The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA⁺hlyA⁺) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.

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The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR^TM vector. These clones were sequenced. The fimA sequences were found to be identical between V. cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V. cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.

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In Salmonella typhimurium, the transcription of several virulence genes including spvB is regulated by the PhoP/PhoQ regulatory system. To further examine the relationship between the PhoP/PhoQ and Spv systems for virulence in mice, we examined a non-polar phoP mutation combined with different virulence plasmid genotypes for effects on virulence of S. typhimurium in the mouse model. PhoP^-/Spv⁺ and PhoP^-/Spv^- mutants were not detectably recovered from the spleens of subcutaneously or orally inoculated mice. The phoP gene constitutively expressed from the lacZ promoter of a low copy number vector (phoP^C) only partially complemented the non-polar phoP mutation for mouse-virulence in both the Spv⁺ and Spv^- backgrounds; both PhoP^C strains exhibited virulence equal only to a PhoP⁺/Spv^- strain. Interestingly, in a PhoP⁺ background, the phoP^C gene reduced splenic infection of the Spv⁺ but not Spv^- salmonellae after subcutaneous or oral inoculation compared with the PhoP⁺ parents. Additionally, the phoP^C gene in an Spv⁺ background reduced the net growth of salmonellae in macrophages in vitro; phoP^C in an Spv^- background was without effect. These data suggest that the constitutive expression of the phoP gene attenuates the virulence of S. typhimurium in mice in an Spv-dependent manner.

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The phylogenetic and taxonomic heterogeneity of a rare opportunistic yeast pathogen, Cryptococcus humicolus, was revealed by analysis of the sequence of the internal transcribed spacer (ITS) region. Sixteen strains of C. humicolus showed a wide diversity in their ITS sequences. In addition, their 18S rDNA sequences were determined and used to analyze the phylogenetic relationships among C. humicolus and related yeasts. On trees constructed by the Neighbor-Joining and Maximum Parsimony methods, C. humicolus strains were phylogenetically closely related to each other with the exception of one strain, and they clustered with C. curvatus and Trichosporon species with high bootstrap values. Three C. humicolus strains obtained from humans belonged to the group of Trichosporon serotype I species. The results suggest that C. humicolus is a genetically heterogeneous species which should be reclassified on the basis of DNA sequence data.

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The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10- to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of A11, may be important segments for Atl to bind to the cell surface.

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Antibodies specific to Salmonella enteritidis (S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer-cultured human intestinal epithelial cells, Caco-2 and T84. The Salmonella adherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL-8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti-S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.

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Heat-stable enterotoxin Ip (STIp) of Escherichia coli is synthesized as a precursor form consisting of pre- (amino acid residues 1 to 19), pro- (amino acid residues 20 to 54) and mature (amino acid residues 55 to 72) regions. Mature STIp (bioactive STIp) is formed in the periplasmic space after the precursor is proteolytically processed and the mature STIp translocates across the outer membrane through the secretory system including TolC, an outer membrane protein of E. coli. However, it remains unknown how the mature STIp is recognized by this secretory system. In this study, we investigated the amino acid residues of STIp involved in its translocation across the outer membrane. We prepared mutant STIp genes by site-directed mutagenesis and analyzed translocation of the mutant STIps across the outer membrane. Deletion of the Phe or Tyr residue at position 3 or 18, respectively, decreased the efficiency of translocation of STIp across the outer membrane. To confirm the involvement of these amino acid residues, we further mutated the codons for these amino acid residues to that for Gly. These mutations also decreased the efficiency of extracellular secretion of STIp. In contrast, substitution of Phe-3 and Tyr-18 with Tyr and Phe, respectively, did not affect the efficiency of translocation of the toxin. These results indicated that the aromatic amino acid residues at positions 3 and 18 in the mature region are important for the ability of STIp to cross the outer membrane.

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HIV-1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 strains using a baculovirus expression system and investigated the association of CD4-gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co-down-modulations of the CD4-gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down-modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell-type and HIV-1 strain-specific differences. We found that T-tropic gp120s were capable of priming co-down-modulation with tailless CD4 by interacting with CXCR4, whereas M-tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T-tropic HIV-1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co-down-modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co-down-modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down-modulation of the CD4/gp120 complexes.

