Cytolysin Activity Research Articles

Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity

Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5°C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.

Metalloprotease is not essential for Vibrio vulnificus virulence in mice.

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of Vibrio vulnificus, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of Vibrio vulnificus by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.

A novel means of self-protection, unrelated to toxin activation, confers immunity to the bactericidal effects of the Enterococcus faecalis cytolysin.

Enterococcus faecalis has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of E. faecalis is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids. E. faecalis strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3' end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3' to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn917 insertion.

Antiparasite activity of sea-anemone cytolysins onGiardia duodenalis and specific targeting withanti-Giardia antibodies

The killing activity of sea-anemone cytolysins on Giardia duodenalis wasinvestigated. Three different toxins, sticholysin I and II from Stichodactyla helianthus (St I and St II) and equinatoxin II from Actinia equina (EqtII) were all found to beactive in an acute test, with a C 50 in the nanomolar range (St I, 0.5 nM; St II, 1.6 nM; and EqtII, 0.8 nM). A method to target the cytolysin activity more specifically towards theparasite cells by using anti- Giardia antibodies was then investigated. Parasite cells weresensitised with a primary murine monoclonal or polyclonal antibody followed by a biotinylatedsecondary anti-mouse-IgG monoclonal antibody. Subsequently, avidin and a biotinylated EqtIImutant were added, either in two separate steps or as a pre-formed conjugate. When themonoclonal antibody was used, the C 50 of biotinylated EqtII was 1.3 nM withsensitised cells and 5 nM with non-sensitised cells, indicating a four-fold enhancement of activitywith the cell treatment. Treatment with the polyclonal antibody was somehow more effective thanwith the monoclonal antibody in an acute test. This indicates that sea-anemone cytolysins canefficiently kill Giardia cells, and that it is possible to improve, to a certain extent, theanti-parasite specificity of these toxins with anti- Giardia antibodies. However, thefeasibility of this approach in vivo remains to be demonstrated. © 1999 Australian Society forParasitology

Inhibition of haemolytic activity of cytolysins from sea anemone using polyclonal antibodies

Inhibition of haemolytic activity of cytolysins from sea anemone using polyclonal antibodies

Stimulation of Haemolytic Activity of Sea Anemone Cytolysins by 8-Anilino-1-naphthalenesulphonate

This study reports for the first time stimulation of protein activity by the hydrophobic probe, 8-anilino-1-naphthalenesulphonate (ANS). Magnificalysin (HMg) I and II and equinatoxin (EqTx) II and III are cytolysins isolated from the sea anemoneHeteractis magnificaandActinia equina,respectively. The haemolytic activity of these cytolysins could be stimulated by treatment with ANS. Their activation involved conformational changes following ANS treatment as shown by fluorescence spectra. ANS-induced conformational changes were reversible upon removal of ANS. ANS-stimulated activity of HMg I was inhibited by sphingomyelin and antiserum but not affected by bromosuccinimide (NBS) which oxidises tryptophan residues. However, toxin pre-treated with NBS could no longer be stimulated by addition of ANS. Energy transfer from tryptophan to ANS was observed by a fluorescence scan. Hence the tryptophan residues appear to be involved, at least partially, in ANS-binding. ANS-induced conformational change may be responsible for the activation of the cytolytic activity of these cytolysins.

Pathogenesis ofActinobacillus pleuropneumoniae infections

Pathogenesis ofActinobacillus pleuropneumoniae infections

A natural killer cell granule protein that induces DNA fragmentation and apoptosis.

We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin," was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that fragmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca2+ dependent. A role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD N alpha-CBZ-L-lysine thiobenzyl esterase, which copurified with fragmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis.

Characterization of non-lytic cytolysin-membrane intermediates

In order to understand the nature of cytolysin-membrane interactions, the characteristics of stable, non-lytic cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K 562) tumor targets formed cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for cytolysin binding.

A common cytolytic region in myotoxins, hemolysins, cardiotoxins and antibacterial peptides.

Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure-function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.

Possible involvement of CTL granule proteases in target cell DNA breakdown.

