Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The β-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing β-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing β-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca chelation, except for jun. These results suggest that SLS caused Ca influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.


A simple method to differentiate three classes of cholesterol-dependent cytolysins.

Cholesterol-dependent cytolysins (CDCs) are proteinaceous toxins widely distributed in gram-positive pathogenic bacteria. CDCs can be classified into three groups (I-III) based on the mode of receptor recognition. Group I CDCs recognize cholesterol as their receptor. Group II CDC specifically recognizes human CD59 as the primary receptor on the cell membrane. Only intermedilysin from Streptococcus intermedius has been reported as a group II CDC. Group III CDCs recognize both human CD59 and cholesterol as receptors. CD59 contains five disulfide bridges in its tertiary structure. Therefore, we treated human erythrocytes with dithiothreitol (DTT) to inactivate CD59 on membranes. Our data showed that DTT treatment caused a complete loss of recognition of intermedilysin and an anti-human CD59 monoclonal antibody. In contrast, this treatment did not affect the recognition of group I CDCs, judging from the fact that DTT-treated erythrocytes were lysed with the same efficiency as mock-treated human erythrocytes. The recognition of group III CDCs toward DTT-treated erythrocytes was partially reduced, and these results are likely due to the loss of human CD59 recognition. Therefore, the degree of human CD59 and cholesterol requirements of uncharacterized group III CDCs frequently found in Mitis group streptococci can be easily estimated by comparing the amounts of hemolysis between DTT-treated and mock-treated erythrocytes.


Human serum albumin stabilizes streptolysin S activity secreted in the extracellular milieu by streptolysin S-producing streptococci.

Anginosus group streptococci (AGS) are opportunistic pathogens of the human oral cavity; however, their pathogenicity has not been discussed in detail. Oral streptococci live in the gingival sulcus, from where they can easily translocate into the bloodstream due to periodontal diseases and dental treatment and cause hazardous effects on the host through their virulence factors. Streptolysin S (SLS), a pathogenic factor produced by β-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of β-hemolytic Streptococcus anginosus subsp. anginosus (SAA), focusing on different growth conditions such as in the bloodstream. Consequently, SLS-dependent hemolytic activity/cytotoxicity in the culture supernatant of β-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from β-hemolytic SAA, but also from other SLS-producing streptococci, is stabilized by HSA. As HSA is the most abundant protein in human plasma, the results of this study provide new insights into the risk of SLS-producing streptococci which can translocate into the bloodstream.


Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from strain Nm-76.

Some strains of exhibit β-hemolysis due to the β-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of strain Nm-76. Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in . The gene encoding CDC found in Nm-76 and its homolog are distributed among many strains. This CDC is phylogenetically different from other previously characterized CDCs, such as -derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in .


Hapten-labeled fusion-polymerase chain reaction of multiple marker genes for the application of immunochromatographic test.

A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.

Journal of Bioscience and Bioengineering


Charge-independent protein adsorption characteristics of epitaxial graphene field-effect transistor on SiC substrate


Synthesis of D-π-A type benzothiazolepyridinium salt composite and its application as photo-degradation agent for amyloid fibrils

We have synthesized a cyan fluorescent benzothiazole-pyridinium salt composite based on D-p-A architecture. This salt was found to work as not only a two- and three-photon excitable fluorophore but also a degradation agent against amyloid fibrils under LED irradiation conditions.

Bioorganic & Medicinal Chemistry Letters


Two-photon excitable boron complex based on tridentate imidazo[1,5-a]pyridine ligand for heavy- atom-free mitochondria-targeted photodynamic therapy


Molecular characteristics of an adhesion molecule containing cholesterol-dependent cytolysin-motif produced by mitis group streptococci

Streptococcus pseudopneumoniae (SPpn) is a relatively new species closely related to S. pneumoniae (SPn) and S. mitis (SM) belonging to the Mitis group of the genus Streptococcus (MGS). Although genes encoding various pneumococcal virulence factors have been observed in the SPpn genome, the pathogenicity of SPpn against human, including the roles of virulence factor candidates, is still unclear. The present study focused on and characterized a candidate virulence factor previously reported in SPpn with deduced multiple functional domains, such as lipase domain, two lectin domains, and cholesterol-dependent cytolysin-related domain using various recombinant proteins. The gene was found not only in SPpn but also in the strains of SM and SPn. Moreover, the gene product was expressed in the gene-positive strains as secreted and cell-bound forms. The recombinant of gene product showed lipase activity and human cell-binding activity depending on the function of lectin domain(s), but no hemolytic activity. Thus, based on the distribution of the gene within the MGS and its molecular function, the gene product was named mitilectin (MLC) and its contribution to the potential pathogenicity of the MLC-producing strains was investigated. Consequently, the treatment with anti-MLC antibody and the mlc gene-knockout significantly reduced the human cell-binding activity of MLC-producing strains. Therefore, the multifunctional MLC was suggested to be important as an adhesion molecule in considering the potential pathogenicity of the MLC-producing strains belonging to MGS, such as SPpn and SM.


Complete Genome Sequence of Streptococcus mitis Strain Nm-65, Isolated from a Patient with Kawasaki Disease

Nm-65 is a human commensal streptococcal strain of the mitis group that was isolated from the tooth surface of a patient with Kawasaki disease. The complete genome sequence of Nm-65 was obtained by means of hybrid assembly, using two next-generation sequencing data sets. The final assembly size was 2,085,837 bp, with 2,039 coding sequences.


Synthesis and Optical Property of Quadrupolar Pyridinium Salt and Its Application as Bioimaging Agent


Two- and three-photon excitable quaternized imidazo[1,2-a]pyridines for mitochondrial imaging and potent cancer therapy agent


Cytotoxic property of Streptococcus mitis strain producing two different types of cholesterol-dependent cytolysins


A photometric pH assay for microplate bacterial cultures.

A photometric pH assay for sugar-fermenting bacterial culture on a 96-well plate was developed. This assay can save time and effort in repeat handlings. Its use could decrease the risk of bacterial contamination in measurement devices and leakage into the environment. The assay's pH estimation range was pH 4.2-7.6.


Change in membrane potential induced by streptolysin O, a pore-forming toxin: flow cytometric analysis using a voltage-sensitive fluorescent probe and rat thymic lymphocytes

Streptolysin O (SLO) is a bacterial pore-forming toxin that is employed to permeabilize cell membranes in some biological experiments. SLO forms various types of pores with different shapes, increasing membrane ion permeability and subsequently inducing changes in membrane potential. To characterize the pores formed by SLO, the changes in membrane potential induced by SLO in rat lymphocytes were considered using flow cytometry with a voltage-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol). SLO caused three types of membrane potential responses accessed with Oxonol. One type induces a great decrease in Oxonol fluorescence (large hyperpolarization) that may be elicited via the increase of Ca -dependent K permeability by SLO-induced influx of external Ca . A second type is an increase in Oxonol fluorescence (depolarization) that may be caused by a nonspecific increase in membrane cation permeability. The third type is a small decrease in Oxonol fluorescence (small hyperpolarization), probably via an increase in Cl permeability. That SLO transitionally changes membrane ion permeability may have implications in the pathology of pyogenic group streptococci infections in which SLO is thought to be one of the key virulence factors.


Fluorescent Imidazo[1,5-a]pyridinium Salt for a Potential Cancer Therapy Agent


β-Hemolytic Streptococcus anginosus subsp. anginosus causes streptolysin S-dependent cytotoxicity to human cell culture lines in vitro

Streptococcus anginosus subsp. (SAA) is one of the opportunistic pathogens in humans that inhabits the oral cavity. The type strain of SAA, NCTC10713, showed clear β-hemolysis on blood agar plates, and the sole β-hemolytic factor revealed two streptolysin S (SLS) molecules. SLS is well known as the peptide hemolysin produced from the human pathogen and shows not only hemolytic activity on erythrocytes but also cytotoxic activity in cell culture lines and , such as in a mouse infection model. However, no cytotoxic activity of SLS produced from β-hemolytic SAA (β-SAA) has been reported so far. In this study, the SLS-dependent cytotoxicity of the β-SAA strains including the genetically modified strains was investigated . The SLS-producing β-SAA showed cytotoxicity in human cell culture lines under the co-cultivation condition and it was found that this cytotoxicity was caused by the SLS secreted into the extracellular milieu. The results from this study suggest that the SLS produced from β-SAA might indicate the cytotoxic potential similar to that of the SLS from and the SLS-producing β-SAA would be recognized as "a wolf in sheep's clothing" More attention will be paid to the pathogenicity of β-hemolytic Anginosus group streptococci.


Recognizability of heterologous co-chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK

Streptococcus intermedius DnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.


