The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars.

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.

Haemolysin test for characterization of immune ABO antibodies.

BACKGROUND & OBJECTIVES: Antibodies with haemolytic properties are common within the ABO system. These lytic antibodies are immunoglobulin G (IgG) and in high titres cause haemolysis during blood transfusion. Information on Immunoglobulin types and concentration of ABO haemolysins in Indian population is lacking. The present study was undertaken to know the usefulness of haemolysin test for characterization of immunoglobulin class of ABO antibodies. METHODS: Serum samples from 187 O group blood donors were screened for A and B haemolysins. Thirty five samples were treated with dithiothretiol (DTT) for characterization of Ig class. Antibody titre was compared with grade of haemolysis. RESULTS: Of the 51 strongly haemolytic serum samples, 32 (62.8%) had IgG titres of > or = 64 after treatment with DTT. There was significant association (P<0.05) between grade of haemolysin and anti B IgG titre. INTERPRETATION & CONCLUSION: Haemolysin test was found to be a useful screening test to identify group O donors with high levels of IgG anti A and/or anti B for blood transfusion purposes.

Purification & characterisation of haemolysin from Serratia marcescens B-91.

BACKGROUND & OBJECTIVES: Serratia marcescens an opportunistic human pathogen, is frequently encountered in a variety of debilitating diseases. Relatively little is known about its virulence traits though most clinical isolates secrete a distinct haemolysin which is considered as a useful marker for pathogenicity of Serratia. In this study purification and characterisation of S. marcescens B-91 haemolysin have been attempted. METHODS: S. marcescens B-91 haemolysin was purified to homogeneity from the growth medium using ammonium sulphate fractional precipitation and gel filtration through Sephadex G-75 column. Homogeneity was determined by gel electrophoresis and purified haemolysin was tested for its stability and other characteristics. RESULTS: The haemolysin was characterised to be a 45 kDa molecular weight protein on SDS-polyacrylamide gel electrophoresis. It was inactivated at 60-100 degrees C within 30 min, and on overnight treatment with 2 per cent formaldehyde. It was also susceptible to the action of pronase, protease and trypsin. INTERPRETATION & CONCLUSIONS: The results indicate that the fragile stability of S. marcescens haemolysin is dependent on the storage temperature. The purified haemolysin can be used for understanding the role of haemolysin in the pathogenesis of S. marcescens and also for evaluation of immunoprophylactic activity.

A hemolytic protein from cultured mycelia of mushroom, Termitomyces clypeatus.

A hemolytic protein was purified from cultured mycelia of T. clypeatus. Some of the physico-chemical properties of the hemolysin were studied. The protein was analysed to be a lipoprotein and delipidation removed its hemolytic property. The monomeric protein subunit of the lipoprotein had a molecular weight of 64,000. Mode of action of the hemolysin were studied by observing protections of sugar and lipid components to hemolysin mediated lysis of red blood cells. It was observed that the hemolysin possibly interacted with the phospholipid components of the blood cells causing lysis.