We have carried out experiments to test whether the granule exocytosis model for lymphocyte cytotoxicity can account for the rapid target DNA breakdown seen during CTL-induced cytotoxicity. Dense granules isolated from cloned mouse CTL and from rat NK tumor cells cause target DNA breakdown during granule cytolysin-mediated lysis of tumor cells, while the purified granule cytolysin caused lysis without DNA breakdown. When target cells are permeabilized with detergent, granule extracts have the ability to release 125I-DNA from nuclei in the absence of detectable cytolysin activity. This activity formed the basis for a nuclear DNA release (NDR) assay; this activity was a property of dense granules of cytotoxic lymphocytes but generally not of other types of lymphoid cells. NDR activity in NK tumor granules had a pH optimum of 7 and was inhibited by micromolar levels of Zn+2, and could be purified away from the granule cytolysin by column chromatography. NDR activity in CTL dense granules could be inactivated by submillimolar concentrations of the protease inhibitors PMSF and DFP (but not soybean trypsin inhibitor or TLCK). In support of the relevance to CTL cytotoxicity of these findings with the NDR assay, pretreatment of CTL with PMSF in the presence of agents raising the intragranular pH inactivated 125I-DNA release from target cells (but not the 51Cr release). These results suggest that a CTL granule component(s), probably a protease, is required for target DNA breakdown.

Inhibition of cytolysin activity in large granular lymphocyte granules by lipids: Evidence for a membrane insertion mechanism of lysis

The ability of various compounds to inhibit the lytic activity of purified cytoplasmic granules from LGL tumors was tested. The lytic activity of granule cytolysin was modestly inhibited by inorganic phosphate, and various monophosphoesters, with I 50 values in the range of 8–20 m M. Among monophosphoesters, choline phosphate was exceptionally potent, with an I 50 of 1.4m M. In contrast to the inhibition by phosphate esters, the parent compounds such as neutral sugars, glycerol, and choline, showed no detectable inhibition at 50 m M. A lysolipid bearing a short aliphatic chain and some detergents were inhibitory with I 50 values in the range of 1 m M. Lysolipids with longer aliphatic chains, phospholipids as liposomes, and related lipid compounds were found to display potent inhibition of the hemolytic activity of LGL granule cytolysin, with I 50 values in the range of 0.2–30 μ M. Soluble globular proteins inhibited LGL granule cytolysin hemolytic activity with I 50 of 0.07–0.4 mg/ml. However, lipoproteins were 2–3 orders of magnitude more potent inhibitors, with L 50 values of less than 1 μg/ml. The observed potent inhibition by lipid compounds lends support to a model of the lytic action of granule cytolysin in which soluble cytolysin molecules insert into the membrane lipid bilayer during the course of the lytic event.

Cytolytic and biochemical properties of cytoplasmic granules of murine lymphokine-activated killer cells.

The incubation of murine spleen cells in the lymphokine interleukin 2 (IL 2) gives rise to lymphokine-activated killer (LAK) cells capable of lysing fresh tumor cells in short-term lytic assays. During the course of cultures used to generate LAK cells, cytoplasmic granules were prepared and were analyzed for the presence of the cytolysin previously described in large granular lymphocytes (LGL) and cytotoxic T lymphocytes (CTL). Such cytolysin activity is initially undetectable, appears after 2 days of culture, and continues to increase until day 7. The LAK cytolysin has properties similar to those of previously described cytolysins with respect to nonspecific killing of various target cells, rapid kinetics, and absolute dependence on calcium. Antibodies raised against purified LGL tumor granules neutralized the activity of the LAK cytolysin. The precursors of both the LAK cells and the cells bearing the cytolysin are eliminated by treatment with anti-asialo-GM1 and complement, strongly suggesting that the actual LAK effector cells and the cytolysin-bearing cells are identical. Biochemical analysis of the LAK granules indicate that they contain the lysosomal enzyme arylsulfatase. The protein content of granules isolated from various days of culture with r-IL 2 undergoes a dramatic change, with major protein bands around 30,000 daltons becoming prominent, as well as the cytolysin protein band at 70,000 daltons. These data suggest that the mechanism of cell lysis by LAK cells is similar to that of CTL and natural killer-mediated lysis, and each of these forms of lymphocyte-mediated cytolysis is based on a granule exocytosis mechanism.