Fluorescent N-Heteroarenes Having Large Stokes Shift and Water Solubility Suitable for Bioimaging


Construction of Anti-HER2 Recombinants as Targeting Modules for a Drug-delivery System Against HER2-positive Cells

Recombinant antibodies have been investigated and used in applications such as targeting modules of drug-delivery systems (DDS) against cancers. This study aimed to prepare recombinant antibodies against HER2, containing sortase A (SrtA) recognition sequence, that are applicable as targeting modules in DDS after linkage with the drug-carrier containing oligoglycine-acceptor peptide by SrtA transpeptidation. The recombinant trastuzumab fragment antibodies (scFvs and Fab) with the SrtA-recognition motif (LPXTG) at their C-terminal were constructed and expressed in Escherichia coli and Chinese hamster ovary (CHO) cells, respectively. The reactivity of the purified recombinant antibodies towards HER2-expressing cells was also evaluated via immunofluorescent staining. Fab demonstrated higher yield and purity and better reactivity towards HER2-expressing cells (HCT-15 and HeLa) when compared to scFvs. The CHO expression system possesses superior yield and purity when compared to the E. coli expression system with respect to the preparation of recombinant antibodies applicable in targeting modules for DDS (DDS-TM). Moreover, a Fab variant prepared in this study demonstrated the potential to be a DDS-TM against HER2-expressing cancer cells.


Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene.

Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY-high producers with loss-of-function mutations in LacR.


A novel plasmid, pSAA0430-08, from Streptococcus anginosus subsp. anginosus strain 0430-08

Mobile genetic elements (MGEs) are the genetic material often involved in the interspecies and intraspecies genetic transduction in bacteria. However, little is known about MGEs in the Anginosus group of streptococci (AGS), one of the streptococcal groups found in the oral cavity of humans. We looked for the presence of MGEs in Streptococcus anginosus subsp. anginosus (SAA), a representative species belonging to AGS, and found a novel plasmid from SAA strain 0430-08. This plasmid was 7038bp and ~31% G/C content which we named pSAA0430-08, and examined its genetic structure and characteristics. Open reading frame (ORF) prediction revealed that pSAA0430-08 was composed of 10 ORFs including a putative plasmid replication protein (ORF1) and a putative toxin-antitoxin system (ORF9 and ORF10). Between ORF10 and ORF 1, four tandem repeats of 22bp each, generally termed as iteron, were also observed. Using variant plasmids of pSAA0430-08, we confirmed that both ORF1 and iteron were necessary for replication in host cells. Interestingly, the region from ORF4 to ORF7 showed homology with a genomic DNA segment of S. gordonii strains. Thus, this plasmid may travel between the different species in Streptococci, i.e., S. gordonii and S. anginosus.


Effects of the Nonsteroidal Anti-inflammatory Drug Celecoxib on Mitochondrial Function

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and pain. In the present study, we examined the effects of celecoxib, a cyclooxygenase-2 (COX-2)-selective NSAID, on rat liver mitochondrial function. Celecoxib dose-dependently induced mitochondria swelling, which was not suppressed by cyclosporine A (CsA). The oxygen consumption rate in mitochondria-suspended solution was facilitated by the addition of celecoxib, and its uncoupling activity was observed. Celecoxib also suppressed SF6847-induced uncoupling, and appeared to exert inhibitory effects on the electron transport chain. Celecoxib suppressed the state 3 oxygen consumption rate in the presence of ADP. Protein release from the mitochondrial matrix was detected following the addition of celecoxib, and aldehyde dehydrogenase 2 (ALDH2) and hydroxymethylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2) bands were confirmed in a Western blot analysis. On the other hand, protein release of cytochrome C (CytC), which is an inducer of apoptosis, from the intermembrane space was not observed. Celecoxib enhanced the membrane permeability of human erythrocytes and synthesized liposomes dose-dependently. It then induced the membrane-involving mitochondrial swelling and suppressed mitochondrial function.


Positive- and Negative-Control Pathways by Blood Components for Intermedilysin Production in Streptococcus intermedius.

is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants -null and -null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM -acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of .


Evaluation of the effectiveness of the potent bis-quaternary ammonium compound, 4,4'-( , -hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) on Pseudomonas aeruginosa.

Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-( , -hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.


In Vitro Evaluation of the Biocompatibility of Newly Synthesized Bis-Quaternary Ammonium Compounds with Spacer Structures Derived from Pentaerythritol or Hydroquinone

With the object of developing new biocides milder for human use than the current antiseptics, we synthesized a series of bis-quaternary ammonium compounds (bis-QACs). The antimicrobial activity of the newly synthesized bis-QACs and common biocides used as antiseptics was compared by examining minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). Moreover, the cytotoxicity of these compounds against human cells was determined to calculate the biocompatibility index (BI) of these compounds. BI was the ratio of the concentration of a biocide giving a 50% lethal effect on normal human epidermal keratinocytes to its MBC against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The commonly used antiseptics tested were benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB). In comparison with these antiseptics, it was shown that some of new bis-QACs exhibited a wider and more potent antimicrobial spectrum than OCT. The cytotoxicity of these bis-QACs was equal or lower compared to that of the quaternary ammonium compounds (BAC and OCT), although these bis-QACs showed higher toxicity than the biguanide-based compounds (CHG and PHMB). Finally, the comparison of BIs revealed that new bis-QACs such as N-dodecyl {4,4'-(2,4,8,10-tetraoxaspiro[5.5]undecan-3,9-diyl) }dipyridinium dibromide (4TOSU-12), 3,3'-[1,4-Phenylenebis (oxy)]bis (1-dodecylpyridinium) dibromide (3PHBO-12) and 3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(1-dodecylpyridinium-3-yl) oxy]methyl}propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) had equal or greater biocompatibility than the commonly used biocides tested. Thus, these results strongly suggested that 4TOSU-12, 3PHBO-12 and 3HHDMP-12 could be useful as antiseptics for topical application to the skin.


Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity

Cardiotoxin (CT) is a well-known cell lytic protein and has been purified from cobra venom. Cardiotoxin-like basic protein (CLBP) has two amino acid insertions and does not exhibit cell lytic activity. The molecular features of these CT family proteins were examined in the present study using molecular modeling and molecular simulation techniques. Molecular models of CT and CLBP were constructed based on the X-ray data of Naja mossambica mossambica CT VII4 (Protein Data Bank ID: 1CDT). The structural features of these models were examined using molecular orbital and electrostatic potential parameters. The stereo-hydrophobicities and molecular torsions of CT and CLBP, which are indexes of structural features, were similar. Electrostatic potential fields (ESP) differed between CT and CLBP and this was considered one of the critical factors in molecular titer. The distribution of ESP fields may affect the cytolytic activity of the CT family.


Development of a Sortase A-mediated Peptide-labeled Liposome Applicable to Drug-delivery Systems

In order to develop an efficient drug-delivery system (DDS), a lipopeptide-loaded liposome that functions as a platform for the transpeptidase reaction mediated by sortase A (SrtA) was constructed and its stability, as well as cell-specific targeting were evaluated in the present study. Several lipopeptides possessing an acceptor peptide sequence (oligoglycine ≥ three residues) or donor peptide sequence (LPETG) for the SrtA-mediated reaction were chemically synthesized and then inserted into the liposome membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (DPPC-Chol-lipo) to obtain the lipopeptide-loaded liposomes. The transpeptidase reaction mediated by recombinant SrtA (His-ΔN59SrtA) was employed to modify the peptide moiety on the liposomal surface using a fluorescently-labeled substrate peptide corresponding to the species of each loaded lipopeptide. Furthermore, lung tumor-binding peptide (LTBP)-labeled liposomes, prepared by this transpeptidase reaction, were investigated for selective targeting to lung cancer cells in vitro. The His-ΔN59SrtA-mediated transpeptidation of fluorescently-labeled peptide on the lipopeptide-loaded DPPC-Chol-lipo was confirmed. The selective targeting of LTBP-labeled liposomes to the lung cancer cell line A549 was also observed in vitro. These results suggest that the labeling of acceptor or donor lipopeptide-loaded liposomes with the transpeptidase SrtA could be a useful method for developing a platform applicable to a cancer-targeting DDS.


5-O-Acyl plumbagins inhibit DNA polymerase activity and suppress the inflammatory response

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase (pol) γ. In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins displayed enhanced mammalian pol inhibition, with plumbagin conjugated to docosahexaenoic acid (C22:6-acyl plumbagin) exhibiting the strongest inhibition of pol λ among the ten 5-O-acyl plumbagins synthesized. C22:6-acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols or DNA metabolic enzymes tested. The inhibition of pol λ, a DNA repair/recombination pol, by these compounds was significantly correlated with both their suppression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) production by mouse RAW264.7 macrophages and the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. These data indicate that 5-O-acyl plumbagins act as anti-inflammatory agents by inhibiting mammalian pol λ. These results further suggest that C22:6-acyl plumbagin is a promising anti-inflammatory candidate and that acylation could be an effective chemical modification to improve the anti-inflammatory activity of vitamin K3 derivatives, such as plumbagin.


Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equal was tenfold more effective than daidzein. These differentiation promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.


