Tomoko Yamamoto

Induction of Yersinia enterocolitica Stress Proteins by Phagocytosis with Macrophage

Yersinia enterocolitica is a facultative intracellular pathogen which invades to epithelial cells and survives in phagocytes. Since the internal environment of phagocytes should be stressful conditions for the phagocytosed Yersinia, the bacteria should respond to protect themselves from otherwise lethal results. We analyzed the stress‐induced proteins which possibly contribute to survival of Yersinia within the phagocytes. Y. enterocolitica was radiolabeled during the growth in macrophage‐like J774‐1 cells, and the bacterial proteins were analyzed by two‐dimensional gel electrophoresis. At least 16 proteins were selectively induced in response to phagocytosis, and several out of 16 proteins were also induced by heat shock at 42 C or oxidative stresses in vitro. Of those, two major stress proteins were identified to be homologues of DnaK and CRPA by immunoblotting analysis. These results have indicated that Y. enterocolitica exhibits a global stress response to the hostile environment in the phagocytosed macrophage.

Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat-Shock Protein 60

The H20mAb recognizing the 60‐kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat‐shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr‐incubation with the mAb induced no significant inhibition of H. pylori growth. The 24‐ and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.

Detection and characterization of antibodies to bacterial heat-shock protein 60 in sera of patients with primary biliary cirrhosis

The enzyme‐linked immunosorbent assay (ELISA) with bacterial heat‐shock protein 60 (HSP60) purified from Yersinia enterocolitica (Ye) revealed that the antibodies directed against YeHSP60 existed in sera of patients with primary biliary cirrhosis (PBC). To characterize the epitope specificity of the antibodies in patients, the epitope mapping of HSP60 by means of the antibodies was performed. The results have suggested that the epitope recognized with anti‐HSP60 antibodies in PBC relates to the amino acid sequence of YeHSP60 molecule as follows: DLGQA‐KRVVINKDTTIIIDGVGDEAAIQGRLAQIRQQIEEATSDYDKEK.

Cloning and nucleotide sequence analysis of immunodominant heat-shock protein of Yersinia enterocolitica.

Yersinia enterocolitica, a highly antigenic 60-kDa protein, designated cross-reacting protein antigen (CRPA), is a member of the chaperonin-60 family of molecular chaperones. The gene encoding CRPA was cloned, expressed and sequenced. A partial library from Y. enterocolitica 0:3 genomic DNA was constructed in vector pUC19 and was screened by the immunoreactivity to monoclonal antibody 1A4, which has specificity for a species-specific epitope on the CRPA molecule. The crpA gene region consists of a putative two-cistron operon encoding proteins of 549 and 97 amino acids. The operon structure was led by a consensus heat-shock promoter sequence. Homology searches using the derived amino acid sequence have revealed that CRPA is 88.2% identical to GroEL of Escherichia coli. CRPB, another protein encoded by the operon, shows extensive sequence homology, 91.8% identical to GroES of E. coli which is a member of chaperonin-10.

Epitope homology between bacterial heat shock protein and self‐proteins in the host cell

Mouse monoclonal antibodies (MAbs) showing distinct reactivity against the 60‐kilodalton (kDa) antigen heat shock protein of Yersinia enterocolitica, designated cross‐reacting protein antigen (CRPA), have previously been established. The reactivities of these MAbs (5C3 and 3C8) against mouse and human host cells were studied by Western blotting and flow cytometric analysis. The results indicated that epitopes on the bacterial 60‐kDa heat shock protein are present on various molecules in mouse spleen cells and human B cells. An epitope recognized by MAb 5C3 was expressed on the mouse and human host cell surface, and an epitope recognized by MAb 3C8 was also expressed on the human host cell surface.

Immunocytochemical localization of 60-kDa heat shock protein in Vibrio cholerae.

The immunocytochemical localization of the 60-kDa heat shock protein (HSP) in Vibrio cholerae strain 569B was studied by transmission electron microscopy using a combination with the antigen-specific monoclonal antibody (5C3) and immunogold labelling. The labelling with gold particles in V. cholerae detected 2 types; the gold particles were exclusively detected in the cytoplasm for one type and in the periplasmic space for another type, suggesting that the 60-kDa HSP of V. cholerae corresponding immunologically to Escherichia coli GroEL may translocate from the cytoplasm to the periplasmic space in the V. cholerae cell.

Flow cytometric analysis for adhesion of Vibrio cholerae to human intestinal epithelial cell

The adhesion of Vibrio cholerae O1 strains to human intestinal epithelial cell, Intestine 407, was analyzed by flow cytometer. According to positive percentages of Intestine 407 cells adhered by V. cholerae, two groups of V. cholerae strains were classified as follows: more adhesive (more than 50%), less adhesive (less than 50%) strains. In addition, the fluorescence intensity after attachment of V. cholerae was directly correlated to the number of the microorganisms. It was concluded that flow cytometry is a useful and objective method for analyzing adhesion of V. cholerae to cultured cells.

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat‐Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat‐shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316‐342, p327‐359, p340‐366, p316‐326, p316‐321, p319‐323, and p321‐326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316‐342, p316‐326 and p321‐326, and 3C8 mAb p316‐342 and p316‐326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316‐326. The sequence homology between p316‐326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.