Shinji Yamasaki

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT‐I), VT2 (or SLT‐II), VT2vha, VT2vhb, SLT‐IIv (or VT2vp1, VTe) and SLT‐IIva (or VT2vp2). A total of 80 Verocytotoxin‐producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin‐producing Escherichia coli (VTEC), a sensitive bead‐enzyme‐linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo‐ and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Importance of arginine at position 170 of the A subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia coli for toxin activity

Comparison of the primary structures of the A subunits of Vero toxin 1 (VT1), Vero toxin 2 (VT2), and two variants of VT2 (VT2vp and VT2vh) and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1). All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis. To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis. A total of 22 mutants were prepared to examine 14 conserved residues, and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1. Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically. These results suggest that, in addition to the glutamic acid at position 167 reported previously, arginine at position 170 also plays an important role in the toxin activity of VT1. A possible chemical mechanism of the enzymatic (N-glycosidase) activity of VT1 is proposed based on the relative activities of various mutants.

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT‐IIv (VT2vp1) and SLT‐IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin‐producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.

Detection of Cholera Toxin by a Highly Sensitive Bead-Enzyme Linked Immunosorbent Assay

A bead‐enzyme linked immunosorbent assay (bead‐ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead‐ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead‐ELISA. Evaluation of the sensitivity and specificity of the bead‐ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead‐ELISA was more sensitive than the RPLA. Quantification of CT by the bead‐ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead‐ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae 0139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide‐directed site‐specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q‐R170L (glutamic acid at position 167 and arginine at position 170 from N‐terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q‐R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild‐type VT2vp1. Since the antigenic property of the E167Q‐R170L was demonstrated to be similar to that of the wild‐type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q‐R170L, as a toxoid is discussed.

Examination by site-directed mutagenesis of the amino acid residues of the thermostable direct hemolysin of Vibrio parahaemolyticus required for its hemolytic activity☆☆☆

The amino acid sequences of eight variants of thermostable direct hemolysin (TDH) and a TDH-related hemolysin, deduced from nucleotide sequences, were compared. Fourteen amino acid residues, mainly within conserved regions, were chosen to prepare mutant TDHs with one or two amino-acid replacements by site-directed mutagenesis. Of the 25 mutant TDHs prepared, those with replacements of Trp65 (Trp65-His65, Trp65-Tyr65, Trp65-Leu65) or Leu66 (Leu66-Ser66) showed very significant reduction (more than 150-fold) of hemolytic activity. Several other mutant TDHs with replacements at Lys45, Arg46 and Gly90 showed weaker hemolytic activity than the wild-type TDH, but extents of reduction in hemolytic activity were much less than for mutant TDHs with replacements at Trp65 and Leu66. These results indicate that Trp65 and Leu66 are very important for the hemolytic activity of TDH.

Specific Detection of a Verotoxin 2 Variant, VT2vp1, by a Bead‐Enzyme‐Linked Immunosorbent Assay

A bead‐enzyme‐linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vpl, was developed. The sensitivity of the bead‐ELISA was 200 pg/ml of the purified VT2vpl and it did not react with 20 ng/ml of the purified VT2. The specificity of the bead‐ELISA was examined with 107 strains of Verocytotoxin‐producing Escherichia coli that include VT1‐, VT2‐, VT2vha‐, VT2vhb‐ and VT2vp1‐producing E. coli, and only VT2vp1‐producing E. coli that were confirmed by VT2vp1‐specific polymerase chain reaction gave positive results. It was noted that all 58 VT2vp1‐producing E. coli strains were from pigs, but not from cows and humans.

Heat-Stable Enterotoxins Produced by Enteric Bacteria

Enterotoxigenic Escherichia coli produces two types of enterotoxin that cause diarrhea in humans and cattles. One is a high molecular weight heat-labile enterotoxin (LT) and the other is a low molecular weight heat-stable enterotoxin (ST). Two different kinds of ST have been described. One is STa or STI that is methanol soluble and active in suckling mice and piglets, but inactive in weaned pigs. The other is STb or STII that is methanol insoluble and active in weaned pigs, but inactive in suckling mice. At least two genetically distinct STa’s, that is STIa and STIb, have been reported. STIa and STIb also have been called STp and STh, respectively, because STIa and STIb were originally reported in E. coli isolated from pigs (and cows) and humans, respectively, although human isolates produce both STh and STp.