Molecular Analysis of Streptococcus anginosus-derived SagA Peptides

SagA1 and SagA2 molecules produced from beta-hemolytic Streptococcus anginosus subsp. anginosus are composed of a leader peptide and a propeptide, and their mature form has hemolytic activity as a well-known Streptococcal peptide toxin, streptolysin. The function of these SagA molecules is thought to be dependent on intra-molecular heterocycle formation. In this study, we examined the heterocycle-involved molecular features of SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing on their heterocycle formation. Molecular models of SagA1, SagA2, and SPySagA were constructed using a molecular modeling technique. Molecular dynamics and molecular mechanic analyses of the modeled SagA molecules were performed to obtain their energy profiles. Total energy of the modeled SagA1, SagA2, and SPySagA decreased with heterocycle formation, and the border between the leader peptide and propeptide was clearly observed after heterocycle formation. The flexibility of SagA molecules was changed by intramolecular heterocycle formation, and their function (e.g. hemolytic activity) seems to be regulated by structural transition with heterocycle formation.


Investigation on the Reaction Conditions of Staphylococcus aureus Sortase A for Creating Surface-modified Liposomes as a Drug-delivery System Tool

This study aimed to determine the preferred conditions for the transpeptidase reaction of sortase A from Staphylococcus aureus, for the purpose of creating functional liposomes useful for a drug-delivery system (DDS). His-tagged recombinant sortase A with 59 amino acids deleted from the N-terminus (His-ΔN59SrtA) was prepared using an Escherichia coli expression system. The pH dependency and sorting signal sequence dependency of the transpeptidase reaction of His-ΔN59SrtA were analyzed by monitoring the transfer of model donor-substrates (i.e. His-tagged mutant green fluorescent proteins with a C-terminal LPxTG sorting signal) to model acceptor-beads with a GGGGGC peptide. In addition, using preferred conditions, the sortase A reaction was used to modify liposome surface. The transpeptidase reaction of His-ΔN59SrtA was enhanced under weakly acidic conditions. Transfer efficiency, based on sorting signal recognition by His-ΔN59SrtA, was similar to or higher than that obtained using several substrates with amino acids other than Glu in the sorting signal position "x". Furthermore, liposomes containing GGGGGC peptide-linked dipalmitoylphosphatidylethanolamine were successfully modified using the preferred conditions for His-ΔN59SrtA determined in this study. Preferred conditions for the transpeptidase reaction of His-ΔN59SrtA, especially in a weakly acidic environment to enhance reaction, was established and successfully used to create functional liposomes applicable to DDS.


Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.


A streptolysin S homologue is essential for beta-haemolytic Streptococcus constellatus subsp. constellatus cytotoxicit

Streptococcus constellatus is a member of the Anginosus group streptococci (AGS) and primarily inhabits the human oral cavity. S. constellatus is composed of three subspecies: S. constellatus subsp. constellatus (SCC), S. constellatus subsp. pharyngis and the newly described subspecies S. constellatus subsp. viborgensis. Although previous studies have established that SCC contains β-haemolytic strains, the factor(s) responsible for β-haemolysis in β-haemolytic SCC (β-SCC) has yet to be clarified. Recently, we discovered that a streptolysin S (SLS) homologue is the β-haemolytic factor of β-haemolytic Streptococcus anginosus subsp. anginosus (β-SAA), another member of the AGS. Furthermore, because previous studies have suggested that other AGS species, except for Streptococcus intermedius, do not possess a haemolysin(s) belonging to the family of cholesterol-dependent cytolysins, we hypothesized that, as with β-SAA, the SLS homologue is the β-haemolytic factor of β-SCC, and therefore aimed to investigate and characterize the haemolytic factor of β-SCC in the present study. PCR amplification revealed that all of the tested β-SCC strains were positive for the sagA homologue of SCC (sagA(SCC)). Further investigations using β-SCC strain W277 were conducted to elucidate the relationship between sagA(SCC) and β-haemolysis by constructing sagA(SCC) deletion mutants, which completely lost β-haemolytic activity. This loss of β-haemolytic activity was restored by trans-complementation of sagA(SCC). Furthermore, a co-cultivation assay established that the cytotoxicity of β-SCC was clearly dependent on the presence of sagA(SCC). These results demonstrate that sagA(SCC) is the factor responsible for β-SCC β-haemolysis and cytotoxicity.


The diversity of receptor recognition in cholesterol-dependent cytolysins

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59.


LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.


Profiles of ILY, VLY and Sm-hPAF interaction with human CD59

The molecular features of a new member of the bacterially -derived cytolysin family were examined. In particular, the interactive mechanisms of intermedilysin (ILY), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) with human CD59 (hCD59) were analyzed. Molecular models of VLY and Sm-hPAF were constructed based on X-ray data of ILY (protein data bank ID=1S3R), and their interactive profiles with hCD59 were examined using molecular simulation. Non-binding (NB) energy between ILY and hCD59 was three orders of magnitude higher than the energy between VLY and hCD59. NB energy between Sm-hPAF and hCD59 was similar to that between VLY and hCD59. A hydrogen bond (ILY Arg432-hCD59 Glu76) was observed between ILY and hCD59, and a stronger interaction was formed by flexible adjustment between them.


Construction of an improved drug delivery system tool with enhanced versatility in cell-targeting.

His-Z-CDC(ss)(IS) was revealed to be an improved DDS tool with enhanced versatility in cell targeting.


Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus.

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.


Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and β-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and β-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of β-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities.

Journal of Bioscience and Bioengineering


Molecular Profiles of Cholesterol-dependent Cytolysin Family-derived 11mer Regions

Cholesterol-dependent cytolysins (CDCs) are secreted from various types of bacteria and are involved in various diseases (e.g. abscess formation). Traditional CDCs has a conserved 11mer region, which is a key structure in membrane recognition. Based on the X-ray data of traditional CDC perfringolysin O (PFO), molecular models of intermedilysin (ILY), pyolysin (PLO), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) were constructed. The 11mer regions of these models were extracted, and their molecular features were analyzed. The dipole moments of these 11mer regions were classified into four types, and their stereo-hydrophobicity (dGW) was different. It was thought that these results influenced the species specificity and membrane recognition of each cytolysin. Traditional CDCs, ILY, PLO, and VLY consisted of four domains (domains 1 to 4). Domain 0 existed on the N-terminal side in Sm-hPAF in addition to these four domains. The 11mer sequence of Sm-hPAF is the same as that of VLY, but Sm-hPAF has slightly different characteristics (e.g. species specificity, membrane recognition, cholesterol dependency) compared to VLY. Dynamic structure analysis of domain 0 might clarify these differences.


Investigation of a Bacterial Pore-forming Chimera Toxin for Application as a Novel Drug-delivery System Tool

Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins from Gram-positive bacteria. The aim of this study was to investigate the potential of a CDC, intermedilysin, as a drug-delivery system (DDS) for clinical application. Intermedilysin was modified by the addition of a disulfide bridge to regulate pore formation, by swapping domain 4 to provide cholesterol-binding capacity, and by the introduction of a targeting domain. The resultant chimera protein, His-LTBP-CDC(ss)(IP), was investigated for its use as a DDS tool in vitro. His-LTBP-CDC(ss)(IP) exhibited a regulated pore-forming capacity under reducing conditions. This chimera protein was able to deliver a drug-carrier liposome specifically to the target cell, to be endocytosed into the cell with subsequent release of the components into the cytoplasm. A chimera protein derived from the bacterial pore-forming toxin intermedilysin (His-LTBP-CDC(ss)(IP)) forms the basis for a novel DDS tool.


Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor ς(32) in E. coli, although S. intermedius does not utilize ς(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.


Characterization of recombinant Streptococcus mitis-derived human platelet aggregation factor

We previously purified Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) from the culture supernatant of S. mitis strain Nm-65, isolated from the tooth surface of a patient with Kawasaki disease. Here we produced recombinant Sm-hPAF protein (rSm-hPAF) in Escherichia coli, to determine whether rSm-hPAF conserves its platelet aggregation activity. rSm-hPAF precursor (665 amino acids) shows up to 36-56% identity with the family of cholesterol-dependent cytolysins (CDCs), and rSm-hPAF displayed potent hemolytic activity toward mammalian erythrocytes, including human erythrocytes with platelet aggregation activity. The 162-amino acid amino-terminal domain of rSm-hPAF was found in no other CDCs except lectinolysin; this domain is homologous to a portion of pneumococcal fucolectin-related protein. Interestingly, suilysin (SLY) and pneumolysin (PLY) of CDCs also exhibit substantial human platelet aggregation activity, similar to rSm-hPAF, and the platelet aggregation by rSm-hPAF, SLY, and PLY was morphologically confirmed using light and electron microscopy.


Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

Biochemical and Biophysical Research Communications


Sialidase of Streptococcus intermedius: a putative virulence factor modifying sugar chains

A sialidase gene of Streptococcus intermedius was cloned. It was most similar to nanA, a major sialidase gene in Streptococcus pneumoniae, and was expressed in Escherichia coli. Since the gene-knockout S. intermedius strain lost detectable sialidase activity, the gene might code, either solely or mainly, the glycosidase in the bacterial genome. Polymerase chain reaction using the primers for the nanA homologue in S. intermedius (described as nanA below) showed that this sialidase gene was commonly distributed within the isolates of S. intermedius, but not found in the strains of other species among the anginosus group. In biofilm formation assay under cultivation with mucin, the nanA-deleted S. intermedius maintained the amount of biofilm for 72 hr, while that of the parent strain decreased during incubation from 24 to 72 hr. Since sialidase activity in the parent strain increased during that time period, sialidase might contribute to the degradation of biofilm under sialic acid-rich conditions. When S. intermedius was added into the HepG2 hepatoma culture, the calculated disassociation constant (K(d)) of EDTA-releasable bacterial adhesion to the cells was higher in the nanA-deleted strain than in the parent. Furthermore, the rate constant, assuming endocytosis of the bacterium mediated by ASGP-R in HepG2 cells, seemed to be increased by sialidase pretreatment of the bacterial cells before addition to the cell culture. According to the results, modification of sugar chains by sialidase on the bacterial surface and in the surrounding environment might influence both bacterial interaction and host-bacterial interaction in S. intermedius.


Role of catabolite control protein A in the regulation of intermedilysin production by Streptococcus intermedius.

Streptococcus intermedius is an opportunistic pathogen of humans that causes purulent infections, including brain and liver abscesses. This pathogen secretes a human-specific cytolysin, intermedilysin, which has been recognized as a major virulence factor. However, most of the expressional control mechanisms of ily are still unknown. To determine these mechanisms, we analyzed the nucleotide sequence of the ily promoter region. We found a highly homologous region to the catabolite-repressible element (cre) in the ily promoter region and observed a considerable decrease in the amount of secreted intermedilysin when cells were grown in a culture medium containing high concentrations of glucose/utilizable carbohydrates. Disruption of the ccpA gene, which encodes catabolite control protein A, did not induce catabolite repression of ily by glucose/utilizable carbohydrates. In cre mutants, catabolite repression of ily was partially restored, and purified catabolite control protein A bound to an oligonucleotide containing the cre consensus sequence in the ily promoter region. In addition, a prolonged lag phase and slower doubling time of the ccpA mutant cells were observed. Our data show that S. intermedius can modulate ily expression and growth rate through catabolite control protein A-mediated monitoring of the extracellular glucose/utilizable carbohydrate concentration.


Investigation of the chaperone function of the small heat shock protein AgsA.

A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heat-aggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N- or C-terminal truncated mutants and compared their properties with wild type AgsA. AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23rd and C-terminal 11th amino acid (AA) from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50 degrees C. Destabilization of oligomeric complexes was observed for N-terminal 11- and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH) and citrate synthase (CS) but did not prevent the aggregation of insulin at 25 degrees C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25 degrees C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities), good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25 degrees C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25 degrees C. Our data demonstrate that AgsA has several regions necessary for efficient chaperone activity: region(s) important for lysozyme chaperone activity are located outer surface of the oligomeric complex while those region(s) important for insulin are located inside the oligomeric complex and those for MDH are located within the N-terminal arm. In addition, the equilibrium between the oligomer and the dimer structures appears to be important for its efficient chaperone activity.


Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). We earlier reported that the bacterial lipoteichoic acid was detected at the sites of inflammation around damaged bile ducts in the livers of PBC, and PBC patients' sera showed high titers against streptococcal histone-like protein. Here, we investigated whether chronic bacterial exposure could trigger PBC-like epithelial cell damage in normal mouse. BALB/c mice were repeatedly inoculated with various bacteria for 8 weeks. At 1 week (Group 1) and 3, 4, or 20 months (long term; Group 2) after the final inoculation, mice were killed to obtain samples. In the livers of the Streptococcus intermedius (S.i.)-inoculated mice in Group 1, cellular infiltration was predominantly observed around the bile ducts over the hepatic parenchyma. In the S.i.-inoculated mice in Group 2, portal but not parenchymal inflammation was observed in the livers, and periductal cellular infiltrates were detected in the salivary glands. Both S.i.-inoculated Groups 1 and 2 BALB/c mice sera had antibodies against HuCCT1 biliary epithelial cells, anti-nuclear antibodies, and anti-gp210 antibodies, but not anti-mitochondrial antibodies. Immunoreactivity to histone-like DNA-binding protein of S.i. (S.i.-HLP) was detectable around the sites of chronic nonsuppurative destructive cholangitis in the portal area in the livers of both S.i.-inoculated Groups 1 and 2 BALB/c mice. Furthermore, anti-S.i.-HLP antibody bound to synthetic gp210 peptide, as well. Bacteria triggered PBC-like cholangitis, multifocal epithelial inflammation, and autoantibody production. Bacteria are likely involved in the pathogenesis of PBC and of associated multifocal epithelial inflammation.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Inhibition of anti-fluorescent probe monoclonal antibody by long-chain amphiphiles

Inhibitions of amphiphiles with a long chain (alcohols with carbon number of 10, 12, 14, 16 and the corresponding fatty acids) on the antigen-antibody reaction between fluorescent probe (fluorescein (FL)) and anti-fluorescent probe monoclonal antibody (immunoglobulin G (IgG)-49) were investigated by fluorescence spectroscopy. The resulting inhibition modes were compared with those of specific inhibitors, 5- and 6-aminofluoresceins (5- and 6-FLNH(2)). In the presence of all inhibitors, the fluorescence quenching of FL as the result of inhibitor binding to IgG-49 was hindered in an inhibitor concentration-dependent manner. The values of IC(50) and Hill number for the inhibitors were determined from dose-response curves of the quenching inhibition. The IC(50) values of the alcohols were much more dependent on the chain length than those of the fatty acids although all the values were in orders of microM. The Hill numbers of both kinds of inhibitors became larger values than 1, but they had different values for a series of alcohols and fatty acids, respectively. By contrast, the IC(50) values of 5- and 6-FLNH(2) were in orders of nM and the Hill numbers became smaller values than 1. Since the Lineweaver-Burk plots produced an intersection point on the intercept of the X-axis for the long-chain inhibitors with carbon number of 14 while did that on the intercept of the Y-axis for 6-FLNH(2), respectively, we judged the inhibition modes of the former as non-competitive ones and those of the latter as a competitive one. Further, the effect of pressure on the inhibition by C(14)OH, C(13)COOH and 5-FLNH(2) was examined, and the standard volume changes of IgG-49 for the inhibitor binding were calculated from the Johnson-Eyring plots. The volume change for 5-FLNH(2) was smaller than those for C(14)OH and C(13)COOH although they became all negative. This suggests that the volume of IgG-49 shrinks by the addition of these inhibitors, and IgG-49 is not relevant to a model protein for the molecular mechanism of anesthesia.


Identification and molecular analysis of betaC-S lyase producing hydrogen sulfide in Streptococcus intermedius.

Hydrogen sulfide (H(2)S) is a toxic gas that induces the modification and release of haemoglobin in erythrocytes; however, it also functions in methionine biosynthesis in bacteria. betaC-S lyase, encoded by the lcd gene, is responsible for bacterial H(2)S production through the cleavage of l-cysteine. In this study, 26 of 29 crude extracts from reference and clinical strains of Streptococcus intermedius produced H(2)S from l-cysteine. The capacities in those strains were not higher than those in strains of the other anginosus group of streptococci, Streptococcus anginosus and Streptococcus constellatus, but were much greater than those in strains of Streptococcus gordonii, which is known to have an extremely low capacity for H(2)S production. Incubation of the remaining three extracts with l-cysteine did not result in H(2)S production. Sequence analysis revealed that the lcd genes from these three strains (S. intermedius strains ATCC 27335, IMU151 and IMU202) contained mutations or small deletions. H(2)S production in crude extracts prepared from S. intermedius ATCC 27335 was restored by repairing the lcd gene sequence in genomic DNA. The kinetic properties of the purified recombinant protein encoded by the repaired lcd gene were comparable to those of native proteins produced by H(2)S-producing strains, whereas the truncated protein produced by S. intermedius ATCC 27335 had no enzymic activity with l-cysteine or l-cystathionine. However, real-time PCR analysis indicated that the lcd gene in strains ATCC 27335, IMU151 and IMU202 is transcribed and regulated in a manner similar to that in the H(2)S-producing strain.


The essentiality and involvement of Streptococcus intermedius histone-like DNA-binding protein in bacterial viability and normal growth

Streptococcus intermedius histone-like DNA-binding protein (Si-HLP) is a homodimeric protein and, conserved with Escherichia coli HU, a well-documented nucleoid-associated protein (NAP). In E. coli, HU plays important roles as both structural and regulatory factors, but it is not essential for E. coli viability. Streptococcal HLP has been found to bind host cells and induce cytokine production, but its physiological role remains poorly defined. In the present study, using gene insertion knockout and tetracycline-regulated antisense RNA expression techniques, we determined whether Si-HLP is essential for bacterial viability and normal growth in S. intermedius. The Si-HLP-downregulated S. intermedius strain showed alterations in its morphology and surface properties. Downregulation of Si-HLP led to an expanded nucleoid to fill the intracellular space. Transcription levels of several genes, including virulence-associated factors, were found to be activated or repressed in the antisense Si-hlp RNA-expressing strain by real-time PCR and reverse-transcription PCR. Collectively, these data suggest that Si-HLP serves as an essential NAP governing the nucleoid architecture and controlling the gene transcription profile in S. intermedius.


Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via action of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.


Antibacterial activity and characteristics of modified ferrite powder coated with a gemini pyridinium salt molecule

This report describes the synthesis of an antibacterial material consisting of a gemini quaternary ammonium salt (gemini-QUAT) immobilized on ferrite powder, and its antibacterial activity. A gemini-QUAT containing two pyridinium residues per molecule, 4,4'-[1,3-(2,2-dihydroxylmethyl-1,3-dithiapropane)]bis (1-octylpyridinium bromide), was immobilized on ferrite powder by a reaction between the hydroxyl group of the QUAT and trimethoxysilane. Immobilization of the gemini-QUAT on ferrite (F-gemini-QUAT) was confirmed when the dye, bromophenol blue, was released from F-gemini-QUAT-dye after contact between ferrite and the dye. Elemental analysis of the QUAT-ferrite determined the molar amount of QUAT on the ferrite. The antibacterial effect of the ferrite was investigated using a batch treatment system, and this effect was compared with that of another QUAT-ferrite (F-mono-QUAT) binding a mono-QUAT, which possesses one pyridinium residue, prepared by the same immobilization method as F-gemini-QUAT. Results indicated the F-gemini QUAT possessed a higher bactericidal potency and broader antibacterial spectrum compared to F-mono-QUAT. In addition, this study suggested that gemini-QUATs possessed high bactericidal potency without being influenced by immobilization to materials, and the antibacterial activity and characteristics of F-gemini-QUAT could be attributed to the unique structure of the immobilized gemini-QUAT.


Ultrastructural analysis of the membrane insertion of domain 3 of streptolysin O

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO.


Molecular dynamics of human-specific cytolysin: analysis of membrane binding motif for therapeutic application

Intermedilysin (ILY) is a human-specific cytolysin secreted from Streptococcus intermedius. In this study, the dynamic structure of ILY, StreptolysinO (SLO) and their 12mer substituted mutants for 500 ps was analyzed. Several parameters, such as dipole moment and electrostatic potential, were determined to elucidate the molecular mechanism of membrane binding. Molecular models of lLY, SLO and their mutants were constructed using Insightll-Discover with the Homology module. Their molecular dynamics were simulated with the Discover3 (Insight module), and z-matrix data of the membrane-binding 12mer region were extracted to calculate the molecular orbital (MO) parameters (i.e., dipole moment, solvation free energy (dGW)). Cytolysins vibrated like a bow, and the dipole moment direction of ILY 12mer region was different from that of SLO. Certain ILY mutants indicated the SLO-like dipole properties, which had an SLO-type limer cysteine motif amino acid sequence. The ILY 11mer region was more hydrophobic than that of SLO, and seemed to interact easily with the cell membrane without cholesterol. The electrostatic potential field distribution of ILY differed from that of SLO, especially in the 11mer region. In the 11mer region, the dipole moment directions of these cytolysins were constant during molecular movement, and ready to interact with membrane components (i.e., cholesterol, phospholipid).


Construction of a leftover bath water model for microbial testing

In this study, in order to construct a model of leftover bath water, we analyzed one hundred samples of used bath water samples which were provided by twenty-eight volunteer families. It appeared that the number of detected bacteria from such bath water was correlated closely with the number of bathers. Moreover, the pH, acidity, chemical oxygen demand (COD), ion, protein content of the leftover bath water were measured. The number of bathers had no connection with the pH, acidity, COD, and ion content of the leftover bath water. However, the protein content of the bath water correlated with the number of detected bacteria. Based on these results, the model of leftover bath water was constructed. Achromobacter xylosoxidans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa were incubated with the model bath water as indices of bath water contamination. The number of incubated viable cells in the model bath water increased with increasing concentrations of casamino acid. Consequently, it was suggested that varying the concentration of casamino acid based on family size or contamination would be necessary in the efficient use of the constructed model of leftover bath water for microbial testing.


Correlation between the Bacterioclastic Action of a Bis-quaternary Ammonium Compound and Outer Membrane Proteins

Bis-quaternary ammonium compounds (bis-QACs) have the ability to cause a rapid and abundant leakage of the turbid materials from cells, and such a bacterioclastic ability leads to a potent bactericidal activity. In order to clarify the detailed mechanism of the bactericidal action of bis-QACs, the correlation between the bacterioclastic action of 4,4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium bromide) (4DTBP-6,8) and the leakage of outer membrane pore protein E (OmpE) was investigated. Using the antiserum against a fusion protein consisting of GST and the OmpE protein of Escherichia coli encoded by the ompE gene, it was seen that the leakage of OmpE from E. coli cells was caused by treatment with low concentrations (much lower than the critical vesiculation concentration) of 4DTBP-6,8. Furthermore, it was confirmed that 4DTBP-6,8 caused an increase in the turbidity of the cell suspension of Klebsiella pneumoniae, Salmonella typhimurium and Serratia marcescences, and led to the leakage of several proteins which have a high percentage of homology with OmpE of E. coll. By immunoelectron microscopy investigation, it was revealed that the vesiculation from E. coli treated with 4DTBP-6,8 contains OmpE. In addition, the bacteriolytic action of 4DTBP-6,8 was investigated. The results suggested that the lysis of cells by bis-QACs was not an enzymatic action such as that by autolysin but a physical bacterioclastic action. Judging from these results, it is suggested that the leakage of OmpE is one of the major bacterioclastic actions of bis-QACs, and deals the bacterial cells a fatal blow.


Synthesis and biological properties of gemini quaternary ammonium compounds,5,5'-[2,2'-(α,ω-polymethylnedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium iodide) and 5,5'- [2,2'-(ρ-phenylenedicarbonyldioxy)diethyl] bis(3-alkyl-4-methylthiazolium bromide)

We synthesized gemini quaternary ammonium compounds (gemini QACs) having two thiazolium moieties in a molecule, 5,5'-[2,2'-(alpha,omega-polymethylnedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium iodide) (5DEBT-m,n), on which the carbon number of the methylene chain linking the two thiazoles (m) is 2, 6 or 8 and that of the alkyl group (n) is 8, 10, 12, 14 or 16. 5,5'-[2,2'-(p-Phenylenedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium bromide) (5DEBT-P,n) was then synthesized, which is composed of a p-phenylene as the methylene spacer. For five gemini QAC series, in addition to the previously described 5DEBT-4,n to the four new compound series, their antimicrobial activities were determined. 5DEBT-m,10 and -P,10 exhibited a wide and strong bacteriostatic activity against gram-negative and -positive bacteria, fungi and then yeast in comparison with N-tetradecyl-5-(2-hydroxyethyl)-4-methylthiazolium iodide as a mono-QAC. The bactericidal activity of the 5DEBT series against Escherichia coli IFO 12713 and Staphylococcus aureus IFO 12732 was investigated on the basis of the effects of their alkyl chain length and their molecular hydrophobicity. It was found that the effect of theses factors on their activity significantly changes by the difference between the gram-negative and -positive bacteria. Although against the gram-negative bacterium, the change in the activity due to extension of the alkyl group for each compound affected the kind of methylene spacer, against the gram-positive bacterium, it was almost equal in spite of the methylene spacer. This result could be responsible for the bactericidal mechanism of the gemini QACs being influenced by the diversity of the steric structure participating in the methylene chain length and by the bacterium cell surface hydrophobicity.


Syntheses and antimicrobial activities of series of new bis-quaternary ammonium compounds

A series of new bis-quaternary ammonium compounds (bis-QACs), 4,4'-(2,9-dioxadecane) bis(1-alkylpyridinium bromide)s, 3,3'-(2,9-dioxadecane)bis(1-alkylpyridinium bromide)s and 3,3'-(2,7-dioxaoctane)bis(1-decylpyridinium bromide) was synthesized. The compounds were evaluated by their activities against bacteria, molds and yeasts; the activities were expressed as the minimum inhibitory concentrations (MIC) and/or the minimum bactericidal concentrations (MBC). Compound 4,4'-(2,9-dioxadecane) bis(1-decylpyridinium bromide) had MIC values which exceeded those of benzalkoniumchloride and thiabendazole. It was in vitro active against a broad spectrum of Gram-negative and Gram-positive bacteria, against yeasts and some molds, it had high solubility and thermal decomposition temperature. The relationships between structure and biological activity of the tested bis-QACs are discussed.


Antifungal characteristics of N,N'-hexamethylenebis (4-carbamoyl-1-decylpyridinium bromide)

A bis-quaternary ammonium compound (bis-QAC), N,N'-hexamethylenebis(4-carbamoyl-1decylpyridinium bromide) (D-38), exhibited a wide antifungal spectrum and a strong activity against sixteen strains of fungi including nine strains isolated from various kinds of food. The antifungal activity was higher than that of N-dodecylpyridinium Iodide, a typical mono-QAC, and that of the commonly used fungicide, 2-(4-thiazolyl) benzimidazole. The activity of D-38, however, was comparatively low when it was measured by the agar dilution method. It is considered that the hydrophobic D-38 molecules, having two long alkyl chains, interacted with the agar medium, and therefore showed lower activity results. On the other hand, the activity of D-38 against Aspergillus niger IFO 6342 was not significantly influenced by temperature, pH and the initial spore concentration. These results indicate that bis-QACs can exhibit a high antifungal activity whether or not the environmental conditions change.


