Effects of Vibrio vulnificus cytolysin on the numbers of mitochondria and the expression of CD62L in murine liver CD4+T cells / 中华微生物学和免疫学杂志

Objective To understand how Vibrio vulnificus hemolysin (VvhA) affects the viability of murine liver CD4+ T cells as well as its effects on the numbers of mitochondria and the expression of CD62L. Methods The primary murine liver monocytes (MNs) were isolated from C57BL/ 6 mice and then treated with recombinant VvhA (rVvhA) for 6 hours in vitro. The viability of murine liver CD4+T cells and the expression of CD62L were measured by staining with anti-mouse CD4, CD8, CD44, CD62L and cell via-bility fluorescent dye or fluorescent antibody. Moreover, the cells were simply incubated with MitoTracker or JC-1 probes to label mitochondria and mitochondrial membrane potential, which were further analyzed by using flow cytometry analysis. Results With the increase in the doses of rVvhA, the viability of murine liv-er CD4+T cells was decreased from 81. 5% to 15. 8% . The expression of CD62L on the surface of murine liver CD4+T cells was dramatically decreased. Both the murine liver na?ve and effector CD4+ T cells were sensitive to the cytotoxicity of rVvhA. Moreover, treating murine liver CD4+ T cells with rVvhA resulted in significantly decreased numbers of mitochondria and lower mitochondrial membrane potential. Conclusion The cytotoxicity of rVvhA to murine liver CD4+T cells might be achieved through inhibiting the expression of CD62L, decreasing the numbers of mitochondria and lowering mitochondrial membrane potential.

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.

Virulence Changes between EF A7 and EF A7 cyl Mutant in the Mouse Peritonitis Models / 现代检验医学杂志

Objective The purpose of this study was to understand the pathogenic mechanisms of enterococcal hemolysin and establish the foundation to diagnose and control relevant diseases.Methods Take 30 clean grade ICR mice were randomly divided into ten groups,each 6,1 to 4 groups according to 10-fold gradient amount per 1 ml intraperitoneal injection of 5× 107 cfu/ml~5×1010 cfu/ml of EF A7,group 5 as a control group injected 0.5 ml saline,take another 30 clean grade ICR mice,intraperitoneal injection of 5×107 cfu/ml~5×1010 cfu/ml of EF A7 cyl mutant by the same measures,compared to the original strains of mice infected with mutant strain LD50,and a week after infection mortality was observed in mice daily. Another seven groups of mice with the same bacterial concentration (5×109 cfu/ml)were injected intraperitoneally EF A7 and EF A7 cyl mutant,after infection 6 h,12 h and 24 h comparing the number of leukocytes in peripheral blood of mice,re-spectively,the concentration of TNF-αin the acute phase of cytokines.Results In the mouse peritonitis models the 50% le-thal dose (LD50)of EF A7 cyl mutant was 100 times lower than enterococcus faecalis EF A7.The differences of the survival percentage of EF A7 cyl mutant groups and EF A7 groups have significance (P<0.05).As the changes in boold leukocytes numbers and the TNF-αlevel.Conclusion Cyl gene is probably a major virulence factor of Enterococcus and plays an impor-tant role in the mouse peritonitis model.

Role of rVvhA in inducing THP-1 cells damage / 中华微生物学和免疫学杂志

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

Prediction, cloning, expression and identification of the functional motif of the recombinant Ricin B / 中华微生物学和免疫学杂志

Objective To determine the functional motif of the recombinant Ricin B(rRicin B) in Vibrio vulnificus cytolysin (VVC) and understand its molecule pathogenic mechanism.Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG to express rRicin B.The expressed protein was further analyzed by SDS-PAGE and purified by Ni2+-NTA agarose.Renaturation of the rRicin B were also carried out for further analysis.ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells.Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20×103.The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation.FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells.Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm.This feature may play an important role in the activity of pore-forming and the cytotoxicity of Vibrio vulnificus cytolysin.

The role of calcium in the rVvhA induced THP-1 cells apoptosis / 中华微生物学和免疫学杂志

Objective To study the role of Vibrio vulnificus cytolysin(rVvhA) induced THP-1 apoptosis and calcium influx.Methods CCK-8 cell proliferation kit,Fluo3/AM staining and AnnexinV/PI staining were performed to identify the apoptosis and calcium influx induced by rVvhA in THP-1 cells.Results rVvhA could induce THP-1 apoptosis and up-regulate the cellular calcium concentration.BAPTAAM could enhance the calcium influx induced by rVvhA in THP-1.Conclusion rVvhA had cytotoxic to THP-1 cells by inducing apoptosis and triggering extracellular calcium influx.

