Staphylococcus aureus α-toxin: small pore, large consequences.

The small ß-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.

Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action.

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.

Phobalysin, a Small ß-Pore-Forming Toxin of Photobacterium damselae subsp. damselae.

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 ß-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.

A subunit of eukaryotic translation initiation factor 2α-phosphatase (CreP/PPP1R15B) regulates membrane traffic.

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.

Modulation of translation and induction of autophagy by bacterial exoproducts.

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.

Pro-autophagic signal induction by bacterial pore-forming toxins.

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.

Staphylococcus aureus α-Toxin's Close Contacts Ensure the Kill.

The membrane pore-forming α-toxin is an important virulence factor of Staphylococcus aureus. Target cells can remove pores from their surface, but recent work shows that α-toxin may undermine this self-defense by clinging to epithelial cell junctions. The findings could lead to the development of novel remedies against S. aureus infections.

Phobalysin: Fisheye View of Membrane Perforation, Repair, Chemotaxis and Adhesion.

Phobalysin P (PhlyP, for photobacterial lysin encoded on a plasmid) is a recently described small ß-pore forming toxin of Photobacterium damselae subsp. damselae (Pdd). This organism, belonging to the family of Vibrionaceae, is an emerging pathogen of fish and various marine animals, which occasionally causes life-threatening soft tissue infections and septicemia in humans. By using genetically modified Pdd strains, PhlyP was found to be an important virulence factor. More recently, in vitro studies with purified PhlyP elucidated some basic consequences of pore formation. Being the first bacterial small ß-pore forming toxin shown to trigger calcium-influx dependent membrane repair, PhlyP has advanced to a revealing model toxin to study this important cellular function. Further, results from co-culture experiments employing various Pdd strains and epithelial cells together with data on other bacterial toxins indicate that limited membrane damage may generally enhance the association of bacteria with target cells. Thereby, remodeling of plasma membrane and cytoskeleton during membrane repair could be involved. In addition, a chemotaxis-dependent attack-and track mechanism influenced by environmental factors like salinity may contribute to PhlyP-dependent association of Pdd with cells. Obviously, a synoptic approach is required to capture the regulatory links governing the interaction of Pdd with target cells. The characterization of Pdd's secretome may hold additional clues because it may lead to the identification of proteases activating PhlyP's pro-form. Current findings on PhlyP support the notion that pore forming toxins are not just killer proteins but serve bacteria to fulfill more subtle functions, like accessing their host.

Cytotoxin- and Chemotaxis-Genes Cooperate to Promote Adhesion of Photobacterium damselae subsp. damselae.

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.

Repair of a Bacterial Small ß-Barrel Toxin Pore Depends on Channel Width.

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 ß-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 ß-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.IMPORTANCE Previous studies demonstrated that large transmembrane pores, such as those formed by perforin or bacterial toxins of the cholesterol-dependent cytolysin family, trigger rapid, Ca2+ influx-dependent repair mechanisms. In contrast, recovery from attack by the small ß-pore-forming Staphylococcus aureus alpha-toxin or aerolysin is slow in comparison and does not depend on extracellular Ca2+ To further elucidate the scope of Ca2+ influx-dependent repair and understand its limitations, we compared the cellular responses to phobalysin and V. cholerae cytolysin, two related small ß-pore-forming toxins which create membrane pores of slightly different sizes. The data indicate that the channel width of a small ß-pore-forming toxin is a critical determinant of both primary toxicity and susceptibility to Ca2+-dependent repair.

eIF2α Confers Cellular Tolerance to S. aureus α-Toxin.

We report on the role of conserved stress-response pathways for cellular tolerance to a pore forming toxin. First, we observed that small molecular weight inhibitors including of eIF2α-phosphatase, jun-N-terminal kinase (JNK), and PI3-kinase sensitized normal mouse embryonal fibroblasts (MEFs) to the small pore forming S. aureus α-toxin. Sensitization depended on expression of mADAM10, the murine ortholog of a proposed high-affinity receptor for α-toxin in human cells. Similarly, eIF2α (S51A/S51A) MEFs, which harbor an Ala knock-in mutation at the regulated Ser51 phosphorylation site of eukaryotic translation initiation factor 2α, were hyper-sensitive to α-toxin. Inhibition of translation with cycloheximide did not mimic the tolerogenic effect of eIF2α-phosphorylation. Notably, eIF2α-dependent tolerance of MEFs was toxin-selective, as wild-type MEFs and eIF2α (S51A/S51A) MEFs exhibited virtually equal sensitivity to Vibrio cholerae cytolysin. Binding of S. aureus α-toxin to eIF2α (S51A/S51A) MEFs and toxicity in these cells were enhanced as compared to wild-type cells. This led to the unexpected finding that the mutant cells carried more ADAM10. Because basal phosphorylation of eIF2α in MEFs required amino acid deprivation-activated eIF2α-kinase 4/GCN2, the data reveal that basal activity of this kinase mediates tolerance of MEFs to α-toxin. Further, they suggest that modulation of ADAM10 is involved. During infection, bacterial growth may cause nutrient shortage in tissues, which might activate this response. Tolerance to α-toxin was robust in macrophages and did not depend on GCN2. However, JNKs appeared to play a role, suggesting differential cell type and toxin selectivity of tolerogenic stress responses. Understanding their function or failure will be important to comprehend anti-bacterial immune responses.