Characterization and Protein Analysis of Escherichia coli Resistant to a Quaternary Ammonium Compound


Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.


Inhibition modes of amphiphilic ligands with a long chain on the anti-fluorescent probe monoclonal antibody

蛍光プローブ特異的モノクローナル抗体への長鎖両親媒性リガンドの阻害様式

蛍光プローブフルオレセインとその特異的モノクローナル抗体との抗原抗体反応におよぼす長鎖両親媒性リガンド(炭素数10，12，14，16のアルコールおよび対応する脂肪酸)の作用様式を蛍光分光法で調べた．消光阻害の用量応答曲線はアルコール系列および脂肪酸系列でそれぞれ異なり，両リガンドの阻害様式の違いが示唆された．また，上記リガンドの阻害反応に対する圧力効果を調べ，両リガンド存在下ではフルオレセイン特異的モノクローナル抗体の体積は収縮することがわかった．


Bridge-linked bis-quaternary ammonium anti-microbial agents: relationship between cytotoxicity and anti-bacterial activity of 5,5'-[2,2'-(tetramethylenedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazonium iodide)s

論文題目:架橋構造を有するビス型第四運アンモニウム塩抗菌剤，5,5'-[2,2'-(tetramethylenedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazonium iodide)sの抗細菌活性と細胞毒性の相関，5種のビス型化合物について，構造と抗菌，毒性の相関を検討．anti-ester型5DEBT-4,8のコンフォメーション解析から1144個のコンフォーマを得，9サンプルの立体疎水性度を決定したところ他の化合物では極小点が1つであるのに対して，2つの極小点が存在した．これから5DEBT-4,8には疎水性度の異なる2群のコンフォーマが存在し高効率で低毒性なバランスとれた性質の要因と考えられた．


Biological and physicochemical properties of gemini quaternary ammonium compounds in which the positions of a cross-linking sulfur in the spacer differ

Gemini型第四アンモニウム化合物(gemini QAC)の架橋構造のスルフィド結合の位置に着目した．メチレンスルフィド，エチレンジスルフィド，スルフィドがピリジン環に連結したgemini QACの3種のを合成し，抗菌活性，ヒト赤血球溶血毒性，界面活性能に関するスルフィド結合位置の影響について検討した．ピリジン環に連結したスルフィド結合を有する化合物は，抗菌力，溶血毒性，界面活性能いずれにおいても，他の2種薬剤よりも高いことが分かった．その強い抗菌力と溶血毒性は，スルフィド結合の共鳴効果によるアンモニウムカチオン電子密度の上昇，スルフィドがテトラメチレンスペーサ鎖両末端に存在することによるスペーサ鎖の高い伸縮性に起因した小さい分子占有面積，そして高い薬剤分子疎水性に寄与すると考え，スペーサ鎖の立体構造に影響し，スルフィドの位置がアンモニウムカチオン電子密度に影響を及ぼすと結論した．


Synthesis and Antimicrobial Characteristics of a Novel Biocide,4,4'-(1,6-Dioxyhexamethylene)bis-(1-alkylpyridinium halide)

We synthesized a new bis-quaternary ammonium compound (bis-QAC), 4, 4'-(1, 6-dioxyhexamethylene) bis (1-alkylpyridinium halide) s (4DOBP-6-n-X) (alkyl chain length, n=6, 8, 10, 12, 14, 16 and 18, X=Br or I) that has a symmetrical dimeric structure, and is composed of two alkylpyridinium halides connected with a dioxyhexamethylene. We examined the relationship of the hydrophobicity and antimicrobial activity using 4DOBP-6-n-X. The influences of pH, temperature, and hemolytic activity were then measured using 4, 4'-(1, 6-dioxyhexamethylene) bis (octylpyridinium bromide) (4DOBP-6-8-Br) which showed a superior antimicrobial activity among the 4DOBP-6-n-X series. The following three chemical compounds were used as a comparison: 4, 4'-(1, 6-dithiohexamethylene) bis (octylpyridinium bromide) (4DTBP-6-8-Br) which is considered as an excellent antimicrobial agent based on a previous report, alkylbenzyldimethyl ammonium chloride (BAC) and 2-(4'-thiazolyl) benzimida-zole (TBZ). In the results of the experiment, 4DOBP-6-8-Br exhibited a wide antimicrobial spectrum, and had a strong activity against bacteria and fungi comparable to 4DTBP-6-8-Br. The hemolytic activities of 4DOBP-6-8-Br and 4DTBP-6-8-Br were almost same, and lower than that of BAC. 4DTBP-6-8-Br had some defects in that it colored metals or generated a bad smell and a hazardous gas; however, in 4DOBP-6-n-X, these disadvantages were not present. 4DOBP-6-n-X seems to be an environmentally friendly antibacterial agent.


Synthesis and antimicrobial characteristics of novel biocides, 1,1'-(decanedioyl)bis(4-methyl-4-alkylpiperadinium iodide)s with a gemini structure

論文題目:ジェミニ構造を有する新規殺菌剤，1,1-(デカンジオイル)ビス(4-メチル-4-アルキルピペラジニウムアイオダイド)の合成と抗菌特性．ポリメチレンの両端にカルバモイル結合により2分子のピペラジニウムを導入した特徴的構造を有する薬剤の合成方法を確立し，薬剤の殺菌特性について検討した．さらに，pH，温度およびヒト赤血球に対する溶血特性を明らかにした．一連の合成薬剤の大腸菌に対する殺菌活性について薬剤分子疎水性を用いて構造活性相関の手法により解析したところ，ジェミニ型ピリジニウムとは異なる特徴を持っていることを明確にした．


A cell membrane modification technique using domain 4 of intermedilysin for immunotherapy against cancer

ヒト特異的細胞溶解毒素インターメディリシン(ILY)は，そのドメイン4(4D)だけでは細胞膜に強く結合するものの細胞毒性を示さない．この性質を応用し，4DのN末端に導入したCys残基のチオール基や4D分子表面のアミノ基を足場にして胎児性癌抗原(CEA)特異的な抗体の(Fab')2断片を連結した架橋体を作成し，CEA陽性癌細胞に対して活性化リンパ球等の細胞をターゲティングできるモジュールを開発した．この細胞膜機能の改変技術は細胞ターゲティング以外にも様々な利用法が期待できる．


Structural analysis of human specific cytolysin intermedilysin aiming application to cancer immunotherapy

ヒト特異的細胞溶解毒素インターメディリシン(ILY)を癌免疫療法に応用するため，ILYが属する毒素群(CBC)の一種，パーフリンゴリシンOの結晶構造を基にILYの立体構造を分子モデリングした．その結果，ILYは他のCBCと同様のドメイン構成を持ち，ドメイン4はドメイン1-3から立体的に独立していることや，ILYの11アミン酸領域は物理化学的な性質が他のCBCの場合と異なり，コレステロールを受け入れにくい構造であることが示された．


The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

ヒト特異的細胞溶解毒素インターメディリシン(ILY)はストレプトリシンO(SLO)と同様にコレステロール(CHL)結合性細胞溶解毒素遺伝子ファミリーに属するが，CHLとの直接結合能を持たず，そのドメイン4でヒト細胞と結合することを明らかとした．またILYとSLO等の同属毒素間で多数のキメラ毒素を作成してその活性を調べた結果，SLO等ではCHLを結合する11アミノ酸領域は直接ヒト細胞の認識には関わらず，ここを除くドメイン4のC末端側56アミノ酸残基中にヒト細胞の受容体を認識する部位があることを示した．


Vectorial capacity of larvae pupae and adult of housefly (Musca domestica) for Escherichia coli 0157:H7 and the possibility of transmission from source to human

イエバエが腸管出血性大腸菌O157:H7(O157)のベクターとなる可能性を検討するため，O157をイエバエの幼虫に食餌とともに与え，その幼虫及び蛹中での検出を行った．しかし腸内細菌は変態の間に減少する傾向を示し，羽化した成虫の32.6%は腸内細菌を保有していたがO157は検出されず，幼虫や蛹はO157伝搬にあまり寄与しないと考えられた．一方，24時間の伝搬実験の結果から，成虫は食品由来のO157を伝搬して食品を汚染することが示された．また分子疫学的な解析からも，イエバエ由来の株と食中毒患者からの分離株は非常に近縁の株であることが判明した．このように，ヒトでのO157感染にはイエバエが重要なベクターとなることを実証した．


Bacterioclastic Action of a Bis-Quaternary Ammonium Compound against Escherichia coli

