Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

Production of monoclonal antibody against a hemolysin (Vp-TRH) produced by Vibrio parahaemolyticus

The antigenicity of a hemolysin (Vp-TRH: Vp-TDH related hemolysin) produced by Kanagawa phenomenon-negative clinical isolates of Vibrio parahaemolyticus was studied using monoclonal antibodies (MAbs). A total of 12 hybridoma clones which produced MAbs against Vp-TRH were established. All MAbs contained the Kappa light chain and were IgG type. These MAbs were divided into a minimum of 5 different specificity groups, including antibodies specific to Vp-TRH and common to both Vp-TRH and Vp-TDH, a possible pathogenic toxin of Kanagawa phenomenon-positive V. parahaemolyticus. These results clearly show the immunological similarity and dissimilarity (specificity) of Vp-TRH and Vp-TDH.     

Purification of a TDH-related hemolysin produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus 06: K46

A hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus 06: K46 was purified by 55% ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q. The purified hemolysin was physicochemically and immunologically identical with the Vp-TRH (V. parahaemolyticus thermostable direct hemolysin related hemolysin) recently described in V. parahaemolyticus 03: K6 (Honda et al. Infect. Immun. 56: 961-965, 1988). This indicates that V. parahaemolyticus of Kanagawa-negative clinical isolates possessing not only 03: K6 but also different serotypes such as 06: K46 produce Vp-TRH. Production of Vp-TRH by most clinical isolates of Kanagawa-negative V. parahaemolyticus was also demonstrated. These results suggest the importance of Vp-TRH among clinical isolates of Kanagawa-negative V. parahaemolyticus.     

Comparison of Vibrio parahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates

TDH-related hemolysin (Vp-TRH) produced by Kanagawa-phenomenon-negative (KP-) Vibrio parahaemolyticus has been demonstrated to be a possible virulence determinant. Though almost half of KP- isolates examined from diarrhoeal patients produced Vp-TRH, few reports mentioned the ability of environmental isolates to produce Vp-TRH. Considering the route of infection with V. parahaemolyticus, this toxin must be produced by the organisms in the sea or in sea food. To confirm that Vp-TRH produced by V. parahaemolyticus could be involved in sea-food-borne diarrhoeas, Vp-TRH-producing strains were isolated from the environment, identified and hemolysin purified from these strains was compared to hemolysin (Vp-TRH) isolated from diarrhoeal patients. The results showed that the hemolytic activity, antigenicity, reactivity in the rabbit ileal loop test and N-terminal amino acid sequence of Vp-TRH from environmental strains was indistinguishable from the toxin of clinical origin.     

Purification and partial characterization of a Vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin produced by Vibrio hollisae (Vh-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus, was studied. Vh-rTDH was purified by successive column chromatographies on diethylaminoethyl-cellulose and an immunoaffinity column coupled with anti Vp-TDH immunoglobulin. The purified toxin was homogeneous, as demonstrated by conventional and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (PAGE). The molecular weight of Vh-rTDH was slightly smaller than that of Vp-TDH, as determined by sodium dodecyl sulfate--slab gel electrophoresis. Conventional PAGE also showed a difference between Vh-rTDH and Vp-TDH. Vp-TDH and Vh-rTDH showed different lytic activities on erythrocytes from various animals, in particular chicken, sheep, and calf. The hemolytic activity of Vh-rTDH was heat labile when heated at 70 degrees C for 10 min, unlike Vp-TDH. Immunological cross-reactivity between Vh-rTDH and Vp-TDH was demonstrated by both the Ouchterlony test and neutralization test.     

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000-1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000-1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .     

猪链球菌2型溶血素的化学修饰

用DTT、H2O2、DEPC、EDC、NAI、NBS、PCMB、2,3Diacetyl和SA 等9种化学修饰剂,处理猪链球菌2型江苏分离株提纯的溶血素,研究其分子中氨基酸侧链基团与其溶血活性的关系。结果表明,巯基、氨基、羧基和酪氨酸残基等与其溶血活性无关,而色氨酸、组氨酸和精氨酸的化学修饰引起溶血活性的大幅度下降,二硫键的化学修饰引起溶血活性的大幅度增强。显示色氨酸、组氨酸和精氨酸残基是该溶血素的活性必需基团,二硫键的断裂可引起其活性增强。     

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.     

The anti-haemolysin activity of tea and coffee

Extracts of tea and coffee inhibited the haemolytic activities of Staphylococcus aureus α-toxin and Vibrio parahaemolyticus thermostable direct haemolysin (Vp-TDH). Black tea had the strongest anti-haemolysin activity. Green tea was more active than pu-erh tea. Coffee had anti-Vp-TDH activity but not anti-α-toxin activity.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

The nucleotide sequence recognised by the Escherichia coli D type I restriction and modification enzyme.

A type I restriction endonuclease from a new isolate of Escherichia coli (E. coli E166) has been purified and characterised. The enzyme, EcoD, has a recognition sequence similar in overall structure to the previously determined type I enzyme sequences, an exception being that it is degenerate. The sequence is 5'-T-T-A-N-N-N-N-N-N-N-G-T-C-Y-3' 3'-A-A-T-N-N-N-N-N-N-N-C-A-G-R-5' where Y is a pyrimidine, R is a purine and N can be any nucleotide. The enzyme methylates adenosyl residues in both strands of the DNA that are separated by ten base pairs, suggesting that the enzyme interacts with DNA along one face of the helix making contacts in two successive major grooves.     

Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak

Abstract Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995. The components were purified by chromatography on three different columns. Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity. The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined. The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra). The 39 kDa protein showed some similarity to the l₁ protein of haemolysin BL from B. cereus . Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B. cereus . The proteins were different from the B- and L₂-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity.     

Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica

Abstract A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly ( Musca domestica ) as well as other Diptera and Lepidoptera . Crystal δ-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 μg/ml, and β-exotoxin was not detected. Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination. Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cry IA(b), one cry IIA, and two cry IIB genes.     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670(T) isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).     

The isolation of Naegleria italica from Peru indicates that this potentially pathogenic species occurs worldwide

The amoeboflagellate genus Naegleria includes a few species that are virulent in experimental animals. One of these species, Naegleria italica, has been isolated from the environment only in Italy and Australia. I report here the isolation of a strain of N. italica from a water sample collected in Peru. This broadens the occurrence of this species to encompass three different continents. This new N. italica isolate from Peru has the same ITS1, 5.8S rDNA and ITS2 sequence as that of the type strain from Italy and the isolate from Australia. From the same water body in Peru a Naegleria strain was isolated that differs from N. italica by only one additional base pair in the ITS2 sequence. The maximum growth temperature tolerated by this particular isolate is 40 degrees C, which is different from that of N. italica, which is 42 degrees C.