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.(AU)

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Vibrio vulnificus Hemolysin Is Easily Inactivated in Spite of Being Produced at High Levels in Cirrhotic Ascites by a fur Mutation

Vibrio vulnificus produces Hemolysin/cytolysin (VvhA), which is one of the most potent exotoxins capable of killing mice at submicrogram levels. However, V. vulnificus growth and vvhA expression are severely repressed and extracellular VvhA produced at low levels is easily inactivated in human body fluids. This study was conducted to obtain additional unequivocal evidence of the enigmatic characteristic of VvhA. V. vulnificus growth was stimulated, vvhA expression was de-repressed, and extracellular VvhA production was increased in cirrhotic ascites, a human ex vivo experimental system, by a mutation of fur encoding ferric uptake regulator, which acts as a transcriptional repressor. However, regardless of the presence or absence of the fur mutation, extracellular VvhA activity was not detected in cirrhotic ascites. These results indicate that VvhA is easily inactivated even when vvhA expression and extracellular VvhA production are maintained at high levels in cirrhotic ascites.

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97 percent) with the already reported cytolysins from other sea anemone species.(AU)

Hemolytic toxin from the soft coral Sarcophyton trocheliophorum: isolation and physiological characterization

The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60º C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.(AU)

Study on expression of NO and iNOS in J774A. 1 cells induced by rVvhA / 中华微生物学和免疫学杂志

Objective To investigate the effect of recombinant Vibrio vulnificus hemolysin (rVvhA) on the expression of nitric oxide(NO) and induced nitric oxide synthase(iNOS) in J774A. 1 cells.Methods The inhibitory effect of rVvhA on J774A. 1 proliferation was measured by MTF colorimetry technique. The content of nitrite in culture medium was determined by Griess reagent. The expression of iNOS protein and mRNA were measured by immunofluorescence and RT-PCR, respectively. Results The viability of J774A. 1 cells was apparently inhibited when exposed to 0.8 HU/ml rVvhA and up. With the help of IFN-γ, the expression of NO and the activity of iNOS in J774A. 1 cells were remarkably increased when exposed to 0.4 HU/ml rVvhA. Conclusion rVvhA can increase the expression of NO and iNOS, it may play an important role in the pathogenic mechanism of Vibrio vulnificus.

Study on transcription and expression of IL-8 in human Caco-2 cells induced by VvhA recombinant protein / 中华微生物学和免疫学杂志

Objective To investigate the effect of VvhA recombinant protein to the expression of IL-8 in human intestinal epithelial Caco-2 cells. Methods The VvhA recombinant protein(rVvhA) was ex-pressed by prokaryotic expression vector pET-28a (+)-whA in E. coli BL21 (DE3) , purified by Ni~(2+)-NTA affinity chromatography refoldod by stepwise deliquation together with dialysis methods and identified by Western blot. The cytotoxic of rVvhA to human Caco-2 cells was measured by CCK-8. The transcription of IL-8 mRNA in human Caco-2 cells induced by rVvhA was determined by RT-PCR, and the expression of IL-8 in human Caco-2 cells induced by rVvhA was determined by ELISA assay. Results rVvhA was purified with high purity up to 95%. The viability of human Caco-2 cells treated with 1.5 HU/ml rVvhA was inhibi-ted significantly (P < 0.05). The rVvhA can induce human Caco-2 cells to increase the transcription and expression of IL-8 in dose- and time-dependent manner. The transcription of IL-8 gene in human Caco-2 cells treated with 0.6 HU/ml rVvhA in 30 min can be up-regulated significantly, and the expression of IL-8 in human Caco-2 cells treated with rVvhA in 4 h can be increased significantly. Conclusion rVvhA has cy-totoxic to human Caco-2 and can increase the expression of IL-8, it might play a major role in the inflamma-tory reaction of rVvhA-exposed cells.

