Hemolysins of <i>Staphylococcus aureus</i>—An Update on Their Biology, Role in Pathogenesis and as Targets for Anti-Virulence Therapy

Staphylococcus aureus is a dangerous gram positive bacterial pathogen which, not only evades the host’s immune system but also can destroy the leucocytes especially neutrophils. It has an embodiment of virulence factors most of which are secreted. Staphylococcus aureus secretes a number of toxins which cause tissue damage and facilitate spreading and nutrients uptake. Among the toxins, hemolysins α, β, γ, δ and Panton Valentine Leukocidin (PVL) are unique that they drill pores in the membrane, leading to the efflux of vital molecules and metabolites. Hemolysins also help in the scavenging of iron, although many of them also have leucolytic properties. α-hemolysin, also known as α-toxin, is the most prominent cytotoxin which damages a wide range of host cells including epithelial cells, endothelial cells, erythrocytes, monocytes, keratinocytes and it damages cell membrane and induces apoptosis. β-Hemolysin significantly affects human immune cell function. It has Mg2+ dependent sphingomyelinase activity and degrades sphingomyelin of plasma membrane into phosphorylcholine and ceramides. The bi-component leukocidins, which include γ-hemolysin and PVL, attack human phagocytic cells and greatly contribute to immune evasion. Delta toxin is a low molecular weight exotoxin with a broad cytolytic activity. Virulence determinants, quorum sensing and biofilm synthesis provide some attractive targets for design and development of a new group of antimicrobial compounds. This review provides an update on the structure, biological functions of hemolysins and their role in quorum sensing/biofilm synthesis (if any) and as effective therapeutic targets for anti-virulence drug development. We have tried to bring together information available on various aspects of hemolysins and highlighted their distribution among all species of Staphylococcus and other bacteria. We have updated the status of development of candidate drugs targeting the hemolysins for anti-virulence therapy as it offers an additional strategy to reduce the severity of infection and which would, through quorum quenching, delay the development biofilms leading to drug resistance.

Immunopotentiating Effects of Cucurbitacin B in Mice

Cucurbitacin B(CUB)is a major active principle contained in the calyx melo of Cucumis melo L.The immunopotentiating effects of CuB(im,qd×5)were studied.At lower doses, CuB increased the number of peripheral blood T lymphocytes(0.1 mg/kg),the rate of PHA-induced lymphocyte transformation(0.2 mg/kg),the number of plaque forming cells(PFC)of the spleen(0.2 mg/kg)and the level of serum hemolysin(0.4 mg/kg).The phagocytosis of macrophages and the clearance rate of charcoal particles were enhanced only by a large dose(0.8 mg/kg).The results indicate that CuB can potentiate both cellular and humoral immune function.

Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective:To characterize biofilm production by clinical(n=21)and environmental(n=11)isolates of Burkholderia pseudomallei and evaluate the production of proteases,hemolysins and siderophores.Methods:Initially,the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1%glucose(MH-1%glucose)and BHI broth-1%glucose,using the crystal violet staining technique.Subsequently,growing(48 h)and mature(72 h)biofilms were evaluated by confocal microscopy.Finally,the production of proteases,hemolysins and siderophores by planktonic aggregates,growing biofilms and mature biofilms was evaluated.Results:All isolates produced biofilms,but clinical isolates had significantly higher biomass in both MH-1%glucose(P<0.001)and BHI-glucose 1%(P=0.005).The structural analyses by confocal microscopy showed thick biofilms,composed of multiple layers of cells,homogeneously arranged,with mature biofilms of clinical isolates presenting higher biomass(P=0.019)and thickness of the entire area(P=0.029),and lower roughness coefficient(P=0.007)than those of environmental isolates.Protease production by growing biofilms was significantly greater than that of planktonic(P<0.001)and mature biofilms(P<0.001).Hemolysin release by planktonic aggregates was higher than that of biofilms(P<0.001).Regarding siderophores,mature biofilms presented higher production than growing biofilms(P<0.001)and planktonic aggregates(P<0.001).Conclusions:Clinical isolates have higher production of biofilms than their environmental counterparts;protease and siderophores seem important for growth and maintenance of Burkholderia pseu­domallei biofilms.

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica(LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly(p < 0.01). However, the survival time of the two LMW fucoidans was not statistically significant(p > 0.05). In this study, it was shown that both of two LMW fucoidans(LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

Cloning,Expressing,and Hemolysis of tdh,trh and tlh Genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

Detection of 232bp Virulent Gene of Pathogenic <i>Aeromonas hydrophila</i>through PCR Based Technique: (<i>A Rapid Molecular Diagnostic Approach</i>)

The pathogenicity of aeromonads produces due to exotoxins such as cytolytic enterotoxin, hemolysin or aerolysin, lipases and proteases. Rapid detection of A. hydrophila cytolytic enterotoxin (AHCYTONE) gene and their characterization has proven importance so that proper and rapid preventive and control measures could be taken up to reduce mortality and loss in fish culture. The main objective of the present study is to genetic identification of AHCYTONE positive Aeromonas hydrophila. Strains were isolated from fishes from different fish market in West Bengal and water samples from different river and ponds. Initially strains were identified by their phenotypic and biochemical characterization. Due to contradiction of those results, molecular characterization was done by polymerase chain reaction, which is proved a suitable and rapid diagnostic tool for identification and characterization of A. hydrophila. We have also evaluated the potential risk to human health that this finding can represent by determining the presence of the cytolytic enterotoxin gene in such isolates.

Study on hepatoprotective effect of peptide S-8300 from shark liver

AIM: To explore the effects of peptide S-8300 from shark liver (S-8300) on liver function as well as in regulating immune functions in experimental injury models. METHODS: Mice were administered with different doses of S-8300 for four consecutive days, followed by mice injected with tetrachloromethane (CCI4) on d 3. The activity of ALT, AST, LDH, SOD and contents of MDA and GSH in the mice liver were tested. And mice were treated with Cy (100 mg/kg) at the beginning of the experiment to induce immunosuppression model. The S-8300 groups were treated with S-8300 seven days after the beginning of Cy administration. The effects of S-8300 on the formulation of serum hemolysin and the content of IL-2 in serum in the immunosuppression mice were observed. RESULTS: S-8300 obviously decreased the level of ALT (52.2±11.0 U/dL vs135.9±6.5 U/dL, P<0.01), AST (67.5±6.9 U/dL vs 238.8±8.7 U/dL, P<0.01), LDH (155.1±46.8 U/dL vs 240.4±6.0 U/dL, P<0.01) & MDA (0.64?.027 nmol/mg vs 1.06±0.040 nmol/mg, P<0.01) and increased SOD (24.51±1.01 U/mg vs 19.05±0.73 U/mg, P<0.01) & GSH (24.17±0.91 μg/mg vs 14.93±0.45 μg/mg, P<0,01) in mice liver damaged by CCI4. S-8300 also markedly improved the formulation of serum hemolysin (0.094±0.005 A540 vs 0.063±0.006 A540, P<0.01) and increased the level of IL-2 (9.74±1.16 pg/mL vs 5.81±0.87 pg/mL, P<0.01) in serum of the immunosuppression mice. CONCLUSION: The results suggested S-8300 has significant hepatoprotective, immunomodulatory and inhibiting of lipid peroxidation activity.

Studies on Hemolysis of Hemolysin Produced by Synechocystis sp. PCC 6803

Hemolysin produced by various bacteria,may destroy erythrocyte membranes via a pore-forming mechanism,a deter-gent action,or a lipase activity.Previous to this experiment,the mode of action used by cyanobacterial hemolysin had not been re-ported.To characterize the action mode of hemolysin produced by the wild-type strain of Synechocystis sp.PCC6803,hemolysis of erythrocytes originating from human,mouse,sheep,rabbit and goldfish was studied.The erythrocytes of mouse,sheep and rabbit were sensitive,while those of human and fish were resistant,to this hemolysin.Using rabbit erythrocytes,it was shown that hemoly-sis occurred in two steps:a binding step within the first 10 min of treatment and a lytic step after 30 min.Both binding and lysis were highly temperature-dependent.Effects of erythrocyte density on hemolysis suggest that the hemolysin might target erythrocytes via a multiple-hit mechanism.In the osmotic protection experiment,all tested osmotic protectants,with molecular diameters ranging from 0.9 ?5.66 nm,failed to effectively inhibit hemolysis.Scanning electron micrographs showed that the hemolysin caused protuberances or echinocytes in rabbit erythrocytes,and then disrupted and ruptured the erythrocytes.Characteristics of hemolysis showed distinct differences from other pore-forming mechanisms,suggesting that this hemolysin might act through a detergent-like or lipase mecha-nism,rather than a pore-forming mechanism.

The Influence of Polysaccharides Extracted from Radix Isatidis on Immunity

The Radix Isatidis polysaccharides (IIP) injected ip at the dose of 50mg/kg daily for seven days increased the spleen index. the total numbero of leukocytes and lymphocytes,the percentage of ANAE+ lymphocytes and the intensity of delayed type hyperoensitivity in normal mice as well as in immunosuppressed mice.Moreover,IIP could improve the phagocytic function of reticuloendothelial system,promote the yield of hemolysin,and increase the NK activity in norwal mice at the same dose.

Virulence properties of a peptide hemolysin produced by Enterococcus faecalis

The characterization and prevalence of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated. Recently, we described the production of a heat-stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L-arginine). Now, we purified the hemolysin from the culture supernatant by ultra-filtration (PM-10 membrane) and ethanol extraction followed by chromatography in a mBondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteolytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning electron microscopy showed that lyse of erythrocytes occured at once after the exposure to the hemolysin. The mito-chondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results suggest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci infections.

Effect of Radix Isatidis Polysaccharides on Immunological Function and Expression of Immune Related Cytokines in Mice

Objective： To investigate the effect of Radix Isatidis polysaccharides （RIP） on the immunological function and expression of immune related cytokines in mice. Methods： Sixty mice were randomly divided into six groups, the normal group, cyclophosphamide （Cy） model group, Levamisole （positive control） group, RIP low, medium and high dose groups （0.08 g/kg, 0.16 g/kg, 0.32 g/kg, respectively）, ten in each group. By detecting the value of abdominal macrophage phagocytic index, serum hemolysin （HC50）, proliferation of lymphocytes and expression of related cytokines, interleukin （IL-2） and interferon γ, （INF-γ）, the effect of RIP on immunological function and its mechanism were studied. Results： RIP could improve the level of hemolysin in immunological function depressed mice. The results showed that the value of macrophage phagocytic index in the high dose RIP group increased from 1.11 ±0.13 to 1.42±0.26. The level of IL-2 and INF- γ, could be decreased by Cy to 38.12±6.88 ng/L and 139.23±29.87 ng/L, respectively, while RIP in high dose could increase the secretion of IL-2 and INF- γ, to 53.54 ± 14.43 ng/L and 189.91 ± 32.63 ng/L, respectively. Conclusion： RIP could enhance non-specific immunological function, humoral immunity and cellular immunity in mice.

Immunological Effects of Total Flavones from Leaves of Choerospondias axillaris on Mice

ObjectiveTo investigate the effect of total flavones from the leaves of Choerospondias axillaris (TFLCA) on the immune function of normal mice and to provide the experimental basis for the reasonable application of C. axillaris.MethodsThe carbon clearance method, cutaneous delayed hypersensitivity reaction method, serum hemolysin method, and index of immune organs were used to study the effect of TFLCA on the immune function of mice.ResultsTFLCA could enhance the phagocytic function of mononuclear macrophage and the cutaneous delayed hypersensitivity reaction of mice, and increase the content of hemolysin antibody and the thymus index in mice.ConclusionTFLCA could improve the celiac macrophage activity and specific immunity of mice, and TFLCA, consisting with the total flavones of Choerospondiatis Fructus (TFCF), has the effect on the immune function of mice. So both TFLCA and TFCF have the regulatory effects on the immune function of mice.

Oligomerization of Vibrio cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.