Michiko Arita

Amino acid sequence of heat-stable enterotoxin produced by Vibrio cholerae non-01

The amino acid sequence of heat‐stable enterotoxin, produced by Vibrio cholerae non‐01 and isolated from its culture supernatant, was determined by both Edman degradation of native and reductively carboxy‐methylated enterotoxin and also a combination of fast atom bombardment mass spectrometry and carboxy‐peptidase Y digestion of native enterotoxin to be as follows: Ile‐Asp‐Cys‐Cys‐Glu‐Ile‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Phe‐Gly‐Cys‐Leu‐Asn. This sequence is very similar, but not identical, to those of heat‐stable enterotoxins produced by enterotoxigenic Escherichia coli and Yersinia enterocolitica.

vpaH, a Gene Encoding a Novel Histone-Like Nucleoid Structure-Like Protein That Was Possibly Horizontally Acquired, Regulates the Biogenesis of Lateral Flagella in trh-Positive Vibrio parahaemolyticus TH3996

ABSTRACT A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.

Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks.

An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks.We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm # 1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The 0 serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1–170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.

Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains.

Purification and Characterization of the CS2 Pili of Colonization Factor Antigen II Produced by Human Enterotoxigenic Escherichia coli

A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied. Analysis revealed that CS2‐possessing ETEC was predominant among isolates from travellers diarrhea at Osaka, Japan. TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl‐Sepharose CL‐4B. It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili. This suggests that TH61 pili purified in this study are CS2 pili. Subunit (pilin) of the purified pili has a molecular weight of about 16,000. Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili. These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells.

DEVELOPMENT OF A SIMPLE TEST (BIKEN TEST) FOR DETECTION OF LT-PRODUCING ESCHERICHIA COLI AND APPLICATION OF THIS TEST

It has been established that enterotoxigenic Escherichia coli is one of the most important causative bacteria of diarrhea in developing countries and of traveller’s diarrhea (18, 20). Although we need further informations on epidemiology of enterotoxigenic E. coli, they are limited because of difficulties of performing the identification of the organisms.