ビス型第四アンモニウム塩系化合物(bis-QAC)の大腸菌に対する細胞破壊作用について検討した．bis-QAC処理菌体からの濁り物質の増加とともにbis-QACの殺菌活性も上昇したことから，この濁り物質の生成はbis-QACの殺菌機構を解明する上で重要である．bis-QAC処理菌体から漏洩した物質の解析及び処理菌体の表面と内部の形態を電子顕微鏡で観察した結果，薬剤と細菌表層に存在するマグネシウムイオンとの置換反応により細胞膜内に侵入したbis-QACは，細胞表面にブレッブ形成を引き起こす．電子伝達系の呼吸系酵素を阻害し，リポポリサッカライドやポーリンタンパクのような細胞膜構成成分を漏洩させることが示唆された．細胞内部まで侵入したbis-QACがペプチドグリカンを破壊し，細胞表面に形成した大きなホールから細胞内物質を漏洩させることが細菌に対する致命的攻撃であることが示唆された．


Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction

ヒト特異的細胞溶解毒素インターメディリシン遺伝子をS. intermedius菌種マーカーとし，またアンギノーサス群連鎖球菌に分布する2タイプのヒアルロニダーゼ遺伝子とrRNA遺伝子をそれぞれ組み合わせた2段階のPCRシステムを開発した．これを用いると現在知られているアンギノーサス群連鎖球菌の全菌種(3菌種，4亜菌種)を迅速に鑑別でき，本菌群による感染症の臨床診断にも応用可能であることを示した．


Synthesis and Antimicrobial Characteristics of Novel Antimicrobial Agent,1,4-Dialky-1,4-dihydroxyethylpiperadinium Diiodides Immobilized on Inorganic Materials

論文題目:新規殺菌剤1,4-ジヒドロキシエチルピペラジニウムジアイオダイドを無機材料表面に固定化した材料の合成と抗菌特性 本論文は固定化殺菌剤に関するものであり，分子内にアンモニウム残基とヒドロキシエチル基をそれぞれ2個導入した薬剤の新規合成研究およびシランカップリング反応によるフェライト表面およびガラスビーズ表面に固定化する研究およびこれらを用いて各種微生物に対する殺菌作用と殺菌特性を解析したものである．固定化殺菌材の殺菌活性はpHには影響を受けなかったが温度には大きな影響を受けることを明らかにした．しかし固定化殺菌材は非常に広い殺菌スペクトルを有し，5∼6回繰り返し使用には殺菌活性が低下しない優れた特徴を示した．


Antimicrobial characteristics of bis-quaternary ammonium compounds possessing a p-phenylene group in their spacer chains

p-フェニレンスペーサにより連結された2つのピリジニウム塩から成るフェニレン-ビス型四アンモニウム化合物 (Ph-bis-QACs)の抗菌特性および構造活性相関についてメチレンスペーサ鎖により連結されたメチレン-ビス型四アンモニウム化合物(M-bis-QACs)と比較検討した．Ph-bis-QACsは，高い殺菌活性と広い抗菌スペクトルを示した．Ph-bis-QACsの抗菌活性は，M-bis-QACsのメチレンスペーサ鎖をp-フェニレンスペーサ鎖に変換することによってM-bis-QACsの活性より高くなった．M-bis-QACsの活性は，アンモニウム窒素の電子密度に影響を受けるが，Ph-bis-QACsはそれに依存しないことが分かった．従って，bis-QACの抗菌活性は，アンモニウム窒素の電子密度よりもスペーサ鎖の回転軸を減少させること，分子構造の剛直性を上昇させることによって，より強化されると考えられ，bis-QACの立体構造が，bis-QACの抗菌活性の重要な因子であると示唆した．


Kinetic Analysis of the Bactericidal Action of a Bis-quaternary Ammonium Compound against Escherichia coli

ビス型第四アンモニウム塩系化合物(bis-QAC)の大腸菌に対する殺菌機構を解明するためbis-QAC，4,4'-(1,6-ヘキサメチレンジチオ)ビス(1-オクチルピリジニウムブロマイド)の殺菌作用の動力学的特性を解析し，mono-QAC，N-オクチルピリジニウムブロマイドと比較した．mono-QACのアレニウスプロットは薬剤処理温度及び細胞培養温度の影響を受けて二つの直線を示したのに対し，bis-QACのアレニウスプロットは直線性を示した．mono-QACの見かけの殺菌活性化エネルギーは10-30℃で99.8kJ/mol，30-40℃で40.6kJ/molであったが，bis-QACは15.6kJ/molと非常に低い値であった．この結果より，bis-QACの殺菌作用はmono-QACとは異なるメカニズムであり，mono-QACの殺菌作用の律速段階が細菌細胞膜への作用であるのに対して，bis-QACは細胞膜表面への吸着であることが示唆された．


Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR

論文題目:緑膿菌の細胞膜タンパク質OprRの発現と第四アンモニウム塩に対する耐性化の相関 第四アンモニウム塩系消毒剤に耐性化した緑膿菌において顕著に発現量が増加する約25.6kDaの蛋白質を2次元電気泳動で分離し，そのN末端アミノ酸配列の解析によって，それが外膜蛋白質のOprRであることを明らかとした．またその耐性化株でOprRのノックアウト株を作成したところ，第四アンモニウム塩系消毒剤に対する感受性が野生株と同程度まで上昇することから，OprRがこの消毒薬耐性に重要な役割を果たしていることを示した．


Analysis of Structural Features of Bis-Quaternary Ammonium Antimicrobial Agents 4,4'-(α,ω-Polymethylenedithio)bis(1-alkylpyridinium Iodide)s Using Computational Simulation

2個のピリジン環を持ち，その2環を結ぶブリッジ構造が異なる4タイプのビス型第四アンモニウム塩系消毒剤について，その分子構造とNB1RGB細胞に対する細胞毒性の構造活性相関を配座探索と分子軌道計算により検討した結果，他の3タイプの消毒剤はエネルギー極小状態がただ一つであるのに対し，ピリジンの四級窒素にオクチル基を持つチオエーテルブリッジタイプ(チオエーテル間のメチレン鎖数が4(4DTBP-4,8)と6(4DTBP-6,8))の場合だけエネルギー極小状態が2つ存在した．この結果から，4DTBP-4,8と4DTBP-6,8が高い効力と安全性を示すのは，分子の柔軟性と疎水性度が上手くバランスしているためであることを示した．


Bactericidal Action of a Bis-quaternary Ammonium Compound against Vegetative Cells and Spores of Bacillus subtilis

論文題目:Bacillus subtilisの栄養細胞および芽胞に対するビス型第四アンモニウム塩の殺菌活性 ビス型第四アンモニウム塩は4,4'-(1,6-ヘキサメチレンジチオ)ビス(1-オクチルピリジニウムアイオダイド)を用い，枯草菌の栄養細胞と芽胞に対する殺菌活性の測定，芽胞発芽阻害作用の検討，殺菌速度および殺菌活性化エネルギーの測定，pH依存性などについて詳細に検討した．その結果，本薬剤は芽胞に対して発芽阻害作用と発芽後の増殖も阻害することが明らかとなった．また，殺菌活性化エネルギーは非常に低く細胞への吸着段階が殺菌の律速段階であることがわかった．


Microbial Contamination in Home Laundry Operations in Japan

論文題目:日本における家庭洗濯の微生物汚染．家庭洗濯中の衛生レベルは生活様式の変化と共に変遷してきた．諸外国では洗濯中に微生物汚染や感染が報告されている．そこで日本における洗濯中の汚染について様々な着用後の繊維製品に付着している微生物種の同定，洗濯物間の微生物転移による感染の危険性の実態について明らかにした．さらに除菌洗剤と非除菌洗剤を用いて検討し，除菌洗剤の汚染や感染防止効果が高いことを明らかにした．


The Properties of Bacterial Adaptation to Mono-and Bis-Quaternary Ammonium Compounds

論文題目:モノ型およびビス型第四アンモニウム塩に対する細菌の適応の性質．モノ型(デシルピリジニウム塩)およびビス型第四アンモニウム塩(架橋構造がチオール，エステル，カルバモイルおよびアミド)に対する細菌の適応について検討した．最小発育阻止濃度(MIC)測定法により検討した結果，モノ型ではMIC値が上昇し，抵抗性化が見受けられたが，ビス型第四アンモニウム塩では全く耐性化が見られなかった．抵抗性菌の表面疎水性を解析した結果，モノ型の抵抗性化は細胞表面の疎水性が低下したためであるとした．


Characterization of Pseudomonas aeruginosa Resistant to a Quaternary Ammonium Compound

論文題目:第四アンモニウム塩化合物に対して薬剤耐性を示す緑膿菌の特徴 N-ドデシルピリジニウムアイオダイドに対する薬剤耐性化を最小濃度測定法である培地希釈法を用いて行うことに成功した．その結果，感受性菌よりも8倍高い耐性化細菌を得ることができた．電子顕微鏡で観察した結果，耐性化細胞は感受性細胞には見られないバイオフィルム様の凝集が観察された．さらに，耐性化した細胞の表面疎水性について測定した結果，感受性細胞よりも親水性化していることがわかった．


