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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 S. intermedius 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 nanA-null and msgA-null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM N-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 S. intermedius 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 S. intermedius.

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; equol 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.

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 ß-d-galactosidase and N-acetyl-ß-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 ß-d-galactosidase, ß-d-fucosidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-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-ß-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked ß-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had ß-d-galactosidase and ß-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-ß-d-glucosaminidase and N-acetyl-ß-d-galactosaminidase activities. The ß-d-galactosidase and ß-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-ß-d-glucosaminidase and N-acetyl-ß-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 ß-haemolytic Streptococcus constellatus subsp. constellatus cytotoxicity.

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 ß-haemolytic strains, the factor(s) responsible for ß-haemolysis in ß-haemolytic SCC (ß-SCC) has yet to be clarified. Recently, we discovered that a streptolysin S (SLS) homologue is the ß-haemolytic factor of ß-haemolytic Streptococcus anginosus subsp. anginosus (ß-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 ß-SAA, the SLS homologue is the ß-haemolytic factor of ß-SCC, and therefore aimed to investigate and characterize the haemolytic factor of ß-SCC in the present study. PCR amplification revealed that all of the tested ß-SCC strains were positive for the sagA homologue of SCC (sagA(SCC)). Further investigations using ß-SCC strain W277 were conducted to elucidate the relationship between sagA(SCC) and ß-haemolysis by constructing sagA(SCC) deletion mutants, which completely lost ß-haemolytic activity. This loss of ß-haemolytic activity was restored by trans-complementation of sagA(SCC). Furthermore, a co-cultivation assay established that the cytotoxicity of ß-SCC was clearly dependent on the presence of sagA(SCC). These results demonstrate that sagA(SCC) is the factor responsible for ß-SCC ß-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.

Identification of mutations resulting in derepression of the intermedilysin gene by sequential mutagenesis of its promoter region in Streptococcus intermedius.

Streptococcus intermedius secretes the human-specific cytolysin intermedilysin (ILY), a crucial factor in the pathogenicity of this bacterium. Previously, we reported that a lactose phosphotransferase repressor (LacR) represses ily expression, and that its mutation increases ILY production. Interestingly, UNS40, a strain isolated from a liver abscess, produces high levels of ILY despite the absence of mutations in the lacR promoter and coding regions. Our results showed that a G > A mutation at the -90th position from the transcription start point in the UNS40 ily promoter region increased hemolytic activity and decreased the binding ability to LacR. To elucidate the regions involved in the repression of ily expression, we generated mutant strains, in which point or deletion mutations were introduced into the ily promoter region, and then compared their hemolytic activity. Among the point mutations, -120 C > A and -90 G > A and their flanking mutations increased hemolytic activity. These results indicated that these mutations may increase the virulence of S. intermedius.

Interactive Analysis of UTX-114 Family With EGFR-tyk: Molecular Features of Acetyl Glycosylated Gefitinib.

BACKGROUND/AIM: Acetyl glucose adducts (UTX-114, -115, and -116) were prepared from gefitinib, and their characteristics (e.g., anticancer activity, structural property) were analyzed. MATERIALS AND METHODS: Cytotoxicity and radiosensitizing properties of the UTX-114 family were examined using A431 cells. Supramolecular associations between the UTX-114 family compounds and the tyrosine kinase domain of epidermal growth factor receptor (EGFR-tyk) were also examined. The interactive analyses of the UTX-114 family compounds with EGFR-tyk were performed using docking simulation technique. RESULTS: The UTX-114 family showed a similar cytotoxicity as gefitinib, yielding IC50 values of 31.2 µM (gefitinib), 34.3 µM (UTX-114), 36.8 µM (UTX-115), and 39.4 µM (UTX-116). The EGFR-tyk inhibition ratios (IR) of UTX-114, -115, and -116 to gefitinib were 1.515, 0.983, and 0.551, respectively. The EGFR-tyk inhibitory activity of UTX-114 was higher than that of gefitinib. UTX-114 also showed the highest radiosensitizing activity among the tested compounds. UTX-114 expressed 1841 conformers (-8.989~15.718 kcal/mol) with the solvation free energy (dGW) of UTX-114 decreasing with increasing conformational energy, ranging between -354.955~ -260.815 kJ/mol. Interactive energies of gefitinib, UTX-114, -115, and -116 with EGFR-tyk were -123.640, -144.053, -120.830, and -124.658 kcal/mol, respectively. CONCLUSION: UTX-114 yielded the lowest interaction energy with EGFR-tyk among tested compounds. Given the association behavior between UTX-114 and EGFR-tyk, along with its other observed properties, UTX-114 appears to be a viable therapeutic possibility.

Streptolysin S induces proinflammatory cytokine expression in calcium ion-influx-dependent manner.

Anginosus group streptococci (AGS) are opportunistic pathogens that reside in the human oral cavity. The ß-hemolytic strains of Streptococcus anginosus subsp. anginosus (SAA) produce streptolysin S (SLS), a streptococcal peptide hemolysin. In recent clinical scenarios, AGS, including this species, have frequently been isolated from infections and disorders beyond those in the oral cavity. Consequently, investigating this situation will reveal the potential pathogenicity of AGS to ectopic infections in humans. However, the precise mechanism underlying the cellular response induced by secreted SLS and its relevance to the pathogenicity of AGS strains remain largely unknown. This study aims to elucidate the mechanism underlying the host cellular response of the human acute monocytic leukemia cell line THP-1 to secreted SLS. In THP-1 cells incubated with the culture supernatant of ß-hemolytic SAA containing SLS as the sole cytotoxic factor, increased Ca2+ influx and elevated expression of proinflammatory cytokines were observed. Significantly reduced expression of SLS-dependent upregulated cytokine genes under Ca2+-chelating conditions suggests that Ca2+ influx triggers SLS-dependent cellular responses. Furthermore, SLS-dependent enhanced expression of IL-8 was also implicated in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The findings presented in this study are crucial for a comprehensive understanding of the real pathogenicity of SLS-producing ß-hemolytic AGS in the latest clinical situations.

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.