Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

Significance of the lag phase in K1 killer toxin action on sensitive yeast cells

The minimum period between the addition of killer toxin K1 to sensitive yeast cells and the appearance of first cells stained with bromocresol purple indicating membrane damage, is approximately 20 min. The length of this lag phase depends strongly on toxin concentration, extending sharply at toxin levels lower than 60 lethal units (LU) per cell (about one-tenth of the toxin concentration necessary for saturating all surface receptors). As the binding of the toxin to the cell is virtually complete within 1 min, the rest of the lag phase reflects processes different from actual binding,e.g. combination of several toxin molecules to form a membrane ion channel or pore.     

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Zygocin,a secreted antifungal toxin of the yeast Zygosaccharomyces bailii,and its effect on sensitive fungal cells

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.     

MEMBRANE POTENTIAL DEPENDENT BINDING OF SCORPION TOXIN TO THE ACTION POTENTIAL SODIUM IONOPHORE. STUDIES WITH A 3-(4-HYDROXY 3-[125I] IODOPHENYL) PROPIONYL DERIVATIVE

Abstract— A polypeptide toxin purified 80-fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na⁺ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [¹²⁵I] by reaction with N-succinimidyl 3-(4-hydroxy 3-[¹²⁵I] iodophenyl) propionate. The [¹²⁵I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [¹²⁵I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na⁺ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na⁺ ionophore in variant neuroblastoma clones that specifically lack the action potential Na⁺ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na⁺ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm² of cell surface membrane. A single action potential Na⁺ ionophore transports 1 × 10⁸ ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K⁺ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na⁺ medium (130mm -Na⁺, 5mm -K⁺) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na⁺ ionophore. whose conformation is dependent on membrane potential.     

细菌中I型毒素蛋白与细胞膜相互作用机制的研究进展

I型毒素-抗毒素（TA）系统在细菌基因组中广泛存在,在细菌的生长、生存中发挥多种生物学功能,包括抗菌、红细胞毒性、促进持留菌形成、抑制细菌生长或导致细菌休眠等。绝大部分I型毒素蛋白以细胞膜作为靶标,目前已知的一种作用机制是在细胞膜上形成孔洞结构,造成膜电位的下降或细胞膜的破坏,从而抑制ATP的合成或导致细菌死亡;另一种可能的作用机制是毒素蛋白作用在细胞膜上,改变细胞的形状,导致细胞进入休眠状态。I型毒素蛋白-细胞膜作用机制的复杂性和生物功能的多样性远超预期。因此,解析I型毒素蛋白在不同细胞膜中的组装机制及其所形成结构特征就变得非常重要,这也是揭示其结构-功能关系的关键。本文通过综述已报道的I型TA系统的结构特征与生物学功能,结合对其跨膜结构域的预测,探讨了其可能在细胞膜中形成的不同结构及其对功能的影响,分析了影响作用机制的关键因素。这些研究既给耐药细菌的治疗带来机遇,又为新型抗菌药物的研发带来思路。     

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.     

Immunity to K1 killer toxin: internal TOK1 blockade

K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells. Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death. Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin.     

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

Effect of anthrax toxin on the chemiluminescence of human leukocytes

The effect of substrate containing crude anthrax toxin on the phagocytosing leukocyte chemiluminescence has been studied. Preliminary toxin incubation with leukocytes for 60 min blocks cell chemiluminescence in the linear ratio effect concentration in the protein component of the toxin; the minimum concentration of the toxin protein component inhibiting the phagocytosing leukocyte luminescence is 3-5 micrograms per 5 x 10(5) cells. The substrate pure mixture of the oedema factor and protective antigen inhibits the chemiluminescence more intensively than crude toxin does. Inhibiting chemiluminescence activity of the anthrax toxin is directly proportional to its adenylate cyclase activity.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.     

Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.     

The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4's ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4-PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF-SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.     

Viruses activate a genetically conserved cell death pathway in a unicellular organism

Given the importance of apoptosis in the pathogenesis of virus infections in mammals, we investigated the possibility that unicellular organisms also respond to viral pathogens by activating programmed cell death. The M1 and M2 killer viruses of Saccharomyces cerevisiae encode pore-forming toxins that were assumed to kill uninfected yeast cells by a nonprogrammed assault. However, we found that yeast persistently infected with these killer viruses induce a programmed suicide pathway in uninfected (nonself) yeast. The M1 virus-encoded K1 toxin is primarily but not solely responsible for triggering the death pathway. Cell death is mediated by the mitochondrial fission factor Dnm1/Drp1, the K+ channel Tok1, and the yeast metacaspase Yca1/Mca1 encoded by the target cell and conserved in mammals. In contrast, cell death is inhibited by yeast Fis1, a pore-forming outer mitochondrial membrane protein. This virus-host relationship in yeast resembles that of pathogenic human viruses that persist in their infected host cells but trigger programmed death of uninfected cells.     

Enhanced exocytosis of the receptor BT-R(1) induced by the Cry1Ab toxin of Bacillus thuringiensis directly correlates to the execution of cell death

Cry1Ab toxin produced by Bacillus thuringiensis exerts insecticidal action upon binding to BT-R₁, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta. The univalent binding of toxin to receptor transmits a death signal into the cell and turns on a multi-step signal transduction pathway involving adenylyl cyclase (AC) and protein kinase A (PKA), which drives the biochemical events that culminate in oncotic cell death. Here, we report that cell killing by the Cry1Ab toxin is a dynamic episode in which the toxin promotes exocytotic transport of BT-R₁ from intracellular membrane vesicles to the plasma membrane. The resultant dramatic increase in BT-R₁ displayed on the surface of toxin-treated cells effects the recruitment and concomitant binding of additional toxin monomers which, in turn, amplifies the original signal in a cascade-like manner. Blocking the activation of AC/PKA signal transduction by either EDTA or PKAi inhibits exocytotic trafficking of BT-R₁ and prevents cell death. Moreover, the exocytosis inhibitor Exo1 blocks translocation of receptor and progression of cell death alike. Obviously, movement of BT-R₁ is mediated by toxin-induced signal transduction and amplification of this signaling apparently is critical to the execution of cell death.     

Early changes in cytosolic calcium and membrane potential induced by Actinobacillus actinomycetemcomitans leukotoxin in susceptible and resistant target cells

Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.