Synthesis and antimicrobial characteristics of 4,4',4''-(1,2,3-propylene trithio)tris(1-alkylpyridinium iodide)s

トリス型四級アンモニウム化合物 (tris-QAC) 4,4',4''-(1,2,3-propylene-trithio)tris (1-alkylpyridinium iodide)s (4TP-n: アルキル基の炭素数 n = 4,6,8,10,12,14)を合成し，抗菌特性を検討した．4TP-nは，3つのN-alkyl-4-thiopyridinium iodideをプロパンの1,2,3位で結合したトリス型構造を有する．4TP-nの抗菌特性を，ビス型四級アンモニウム化合物 (bis-QAC) 4,4'-(1,6-hexamethylenedithio)bis(1-alkylpyridinium iodide)s (4DTBP-6,n)，モノ型四級アンモニウム化合物 (mono-QAC) N-alkylpyridinium iodide (P-n)，そして商業的な抗菌剤，防黴剤と比較した．4TP-nの静菌，殺菌活性は，4DTBP-6,nと同様に高く，またアルキル鎖長の影響をほとんど受けなかった．4TP-nは，広くかつ強い抗菌スペクトルを有していた．4TP-nの殺菌活性は，pH，温度の影響を受け，その影響を全く受けない4DTBP-6,8とは異なっていた．


Rapid identification of Streptococcus intermedius by PCR with the ily gene as a species marker gene

ヒト特異的細胞溶解毒素インターメディリシン(ILY)遺伝子はアンギノーサス群連鎖球菌ではS. intermedius(SI)のみに特異的に存在したことから，ily遺伝子が本菌種にだけ分布する可能性が考えられた．そこでPCRやサザンドットハイブリダイゼーション法を用いて，連鎖球菌属や近縁菌種におけるily遺伝子の分布を調べ，ily遺伝子がSI特異的な菌種マーカーになり得ることを確認し，それを指標として本菌種による感染症診断に応用可能なPCR検出システムを構築した．


Effect on Polymorphonuclear Cell Function of a Human-Specific Cytotoxin, Intermedilysin, Expressed by Streptococcus intermedius

Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin from Streptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/microl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis of Staphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.

ヒト特異的細胞溶解毒素インターメディリシン(ILY)とS. intermedius(SI)による深部臓器膿瘍の関連性が示されたため，SI感染時にはそれを除去するために働く多形核白血球に対し，ILYがどのような作用を及ぼすかFACSを用いて調べた．その結果，ILYは多形核白血球を死滅させるだけでなく，致死量より少量では細胞表面のCD11b量や貪食能に変化を起こすなど，細胞機能のモジュレーターとなり，SIによる感染を助長する可能性を示した．


Synthesis of 4,4'-(Tetramethylenedicarbonyldiamino)bis(1-decylpyridinium bromide) and Its Antimicrobial and Deodorant Characteristics

論文題目:4,4'-(テトラメチレンジカルボニルジアミノ)ビス(1-デシルピリジニウムブロマイド)． 本論文において4,4'-(テトラメチレンジカルボニルジアミノ)ビス(1-デシルピリジニウムブロマイド)の新規合成方法の確立を行い，さらに一連の合成薬剤の抗菌特性を塩化ベンザルコニウム，チアベンダゾール，ベンツイミダゾールおよびセチルピリジニウムと比較検討した．その結果，細菌類に対して塩化ベンザルコニウムやセチルピリジニウムよりも非常に高い静菌活性を認めた．また，黴にたいしても現行のチアベンダゾールやベンツイミダゾールよりも高い活性を示した．さらに消臭効果やプラスチックに対する抗菌効果などの応用試験を行った．


Synthesis and Antimicrobial Characteristics of N,N'-Hexamethylenebis(4-carbamoyl-1-decylpyridinium bromide)

論文題目:N,N'-ヘキサメチレンビス(4-カルボニル-1-デシルピリジニウムブロマイド)の合成と抗菌特性．ビス型の第四アンモニウム塩であるN,N'-ヘキサメチレンビス(4-カルボニル-1-デシルピリジニウムブロマイドの合成方法を確立し，抗菌特性についてモノ型のアルキルピリジニウムを用いて比較検討した．その結果，非常に優れた殺菌活性を示し，かつ，高い抗黴活性も示した．pH依存性や温度依存性についても検討し，モノ型よりも優れた特性を認めた．静菌活性，殺菌活性および抗黴活性は，塩化ベンザルコニウム，セチルピリジニウムおよびチアベンダゾールよりも高い活性を示した．


Evaluation of the cytotoxic effects of bis-quaternary ammonium antimicrobial reagents on human cells

The cytotoxic effects of newly synthesized bis-quaternary ammonium compounds (bis-QACs) and benzalkonium chloride were investigated on skin and blood cells, namely normal human epidermal keratinocytes of neonatal foreskin, a normal human skin fibroblast cell line NB1RGB, erythrocytes and a lymphoma cell line JM. The bis-QACs tested were 4, 4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium iodide) (4DTBP-6, 8), N,N'-tetramethylenebis(1-dodecyl-4-carbamoylpyridinium iodide) (4BCAP-4,12), N,N'-hexamethylenebis(1-decyl-4-carbamoylpyridinium iodide) (4BCAP-6,10), 4,4'-(1,4-tetramethylenedicarbonyldiamine) bis (1-decylpyridinium iodide) (4DCABP-4,10), 4,4'-(1, 4-tetramethylenedicarbonyldiamine) bis (1-dodecylpyridinium iodide) (4DCABP-4,12), 4,4'-(1, 4-phenyldicarbonyldiamine)bis(1-dodecylpyridinium iodide) (4DCABP-P, 12), and 4,4'-(1, 6-hexamethylenedioxydicarbonyl)bis(1-dodecylpyridinium iodide) (4DOCBP-6,12). All bis-QACs consisted of two identical alkylpyridinium rings and bridge structure linking rings to each other have a methylene bridge but only 4DCABP-P,12 has a phenyl ring as a bridge. Most of the LD(50) values in acute cytotoxic assays of these bis-QACs tended to be lower than those of benzalkonium chloride. However, the comparison of the antibacterial activity against cytotoxic effects on several human cells revealed that bis-QACs, especially 4DTBP-6,8, have wide concentration ranges showing sufficient antibacterial activity and lower cytotoxic effect on human cells (except for 4DOCBP-6,12), although benzalkonium chloride caused significant human cell damage at the concentrations necessary for antibacterial activity. Moreover, judging from the LD(50) value of 4DTBP-6,8 [67 microM, a 6.7-fold higher concentration than the upper value of MIC] obtained in an artificial human skin model TESTSKIN, 4DTBP-6,8 is thought to be a more effective and safer antiseptic reagent for application to the skin surface than benzalkonium chloride. Taken together, bis-QACs are expected to be a promising new generation of antimicrobial QACs.

多用される第四アンモニウム塩系薬剤の塩化ベンザルコニウム(Bz)と抗菌力の強いダイマー型第四アンモニウム塩系薬剤(bis-QACs)のヒト細胞に対する毒性を比較した． 正常表皮角化細胞，皮膚線維芽細胞，赤血球及びTリンパ球株における各薬剤の急性及び亜急性毒性を，10菌種16株に対する最小発育阻止濃度を指標とした抗菌活性と比べたところ，bis-QACsの方がBzよりも使用安全域が広く，bis-QACsが次世代の抗菌剤として期待できることを示した．


Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.

ヒト特異的な細胞溶解毒素インターメディリシン(ILY)遺伝子をクローニングし，アンギノーサス群連鎖球菌におけるily遺伝子の分布を調べた結果，この遺伝子は本菌群内でS. intermedius(SI)のみに普遍的に分布することを示した．またSIのILY分泌量はヒト口腔内常在菌株に比べて深部膿瘍分離株の場合に6-10倍も大量であり，ILYとSI感染による深部臓器膿瘍とは相関したが，他のSIが持つ病原因子候補の量と膿瘍感染症は相関しないことを見出し，SIによる深部膿瘍感染症にILYが重要な病原因子として働くことを示した．


Three-dimensional structural analysis of histone-like protein from streptococci

レンサ球菌のヒストン様蛋白質の構造解析


Synthesis and Antimicrobial Characteristics of 5,5'-[2,2'- (Tetramethylenedicarbonyldioxy) diethyl]bis(3-alkyl-4-methylthiazolium iodide)s

論文題目:5,5'-[2,2'-(テトラメチレンジカルボニルジオキシ)ジエチル]ビス(3-アルキル-4-メチルチアゾリウムアイオダイド)の合成と抗菌特性． 本論文においてビス型のチアゾリウム系抗菌剤および対応するモノ型チアゾリウムを合成し，抗菌特性，静菌スペクトル，静黴スペクトル，pHおよび温度特性などを検討した結果，優れた抗菌特性であることを明らかにした．さらに分子疎水性パラメーターを用い殺菌活性と静菌活性を解析した結果，殺菌活性が分子疎水性に依存しない優れた性質を有することを明らかにした．