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 ß-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 ß-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 ß-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca2+ 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 ß-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca2+ chelation, except for jun. These results suggest that SLS caused Ca2+ 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.

Molecular Aspects of C-glycosides: Interactive Analysis of C-linked Compounds With the SGLT2 Molecular Model.

BACKGROUND/AIM: Interaction analysis between modeled human sodium/glucose cotransporter 2 (hSGLT2) and antidiabetic C-glycoside drugs, such as canagliflozin, dapagliflozin, ipragliflozin, empagliflozin, tofogliflozin, and luseogliflozin was performed. MATERIALS AND METHODS: The hSGLT2 was modeled using the X-ray data of Vibrio parahaemolyticus SGLT2 (protein data bank ID=2XQ2) as a template. Conformational analyses of C-glycosides were performed using CAChe-Conflex. Interactive analyses between hSGLT2 and C-glycosides were performed using Molegro Virtual Docker. RESULTS: Canagliflozin interacted with hSGLT2 via Asn75, Ser287, Lys321 and Gln457 Dapagliflozin interacted with six amino acids (Arg46, Arg49, Ile76, Ser78, Met216 and Ser393). Ipragliflozin (Ala69, Met596 and Gln600), empagliflozin (Ser78, Gly79, Lys154, Asp158 and Ser393), tofogliflozin (Arg49, Met216, Ala389, Ser392 and Ser393), and luseogliflozin (Arg49, Ser74, Ser78, Gly79, His80, Lys154, Asp158 and Ser393) interacted with hSGLT2 via the amino acids described in the parentheses. CONCLUSION: The binding mode of each C-glycoside drug to hSGLT2 was different, and structural features of each compound were revealed. The reactive base points of C-glycosides were the sugar moiety, with the sugar structure being important for hSGLT2 inhibitory action.

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 ß-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of ß-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 ß-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from ß-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 Streptococcus mitis strain Nm-76.

Background: Some strains of Streptococcus mitis exhibit ß-hemolysis due to the ß-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in S. mitis 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 S. mitis strain Nm-76. Methods: 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 Escherichia coli. Results: The gene encoding CDC found in Nm-76 and its homolog are distributed among many S. mitis strains. This CDC is phylogenetically different from other previously characterized CDCs, such as S. mitis-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. Conclusion: DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in S. mitis.

Molecular Interaction Between Boron Tracedrug UTX-51 Derivatives and Bovine Serum Albumin: Application to an Analytical Model of AGEs Destruction by Thermal Neutron Irradiation.

BACKGROUND/AIM: Boron tracedrugs possess global molecular tracking abilities and localized destructive power. We investigated the molecular properties of synthesized boron tracedrugs, including UTX-51, and their interactions with the advanced glycation end-product (AGE)-related protein bovine serum albumin (BSA). MATERIALS AND METHODS: A conformational analysis of the compounds used in the present study was performed using CAChe (Fujitsu Inc., Tokyo, Japan) and the degree of stereo-hydrophobicity of the conformers obtained was verified using Mopac (Fujitsu Inc.). The interactive properties of global minimum conformers of the derivatives tested with BSA were assessed using Molegro Virtual Docker (CLC bio., Aarhus, Denmark). RESULTS: Among the compounds investigated, UTX-51 was confirmed to interact with BSA based on the formation of hydrogen bonds between BSA and UTX-51. CONCLUSION: UTX-51 is a promising boron tracedrug and can be used as the lead structure for developing a therapeutic agent for AGE-related diseases, including cancer.

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.

Diversity of ß-hemolysins produced by the human opportunistic streptococci.

The genus Streptococcus infects a broad range of hosts, including humans. Some species, such as S. pyogenes, S. agalactiae, S. pneumoniae, and S. mutans, are recognized as the major human pathogens, and their pathogenicity toward humans has been investigated. However, many of other streptococcal species have been recognized as opportunistic pathogens in humans, and their clinical importance has been underestimated. In our previous study, the Anginosus group streptococci (AGS) and Mitis group streptococci (MGS) showed clear ß-hemolysis on blood agar, and the factors responsible for the hemolysis were homologs of two types of ß-hemolysins, cholesterol-dependent cytolysin (CDC) and streptolysin S (SLS). In contrast to the regular ß-hemolysins produced by streptococci (typical CDCs and SLSs), genetically, structurally, and functionally atypical ß-hemolysins have been observed in AGS and MGS. These atypical ß-hemolysins are thought to affect and contribute to the pathogenic potential of opportunistic streptococci mainly inhabiting the human oral cavity. In this review, we introduce the diverse characteristics of ß-hemolysin produced by opportunistic streptococci, focusing on the species/strains belonging to AGS and MGS, and discuss their pathogenic potential.

Synthesis of D-π-A type benzothiazole-pyridinium 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-π-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.

Construction of a Drug Release Evaluation System: Application of Mitochondrial Respiration to Monitor Drug Release.

