Myonsun Yoh

Metagenomic Diagnosis of Bacterial Infections

To test the ability of high-throughput DNA sequencing to detect bacterial pathogens, we used it on DNA from a patient’s feces during and after diarrheal illness. Sequences showing best matches for Campylobacter jejuni were detected only in the illness sample. Various bacteria may be detectable with this metagenomic approach.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157: H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay.

A total nine hybridoma cell lines that produced monoclonal antibodies against thermostable direct hemolysin (Vp-TDH), a possible pathogenic toxin, of Kanagawa phenomenon-positive Vibrio parahaemolyticus was isolated and characterized. These monoclonal antibodies (mAbs) were divided into a minimum of five different specificity groups, including mAbs specific to Vp-TDH and common to Vp-TDH and Vp-TRH, a Vp-TDH-related hemolysin produced by Kanagawa phenomenon-negative V. parahaemolyticus. An enzyme-linked immunosorbent assay (ELISA) using mAb-1-D, a mAb specific for Vp-TDH, was developed for specific detection of Vp-TDH. On the other hand, the ELISA using mAb-9-D, and mAb common to both Vp-TDH and Vp-TRH, could be used for detection of both Vp-TDH and Vp-TRH. Thus, by combining these two ELISAs differential detection of Vp-TDH and Vp-TRH can be performed. Hence, the two ELISAs were applied for various strains of V. parahaemolyticus and it was found that most Kanagawa phenomenon-positive and -negative clinical isolates produced Vp-TDH and Vp-TRH, respectively, but all environmental strains, that were Kanagawa phenomenon-negative, produced neither toxin.

Characterization of thermostable direct hemolysins encoded by four representative tdh genes of Vibrio parahaemolyticus

Four tdh genes encoding thermostable direct hemolysin (TDH) cloned from two representative strains of V. parahaemolyticus (tdh 1 and tdh 2 from a hemolytic strain, tdh3 and tdh4 from a non-hemolytic strain) have different nucleotide sequences (the maximum divergence: 3.3%). In this study, each tdh gene product was purified from the lysate of Escherichia coli cells carrying the cloned gene and their properties were compared to investigate the influence of the amino acid substitutions caused by these base changes. The four tdh gene products showed different electrophoretic mobilities under non-denaturing conditions. All the gene products had hemolytic activities for various animal erythrocytes, stimulated vascular permeability in the rabbit skin, and were lethal to mice, although their potencies were slightly different. Antigenicities of the four gene products were indistinguishable. These results indicate that the four tdh genes have evolved to maintain a fundamental molecular structure and biological activities of the gene products and minor structural and/or charge differences of the molecules are perhaps responsible for the slight divergence of their biological activities.

Replacement of Mg2+ by monovalent cations in aminoacyl transfer RNA formation

Abstract 1. The effect of monovalent cations on aminoacyl-tRNA formation in the absence of added Mg2+ has been studied. 2. In valyl-tRNA formation, all monovalent cations studied (NH+4 Li+, Na+ and K+) are stimulatory, with NH+4 and Li+ being the most effective. An optimum concentration of monovalent cations is between 100 and 200 mM. 3. In leucyl-tRNA formation, NH+4, Li+ or K+ stimulates the reaction significantly. Not only the yield of leucyl-tRNA, but also the rate of the reaction is almost the same as those observed in the presence of an optimal amount of Mg2+. 4. In isoleucyl-tRNA formation, only NH+4 is stimulatory. 5. A synergistic effect of Mg2+ and some monovalent cations is observed in valyl-, isolecyul- and phenylalanyl-tRNA formation. 6. In the presence of NH+4, K+ or Na+, PPi-ATP exchange and amino acid hydroxamate formation are not observed, while they do occur in the presence of Li+. 7. It is assumed that in some aminoacyl-tRNA formations certain monovalent cations can replace Mg2+ either completely or partially.

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.

The binding of Vibrio parahaemolyticus 125I-labeled thermostable direct hemolysin to erythrocytes

Thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus was iodinated using chloramine T. The 125I-labeled TDH retained up to 80% of the activity of intact toxin. The binding of 125I-TDH to rabbit erythrocytes was inhibited by addition of nonlabeled TDH. The binding of 125I-TDH to rabbit erythrocytes was completed in the 1st or 2nd min of incubation at 37 degrees C in contrast to that at 4 degrees C. 125I-TDH, which cannot lyse horse erythrocytes as does intact TDH, bound to horse erythrocytes as to those of rabbit. The dissociation constants (KD) derived Scatchard plots were 2.85, 4.39, 4.33 and 5.35 x 10-8M for rabbit, horse, human and sheep erythrocytes, respectively. The lytic sensitivity of various erythrocytes to TDH showed no relationship to the binding capacity.

Effect of environmental conditions on proteins secreted by enterohemorrhagic Escherichia coli O26:H11

Infections due to Shiga toxin‐producing Escherichia coli (STEC) are responsible for severe diarrheal diseases in humans, and these bacteria have recently emerged as a leading cause of renal failure and encephalitis in children and the aged. In this study, we examined the environment‐dependent production of proteins secreted from a strain of STEC O26:H11 by trichloroacetic acid precipitation, SDS‐PAGE, Western blotting and N‐terminal amino acid sequence analysis. Growth of bacteria in essential minimum medium (M9) led to the detection of secreted proteins of 104, 80, 40, 37 and 25 kDa (P104, P80, P40, P37 and P25, respectively). When grown in serum‐free MEM, only P104, P40, P37 and P25 were observed in supernatant fluids. Growth of the bacteria in Luria‐Bertani broth (LB) enhanced the expression of P104, but the productions of the other proteins were remarkably reduced. CO2 increased the secretion of P80 and P37, but reduced the production of P104. N‐terminal amino acid sequencing revealed that P104 was EspP of STEC, which was homologous to EspC of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a subclass of the IgA protease family. P80, which was identified as EspE of STEC, was homologous to Tir of EPEC. P40, P37 and P25 were found to be highly homologous to the similarly sized EspD, EspB and EspA proteins, previously detected in culture supernatants of EPEC. Those proteins are thought to be STEC virulence factors. Sera were obtained from two patients, one with colitis and another with hemolytic uremic syndrome (HUS), caused by STEC O157:H7, to study immune response to secreted proteins. Our results suggested that Tir caused immune response following STEC disease.

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studies using [gamma-32P] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.

Detection of the gene for toxic shock syndrome toxin 1 in Staphylococcus aureus by enzyme-labelled oligonucleotide probes

A colony hybridisation method with enzyme-labelled oligonucleotide probes was developed to detect the gene for toxic shock syndrome toxin 1 (tst). For rapid identification, bacterial colonies were transferred from agar plates directly on to nylon membranes. These procedures took only 3 h. Results obtained by this test correlated well with those obtained by the reverse passive latex agglutination test. Thus, this method is convenient and reliable for the detection of tst in staphylococci, which could be useful for both research and clinical purposes. This method demonstrated that tst was more prevalent in methicillin-resistant Staphylococcus aureus (56%) than in methicillin-susceptible S. aureus (4%).