute loss of ATP or activation of GCN2, but also triggers phosphorylation of eIF2α and inhibits translation. This response appears not to depend on efflux of potassium and requires eIF2α-kinase PKR. Like α-toxin, (S)-3-oxo-C12-HSL increases lipidation of LC3 and accumulation of autophagosomes in cells. Apart from directly affecting host-cell viability, bacterial exoproducts might galvanize bystander cells to prepare for close combat with microbial offenders or inadvertently accommodate some of them.

Photobacterium damselae subsp. damselae (Pdd) is an emerging pathogen of marine animals that sometimes causes serious infections in humans. Two related pore forming toxins, phobalysins P and C, and damselysin, a phospholipase D, confer strong virulence of Pdd in mice. Because infections by Pdd are typically caused following exposure of wounds to sea water we investigated how salinity impacts toxin activity, swimming, and association of Pdd with epithelial cells. These activities were low when bacteria were pre-cultured in media with 3.5% NaCl, the global average salinity of sea water. In contrast, lower salinity increased swimming of wild type Pdd peaking at 2% NaCl, hemolysis, and association with epithelial cells peaking at 1–1.5%. Previously, we have found that hemolysin genes enhance the association of Pdd with epithelial cells, but the underlying mechanisms have remained ill-defined. We here searched for potential links between hemolysin-production, chemotaxis and association of Pdd with target cells at varying salt concentrations. Unexpectedly, disruption of chemotaxis regulator cheA not only affected bacterial swimming and association with epithelial cells at intermediate to low salinity, but also reduced the production of plasmid-encoded phobalysin (PhlyP). The results thus reveal unforeseen links between chemotaxis regulators, a pore forming toxin and the association of a marine bacterium with target cells.