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Since rotavirus vaccines aim to protect children from severe diarrhea, knowledge of the prevailing G serotypes among rotaviruses from hospitalized children is essential. Thus, we determined the G serotypes of rotaviruses collected from children with acute diarrhea in a local referral hospital in Akita, Japan, over the 10-year period between January 1987 and December 1996. Based on the assumption that rotaviruses with an identical electropherotype possess the same G serotype, the G serotypes of 488 rotavirus-positive specimens that were classified into 63 electropherotypes were determined by enzyme-linked immunosorbent assay with a supplementary use of G typing by reverse transcription-PCR. The relative frequencies over the 10-year period were 77.0 (G1), 14.5 (G2), 2.7 (G3) and 5.3% (G4), leaving the possibility that only 0.4% had G serotypes uncommon to human rotaviruses. Of 24, 050 rotaviruses extracted by reviewing 63 serotyping studies in literature, the relative frequencies of the four major G serotypes were 50.6 (G1), 9.3 (G2), 7.2 (G3) and 11.6% (G4). As to uncommon G serotypes, only 0.9% were described as serotypes other than G1-4, and our estimate for potential uncommon serotypes were at most 8.1%. Thus, both this long-term study focusing on the rotaviruses only from severe cases in a single hospital in Japan and the global review of G serotypes published to date indicate that the primary target of any rotavirus vaccines should be rotaviruses possessing serotypes G1-4.

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Otitis media with effusion (OME) is one of the most common diseases in children. Alloiococcus otitidis, a new Gram-positive bacterial species, was isolated from the middle ear fluid of children with OME; however, the pathogenic role of this bacteria is yet unknown. In this study, the ability of cultured epithelial cell lines (Hep-2 and Hela) and monocytic cell lines (THP-1 and U 937) to secrete chemokine interleukin-8 (IL-8) in response to the A. otitidis organism and three bacterial organisms mainly detected from middle ear fluid in OME, and bacterial cell components was investigated. When stimulated with four viable bacterial cells, epithelial cells and monocytes secreted IL-8 in a time-dependent manner. The monocytes produced significantly higher levels of IL-8 than the epithelial cells. Compared with that by viable bacterial cells, IL-8 secretion by stimulated epithelial cells and monocytes was reduced when the bacteria were heated and treated with glutaraldehyde. With bacterial stimulations, cell treatment of interferon-gamma caused monocytes to increase the induction of IL-8 production, however, the induction of monocyte differentiation caused monocytes to reduce the induction of IL-8 production. Furthermore, epithelial cells and monocytes stimulated by four viable bacterial organisms physically separated from cultured cells reduced the induction of IL-8 compared with directly stimulated cells, and monocytes stimulated with soluble extracts prepared from A. otitidis organisms produced IL-8 in a dose-dependent manner. These results suggest that part of the IL-8 stimulation of the A. otitidis organism may exist in a diffusable factor released by the bacteria or soluble components of the bacteria itself.

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Enzyme-linked immunosorbent assays for the quantitation of bacterial superantigens, staphylococcal enterotoxins A, B and C, toxic-shock syndrome toxin-1 and streptococcal pyrogenic exotoxin A, were developed. The assays had sensitivity to quantitate these toxins to 1.4, 5.9, 16.3, 2.5 and 4.3pg/ml, respectively, in a buffer including 50% human plasma. It takes only 150min to complete the assays after plate preparation. Specificity of the assays agreed with those of reverse latex agglutination assay. We also developed enzyme-linked immunosorbent assays to detect antibodies against these five superantigens. The assays are expected to be significant tools for the study of superantigens in several diseases.

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The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.

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This report describes the presence of type 1 fimbriae on Shigella boydii 5 which agglutinate guinea pig erythrocytes and feature mannose-sensitive adherence. Morphologically, the fimbriae were thin, rigid cylinders 2-5μm in length and 3-5nm in diameter, and the organella retained axial holes. This is the first study to have revealed the existence of type 1 fimbriae on S. boydii.

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A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.

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Cells of the human promonocytic cell line U937 were found to be sensitive to hexadecylphosphocholine (HPC), which is a potential anticancer drug. Induction of apoptosis was found in U937 cells after treatment with HPC for 24 to 48hr. The apoptosis in U937 cells exposed to HPC was increased significantly in the presence of interferon-gamma (IFN-γ). The augmentation of HPC-induced apoptosis by IFN-γ is repressed in cells (U937-MP) persistently infected with mumps virus. A persistently infected cell line, U937-MP, showed poor induction of signal transducers and activators of transcription-1α (STAT-1α), STAT-2, p48 and IFN-regulatory factor-1 (IRF-1), which are closely correlated with interferon-α (IFN-α) and IFN-γ signaling pathways. Expression of MHC class-I or class-II was augmented by IFN-α or IFN-γ in U937 cells, but not in persistently infected cells. Therefore, it is suggested that the IFN-γ signaling pathway plays an important role in the augmentation of HPC-induced apoptosis. Mumps virus can suppress the IFN-γ signaling pathway and subsequent development of apoptosis.

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Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40, 000×g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2μM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160, 000×g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.

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