BACKGROUND/AIM: Efficient drug encapsulation and regulation of drug release are important factors for sustained drug release and application for release-controlled anti-cancer and anti-inflammatory drug delivery. In the present study, a direct evaluation system for drug-release from model carrier (e.g., alginate-gel beads) was examined using the mitochondrial oxygen consumption rate as an index. MATERIALS AND METHODS: Alginate-gel beads were coated with the uncoupler SF6847 (SF beads) and used as a model microparticle-type drug. The real-time monitoring of SF6847 release from prepared alginate-gel beads was performed using the mitochondrial oxygen consumption rate. Release profiles of nonsteroidal anti-inflammatory drugs [NSAIDs, mefenamic acid (MEF) and diclofenac (DIC)] from alginate-gel beads as well as SF beads were investigated using the real time monitoring system. RESULTS: SF6847 release from alginate-gel beads was clearly detected using the rat liver mitochondrial oxygen consumption rate. The release features of MEF and DIC from alginate-gel beads were defined by the present trial monitoring system, and these NSAIDs exhibited different release profiles. CONCLUSION: The present drug monitoring system detected released drugs, and the release profile reflected the molecular properties of the test drugs. This system may be applied to the design and development of precise sustained drug release systems (e.g., anti-cancer and anti-inflammatory drugs).

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

Streptococcus mitis 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.

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.

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

We have synthesized a series of quaternized imidazo[1,2-a]pyridines in three steps from commercially available reagents. These compounds exhibit blue fluorescence emission at around 425 nm with good quantum yields. In addition, one specific compound was found to work as not only a two- and three-photon excitable mitochondria imaging agent, but also a therapeutic agent upon continuous irradiation conditions.

Effect of Isomerization of TX-2036 Derivatives on the Interaction With Tyrosine Kinase Domain of EGF Receptor.

BACKGROUND: From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. MATERIALS AND METHODS: The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. RESULTS: R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC50 value of TX-2036 was 1.8 µM. CONCLUSION: R-configured TXs interacted with Lys721 and Thr766 of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile765 and Thr766 (TX-2044), Ser696, Thr766, and Thr830 (TX-2031), Gly772, Cys773, and Thr830 (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.

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

Streptococcus mitis strain Nm-65 secretes an atypical 5-domain-type cholesterol-dependent cytolysin (CDC) called S. mitis-derived human platelet aggregation factor (Sm-hPAF) originally described as a platelet aggregation factor. Sm-hPAF belongs to Group III CDC that recognize both membrane cholesterol and human CD59 as the receptors, and shows preferential activity towards human cells. Draft genome analyses have shown that the Nm-65 strain also harbors a gene encoding another CDC called mitilysin (MLY). This CDC belongs to Group I CDC that recognizes only membrane cholesterol as a receptor, and it is a homolog of the pneumococcal CDC, pneumolysin. The genes encoding each CDC are located about 20 kb apart on the Nm-65 genome. Analysis of the genomic locus of these CDC-encoding genes in silico showed that the gene encoding Sm-hPAF and the region including the gene encoding MLY were both inserted into a specific locus of the S. mitis genome. The results obtained using deletion mutants of the gene(s) encoding CDC in Nm-65 indicated that each CDC contributes to both hemolysis and cytotoxicity, and that MLY is the major hemolysin/cytolysin in Nm-65. The present study aimed to determine the potential pathogenicity of an S. mitis strain that produces two CDC with different receptor recognition properties and secretion modes.

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly.

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

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 Ca2+ -dependent K+ permeability by SLO-induced influx of external Ca2+ . 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.

N,N'-Dimethylated imidazo[1,5-a]pyridinium salt having good water solubility and exhibiting fluorescence emission was found to work as not only a bioimaging agent but also a therapeutic agent under UVA-LED irradiation conditions. Because the continuous UVA-LED irradiation to HeLa cells stained by the synthesized salt resulted in the cell death due to the mitochondrial damage, the salt has a potential application as photodynamic therapy agent against tumor cells.

Correlation Between Radiosensitizing Activity and the Stereo-structure of the TX-2036 Series of Molecules.

BACKGROUND/AIM: The stereo-configuration (R-, S-configuration) of chiral-2-nitroimidazole derivatives alters their radiosensitizing activity. This study aimed at examining the molecular features of these enantiomers by molecular simulation techniques. MATERIALS AND METHODS: A series of 2-nitroimidazole-based radiosensitizer TX-2036 molecules were synthesized, and their profiles were examined using molecular structural analysis such as conformation analysis, molecular orbital analysis, and electrostatic potential analysis. RESULTS: R-configured TXs (TX-2043, -2030, -2036) had a weaker radiosensitizing activity than S-configured TXs (TX-2044, -2031, -2037), and R-compounds had a small minus electrostatic potential (ESP) field in the cyclopentene-1,3-dione region. S-configured TX-2046 had weaker radiosensitizing activity than R-configured TX-2045, and TX-2046 had a small minus ESP field as well as R-configured TX-2043, -2030, - 2036. CONCLUSION: The cyclopentene-1,3-dione involved in the small minus ESP field affected the radiosensitizing activity of the TX-2036 series of molecules.