Effect of apoptosis of rVvhA on J774A.1 and the related mechanism / 中华微生物学和免疫学杂志

Objective To investigate the activity of recombinant Vibrio vulnificus hemolysin (rVvhA) on the apoptosis of J774A.1 cells and the related mechanism. Methods The cytotoxic effect of rVvhA on the growth of J774A.1 cells was identified by MTT, celluar and mitochondrial morphology were observed by transmission electron microscopy, apoptosis or necrosis and mitochondrial membrane potential in J774A.1 cells were measured by flow cytometry, activities of caspase-3 ,-8,-9 were detected by spectrophotometry. Results The viability of J774A.1 cells exposed to rVvhA was inhibited, and it is dependent on dose. Celluar and mitochondrial uhrastructure both occurred to change obviously observed by transmission electron microscopy in J774A.1 treated by 2.0 HU/ml and 3.0 HU/ml rVvhA after 8 hours; and 3.0 HU/ml rVvhA group had a better cytotoxic effect on J774A.1 than that of 3.0 HU/ml rVvhA group. The percentage of apoptosis is (7.80±0.62)%, (12.33±0.12)%, respectively. Besides, the mitochondriai membrane potential also reduced, because the rate of fluorescence which is green increase 1.0% (normal) to 9.8% (2.0 HU/ml rVvhA) and 39.2% (3.0 HU/ml rVvhA). At the same time, the caspase-3, -9 activity increased gradually, but caspase-8 remained unchanging. In J774A.1 cells treated by 3.0 HU/ml rV-vhA + caspase-3 inhibitor(Ac-DEVD-FMK) or caspase-9 inhibitor(Ac-LEHD-FMK), The apoptosis of was reduced to(6.23±3.95)% ,(9.60±3.14)%, and the activity of caspase-3, -9 reduced, too. Conclusion The rVvhA has cytotoxic effect on J774A.1. Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in J774A.1.

The Bacterial Surface Expression of SARS Viral Epitope using Salmonella typhi Cytolysin A

The cytolysin A (ClyA) is a 34 kDa pore-forming cytotoxic protein and expressed by some enteric bacteria including Salmonella typhi. This toxin is transported on the bacterial surface and secreted without posttranslational modification. Using the surface display of ClyA, the expression vectors for 193-aa immunogenic antigen of spike protein (termed S1E) from severe acute respiratory syndrome coronavirus (SARS-CoV) were constructed. The vectors carried a gene encoding S. typhi ClyA conjugated to S1E at the C terminus (termed ClyA-S1E) and asd gene in pGEM-T and pBR322, named pGApLCS1E and pBApLCS1E, respectively. An asd-mutated E. coli transformed with these vectors could grow without diaminopimelic acid (DAP), indicating that they were stably maintained in such mutants. ClyA-S1E recombinant proteins from these vectors were expressed on the surface of the attenuated S. typhimurium deficient of global virulence gene regulator, ppGpp. However, they did not show the hemolytic activity on the blood agar plate and cytotoxicity against HeLa cells. To examine whether bacteria expressing ClyA-S1E induced the immune response against S1E, S. typhimurium deficient of ppGpp and Asd was transformed with these vectors and orally immunized in mice. In the western blotting against GST-conjugated S1E using the immunized mouse sera, it was shown that the significant band was detected in the mouse serum by the bacteria transformed with pGApLCS1E but not with pBApLCS1E. It indicates that the immune response producing antibody was dependent on the expression level of ClyA-S1E. Therefore, ClyA delivery system can be used for SARS vaccine development.

Study on VVC recombinant protein cytolytic activity mechanism of an extracellular cytolytic toxin of Vibrio vulnificus / 中华微生物学和免疫学杂志

Objective To investigate the cytolytic activity of extracellular cytolytic toxin rVVC of Vibrio vulnificus on the apoptosis of human ECV304 cells, and to analyze the activities of Caspase-3,-8 and -9. Methods The cytotoxic effect of refolded rVVC on the growth and apoptosis of ECV304 cells was identified by MTT, Hochest33342/PI fluorescent staining, flow cytometry and DNA agarose electrophoresis analysis, respectively. The activities of Caspase-3, -8 and -9 was measured using a colorimetric method. Results The viability of human ECV304 cells exposed to rVVC was inhibited by rVVC after 24 h. 2.0 HU/ml rVVC groups had a better cytotoxic effect to human ECV304 than that of 0.5 HU /ml rVVC groups. The apoptosis of human ECV304 cells in 2.0 HU/ml rVVC+40 μmol/L Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ml of rVVC groups. After 0.5 h treatment with 2.0 HU/ml of rVVC, the Caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 h (versus control groups, P<0.01). The activity of Caspase-8 and -9 remained unchanging. Conclusion The rVVC has cytotoxic effect on human ECV304 and the cytolysin is probably correlated with Caspase-3.

Apoptosis-inducing effect and intracellular location of Vibrio vulnificus cytolysin to human umbilical vein endothelial cell in vitro / 中华微生物学和免疫学杂志

Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.

Vibrio vulnificus Cytolysin Forms Anion-selective Pores on the CPAE Cells, a Pulmonary Endothelial Cell Line

Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of 37.5 4.0 pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of Na in bath solution with monovalent ions (K, Cs or TEA ) or with divalent ions (Mg2, Ca2 ) did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to 11.8 and 28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at 40 mV was Cl = NO2 > or = Br = I > SCN > acetate > isethionate > ascorbic acid > EDTA2, in descending order. The cytolysin-induced pore current was blocked by Cl channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells.