Cupiennin 1a exhibits a remarkably broad,non-stereospecific cytolytic activity on bacteria,protozoan parasites,insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Evidence for plasmid-mediated toxin production inBacillus cereus BIS-59

A strain ofBacillus cereus, isolated from shrimps pasteurized by radiation, harboured a 7.6mda plasmid. When cells were cured with Acridine Orange, they still produced the haemolytic toxin but not the non-haemolytic one. The results therefore suggest that the plasmid encodes the gene(s) responsible for the non-haemolytic toxin.     

Adherence of a virulent strain of Listeria monocytogenes to the surface of a hepatocarcinoma cell line via lectin-substrate interaction

Listeria monocytogenes was examined for the presence of surface carbohydrates to ascertain whether surface sugars, if present, would interact with eucaryotic surface carbohydrate receptors. We found that a virulent, but not two avirulent strains had a surface -d-galactose residue as determined by agglutination with Griffonia simplicifolia (GS-I) and other lectins. The virulent strain bound to a human hepatocarcinoma cell line (HepG2), which has a well characterized receptor for -d-galactose. This interaction could be blocked by pretreatment of the HepG2 cells with either -d-galactose or neuraminidase, the latter of which will render the galactose receptor functionally inactive. We propose that the attachment of the virulent Listeria to eucaryotic cells occurs as a result of the interaction of the microbial -d-galactose with that of the eucaryotic galactose receptor. This surface carbohydrate may provide an explanation for the mechanism of attachment and penetration of virulent Listeria into host cells during infection. As such, this may allow for amplication of pathogenesis through intracellular multiplication in nonprofessional phagocytes prior to macrophage involvement.Abbreviations ATCC 19113 and ATCC 4428 Listeria monocytogenes, avirulent strains - EDG Listeria monocytogenes, virulent strain - GS-I Griffonia simplicifolia lectin - GepG2 Human, hepatocarcinoma cells - MES buffer 2(N-Morpholino)-ethane sulfonic acid - PBS buffer phosphate buffered saline This work was carried out and submitted in part by JL to the 47th Annual Westinghouse Science Talent Search while a senior at Union High School, Tulsa, OklahomaRecipient of a National Merit Scholarship Award. Presently attending Southwest Missouri State University, Springfield, Missouri     

Polysaccharides purified from the submerged culture of Ganoderma formosanum stimulate macrophage activation and protect mice against Listeria monocytogenes infection

The bioactive components of Ganoderma formosanum have not yet been characterized. We investigated the immunomodulatory activities of the extracellular polysaccharides produced from a submerged mycelial culture of G. formosanum. The polysaccharides were mainly composed of d-mannose, d-galactose and d-glucose. After gel filtration chromatography, three polysaccharide fractions (PS-F1, PS-F2 and PS-F3) were purified. PS-F2 stimulated mouse RAW264.7 macrophages to produce TNF-α and nitric oxide, and enhanced the phagocytic activity of macrophages. PS-F2 challenge in mice triggered an acute inflammatory response characterized by the recruitment of neutrophils and monocytes, which protected mice from subsequent infection of Listeria monocytogenes. The results indicate that the heteropolysaccharides produced by G. formosanum can activate the innate immune response on macrophages.     

Cloning and heterologous expression of a bacteriocin sakacin P from <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sakacin P, a bacteriocin from Lactobacillus sakei, shows strong activity against food-borne pathogens such as Listeria monocytogenes. In L. sakei, the structural gene (sppA) encoding sakacin P is controlled by a strict regulatory mechanism, and the quantity of secreted sakacin P is limited. In this study, the sppA gene was synthesized by splicing overlap extension PCR and cloned into Escherichia coli. After the induction with isopropyl-β-d-thiogalactopyranoside, the recombinant sakacin P was successfully expressed. The collected cells were sonicated, and the activity was detected by agar diffusion method. The results also showed that the low-temperature induction can improve the activity of sakacin P.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Antimicrobial activity of various cationic molecules on foodborne pathogens

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.     

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced ‘transporter associated with antigen processing’-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS. typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys₄₉₂ strain. In contrast, efficacy of i.v. vaccination with either rSalmonella strain did not significantly differ. Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.     

Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen

Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore-forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth. Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfrlngolysln O (PFO). When PFO is secreted by intracellular L. monocytogenes, it is toxic to the infected host cell. PFO-mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria. In this study, we selected for L. monocytogenes mutants in which PFO supported the intracellular growth of L. monocytogenes. Six independent mutants were isolated, each containing a single amino acid change within the PFO protein. Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell. The first class had a severe defect in haemolytic activity. The second class had a change in the pH optimum of PFO. The third class had nearly wild-type levels of haemolytic activity, but had a decrease in protein half-life in the host-cell cytosol. Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole-specific lysin which mediated growth of L. monocytogenes in cultured cells.     

Haemolytic activity within the species Fibrocapsa japonica (Raphidophyceae)

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC₅₀ values ranged between 0.4 × 10⁴ and 1.9 × 10⁴ F. japonica cells ml⁻¹. The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs) was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC₅₀ of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina.In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20–35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC₅₀ values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms.     

Complement-dependent cytotoxic activity of serum mannan-binding protein towards mammalian cells with surface-exposed high-mannose type glycans

Serum mannan-binding protein (S-MBP), a lectin specific for mannose andN-acetylglucosamine, activates complement through the classical pathway. With the help of complement, S-MBP lyses red blood cells which have been coated with yeast mannan and kills bacteria which haveN-acetylglucosamine and/orl-glycero-d-manno-heptose on their core oligosaccharide. In this study, we examined whether mammalian cells, on which S-MBP could bound, are killed by a complement-dependent mechanism. When baby hamster kidney (BHK) cells were treated with an -mannosidase inhibitor, 1-deoxymannojirimycin (dMM), most of the cellular oligosaccharides were transformed from the complex-type to the high mannose-type. S-MBP bound to the dMM-treated BHK cells in the presence of Ca²⁺, and this binding was eliminated by mannose. When dMM-treated cells, labelled with⁵¹Cr, were incubated with complement, radioactivity was released in a dose-dependent manner by S-MBP and complement. This release was not observed with heat-inactivated complement. These observations suggest that S-MBP is able, with the help of complement, to kill not only exogenous microorganisms but also mammalian cells which have high mannose-type oligosaccharides exposed on their surfaces. Abbreviations: S-MBP, serum mannan-binding protein; BHK, baby hamster kidney; dMM, 1-deoxymannojirimycin; FITC, fluorescein-isothiocyanate; TBS, Tris buffered saline; HIV, human immunodeficiency virus; CH₅₀, 50% haemolytic units. The complement nomenclature used was recommended by the World Health Organization (1986).This paper is dedicated to Nathan Sharon.     

Improved Electrochemical Analysis of Neuropathy Target Esterase Activity by a Tyrosinase Carbon Paste Electrode Modified by 1-Methoxyphenazine Methosulfate

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N=2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r=0.990, n=7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.     

Production of thiol-dependent haemolysins by Listeria monocytogenes and related species

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.     

Propofol-induced vascular permeability change is related to the nitric oxide signaling pathway and occludin phosphorylation

The present study was undertaken to elucidate the mechanism of intra-arterial propofol-induced vascular permeability change resulting in tissue edema. The mechanism of propofol-induced hyperpermeability was examined in a rat femoral artery injection model. Vascular permeability was determined by measuring the Evans blue content of the dorsal skin of the infused limb at 15, 30, 45 and 60 min after propofol injection. The total content of the tight junction proteins occludin, ZO-1 and claudin-5 under experimental conditions was also determined by western blotting. Intra-arterial injection with propofol resulted in a marked dose-dependent increase in vascular permeability of the rat hindpaw. Pretreatment with 10 mg/kg of N-nitro-l-arginine methyl ester (l-NAME) but not aminoguanidine significantly inhibited the change in vascular permeability after challenge with propofol. Pretreatment with l-arginine and nitroprusside increased the propofol-induced permeability change. Intra-arterial injection of propofol significantly increased occludin phosphorylation after 15 min, which was consistent with the time profile of the vascular permeability change. l-NAME partially reversed the change in occludin phosphorylation, whereas aminoguanidine had no effect compared with that in the controls. Our observations indicate that nitric oxide (NO) is an important mediator in the induction of vascular permeability induced by propofol. Occludin phosphorylation is a determining factor in the vascular permeability change induced by propofol. NO synthase (NOS) inhibitors might be useful in the treatment of accidental intra-arterial injection of propofol, in the reduction of any adverse effects.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

Haemolytic activity and reactive oxygen species production of four harmful algal bloom species

Based on haemolytic activity and reactive oxygen species (ROS) production of Chattonella marina, Chattonella antiqua, Heterocapsa circularisquama, Alexandrium tamiyavanichii and Karenia mikimotoi, the species were categorized into four types. (1) H. circularisquama: haemolytic activity was detected in both cell suspension and cell-free culture supernatant, but with greater activity in cell suspension than in the supernatant suggesting the presence of both cell surface and secreted haemolytic agents. (2) A. tamiyavanichii: equal haemolytic activities were detected in both the cell suspension and cell-free culture supernatant suggesting the presence of only secreted haemolytic agents. (3) K. mikimotoi: haemolytic activity was detected only in the cell suspension, indicating haemolytic agents occur only on the cell surface. (4) C. marina and C. antiqua: no significant haemolytic activity was detected in either cell suspension or cell-free culture supernatant, but high ROS were detected in the cell suspensions. Heterocapsa circularisquama and K. mikimotoi showed lethal effects on rotifers (Brachionus plicatilis), whereas A. tamiyavanichii, C. marina and C. antiqua had no effect. Our results suggest that H. circularisquama, K. mikimotoi and A. tamiyavanichii produce haemolytic agents with distinct characteristics, whereas C. marina and C. antiqua have an extremely potent ability to produce ROS.     

Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly

A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.