Activation and mechanism of Clostridium septicum alpha toxin

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (AT^pro) which could be activated via a carboxy-terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy-terminus. Proteolytically activated AT^pro had a specific activity of approximately 1.5 × 10⁶ haemolytic units mg^-1. The trypsin-activated toxin (AT^act) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of M_r >210000 on erythrocyte membranes. AT^pro did not exhibit these properties. AT^act formed pores with a diameter of at least 1.3-1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.     

Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin

Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.     

Differences in the activation mechanism between the alpha and beta subunits of human meprin

Meprins are zinc-endopeptidases of the astacin family, which are expressed as membrane-bound or secreted forms in renal and intestinal brush-border membranes of mouse, rat and man. There are two types of meprin subunits, alpha and beta, which form disulfide-bonded homo- and heterodimers; further oligomerization is mediated by non-covalent interactions. Both subunits are translated as proenzymes that have to be activated by removal of an N-terminal propeptide. In the gut, the most probable activator is trypsin. In addition, plasmin has been shown to activate the human alpha subunit in colorectal cancer tissue. In the present study we have overexpressed the human meprin alpha subunit and a His-tagged soluble tail-switch-mutant of meprin beta in Baculovirus-infected insect cells. The recombinant homo-oligomeric proteins were purified by gel filtration and affinity chromatography with yields of up to 10 mg/l cell culture medium and analyzed with regard to their activation mechanism. While both alpha and beta homo-oligomers are activated by trypsin, only meprin alpha homo-oligomers are processed to their mature form by plasmin. These results indicate a different accessibility of the propeptide in meprin homo-oligomers and suggest an explanation for the appearance of meprin hetero-oligomers consisting of active alpha, but latent beta subunits.     

Isoleucine at position 150 of Cyt2Aa toxin from Bacillus thuringiensis plays an important role during membrane binding and oligomerization

Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin. [BMB Reports 2013; 46(3): 175-180]     

Molecular determinants for the interaction of human neutrophil alpha defensin 1 with its propeptide

Human neutrophil α-defensins (HNPs) are cationic antimicrobial peptides that are synthesized in vivo as inactive precursors (proHNPs). Activation requires proteolytic excision of their anionic N-terminal inhibitory pro peptide. The pro peptide of proHNP1 also interacts specifically with and inhibits the antimicrobial activity of HNP1 inter-molecularly. In the light of the opposite net charges segregated in proHNP1, functional inhibition of the C-terminal defensin domain by its propeptide is generally thought to be of electrostatic nature. Using a battery of analogs of the propeptide and of proHNP1, we identified residues in the propeptide region important for HNP1 binding and inhibition. Only three anionic residues in the propeptide, Glu¹⁵, Asp²⁰ and Glu²³, were modestly important for interactions with HNP1. By contrast, the hydrophobic residues in the central part of the propeptide, and the conserved hydrophobic motif Val²⁴Val²⁵Val²⁶Leu²⁸ in particular, were critical for HNP1 binding and inhibition. Neutralization of all negative charges in the propeptide only partially activated the bactericidal activity of proHNP1. Our data indicate that hydrophobic forces have a dominant role in mediating the interactions between HNP1 and its propeptide — a finding largely contrasting the commonly held view that the interactions are of an electrostatic nature.     

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Abstract The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al, 1991) FEBS Lett. 283, 317–320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 Å and 28 Å in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K⁺ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca²⁺ concentration, suggesting a Ca²⁺ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca²⁺ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.     

Structure-function relationships of a novel bacterial toxin, hemolysin E. The role of alpha G

The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (beta tongue) that interacts with target membranes at one pole and an additional helix (alpha(G)) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole. Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with alpha(G). Deletion of amino acids downstream of alpha(G) did not affect activity, but deletions encompassing alpha(G) yielded insoluble and inactive proteins. In the periplasm Cys-285 (alpha(G)) is linked to Cys-87 (alpha(B)) of the four-helix bundle via an intramolecular disulfide. Oxidized HlyE did not form spontaneously in vitro but could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins. Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane. However, gel filtration analyses suggested that immobilizing alpha(G) inhibits oligomerization in solution. Thus once associated with a membrane, immobilizing alpha(G) inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis.

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.     

Reversible Effects of Toxin from Helminthosporium maydis Race T on Oxidative Phosphorylation by Mitochondria from Maize

Host-selective toxin from Helminthosporium maydis race T inhibited oxidative phosphorylation (AT³²P formation) and stimulated ATPase activity by mitochondria from male-sterile (T) but not from normal (N) cytoplasm maize (Zea mays L.). Toxin increased the rate of NADH oxidation, but succinate oxidation was slightly, and malate-pyruvate oxidation was strongly inhibited as the associated ATP formation was abolished. There was a 1-minute lag before toxin gave maximal stimulation of NADH oxidation; the responses to 2,4-dinitrophenol and valinomycin were immediate. There was also a delay in the effect of toxin on ATP formation. T mitochondria were more sensitive than were N mitochondria to uncoupling by nigericin plus K⁺; there was no evidence, however, that the action of toxin is related to that of nigericin or other ionophores. With NADH as the substrate, the degree of uncoupling increased with increases in toxin concentration up to a saturating level; kinetics of the response suggested reversibility. T mitochondria exposed to toxin for 5 minutes regained normal rates of respiration and of ATP formation when they were washed with toxin-free medium, showing that the uncoupling effect is reversible. Evidently HM-T toxin does not bind firmly to its site(s) of action, in contrast to reports for another hostselective toxin.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.     

Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.     

Structural and functional characterization of MERS coronavirus papain-like protease

Backgrounds

A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PL^pro) is expressed, and its structural and functional consequences are elucidated.

Results

Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PL^pro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PL^pro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PL^pro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PL^pro. A natural mutation, Leu105, is the major reason for this difference.

Conclusions

Overall, MERS-CoV PL^pro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PL^pro family, which is applicable to develop strategies inhibiting PL^pro against highly pathogenic coronaviruses.     

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.     

Mechanism of Membrane Damage by Clostridium perfringens Alpha-Toxin

The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-l -α-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT_1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-l -α-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT_1-246 did not. However, the C-terminal domain (AT_251-370) conferred binding to liposomes and the membrane-damaging activity on AT_1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.     

Comparison of the cytostatic and cytolytic activity of tumor necrosis factor-alpha and interleukin 1 alpha in human malignant cell lines.

The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 alpha (IL-1 alpha) share many properties, including in-vitro cytotoxicity. Because cytotoxicity can result from either cytolytic or cytostatic activity, and because differentiating between these activities may have clinical relevance, we determined the cytostatic and cytolytic activity of TNF-alpha and IL-1 alpha for the human cell lines ME-180, SiHa (cervical carcinomas) and A375 (melanoma). Results of these analyses showed that IL-1 alpha mediated cytostatic activity only for A375 cells. IL-1 alpha was not cytolytic in the presence or absence of protein synthesis inhibitors. TNF-alpha was cytostatic for A375 and ME-180 cells, and although TNF-alpha was not cytolytic in the absence of protein synthesis inhibitors, it was cytolytic in the presence of protein synthesis inhibitors. These results suggest that the difference between the cytolytic and cytostatic activities of IL-1 alpha and TNF-alpha may have therapeutic implications for the use of these biological response modifiers in the treatment of gynecological malignancies.     

IL-4 regulation of a protein kinase C independent pathway for the generation of alpha CD3-induced activated killer cells.

alpha CD3 induced the generation of activated killer cells from resting T cells. Pretreatment of the splenic responders with PMA, a phorbol ester, depleted protein kinase C and induced unresponsiveness to the generation of alpha CD3-induced activated killer (CD3-AK) cells. Addition of exogenous IL-4 (1 U/ml) restored the cytotoxic response, with the maximal effect achieved with 30 to 100 U/ml. The phenotypes of CD3-AK cells maintained in IL-2 or in IL-4, with or without PMA, were the same: Thy1+ and CD8+. These results were reproduced with purified T cells and purified CD8+ cells, indicating that both the effectors and precursors were CD8+ cells and IL-4 had a selective effect to upregulate the CD8+ cells. Similar results were obtained by using SSP (staurosporine), another PKC inhibitor. At 2 days prior to testing, switching the lymphokine added to 2-week PMA- and IL-2-maintained CD3-AK cells reversed their cytolytic activity: switching from IL-2 to IL-4 restored cytolytic activity, and switching from IL-4 to IL-2 reduced cytolytic activity. The cytolytic activity of these CD3-AK cells correlated with their ability to produce BLT-esterase. In the absence of PMA, CD3-AK cells cultured in either IL-2 or IL-4 were cytolytic and contained high levels of BLT-esterase. In contrast, in the presence of PMA, only the IL-4-maintained CD3-AK cells were cytolytic and produced significant amounts of BLT-esterase. The effect of IL-4 was abrogated by the alpha IL-4 antibody 11B11, which reduced the cytolytic activity of CD3-AK and the ability to produce BLT-esterase. The requirement of IL-2 was less stringent and its major role appeared to be maintaining the cell growth. These findings indicate that IL-4 may participate in the regulation of a PKC-independent pathway for the generation of CD3-AK cells by regulating the production of cytolytic granules.     

Biological effects of activation of human peripheral blood mononuclear cells by mitogenic oxidizing agents. I Alloantigen-independent activation of alloimmune memory cells

Mechanisms of activation of alloimmune memory cells by immunologically nonspecific signals were investigated utilizing the mitogenic oxidizing agents, neuraminidase and galactose oxidase (NAGO) and sodium periodate (IO₄^?). Direct activation of primary long-term human mixed-lymphocyte culture (MLC) cells (memory cells) with either NAGO or IO₄^? resulted in increased specific secondary cytolytic activity. Kinetics of the proliferative and cytotoxic responses resulting from such treatment of memory cells paralleled those resulting from treatment of memory cells with irradiated cells that were the stimulators in the primary MLC. Indirect activation of memory cells by NAGO or IO₄^?-treated and irradiated syngeneic cells also resulted in increased specific secondary cytolytic activity. In contrast, peripheral blood mononuclear cells (PBM) activated by the mitogenic oxidizing agents did not develop cytolytic activity toward syngeneic or multiple allogeneic target cells, despite extensive proliferative responses. Cytotoxicity generated by either direct or indirect activation of memory cells by IO₄^? was prevented by treatment of the oxidized cells with the reducing agent, sodium borohydride. Incubation of memory cells in supernatants from 24-hr cultures of PBM activated with either NAGO or IO₄^? resulted in proliferation and in an increase in cytolytic activity in memory cells, but not in PBM. These findings indicate that alloimmune memory cells can be activated by immunologically nonspecific lymphocyte-derived signals that do not depend on alloantigen or lectin.