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.

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 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.

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.

[Efficacy of Slightly Acidic Electrolyzed Water against Contamination of Water Line of Dental Units].

OBJECTIVES: The purpose of this study was to evaluate the efficacy of slightly acidic electrolyzed water (SAEW) against the contamination of the water line of dental units and the effects of SAEW on the water line. MATERIALS AND METHODS: The experimental material was a prototype dental unit equipped with a SAEW generator. SAEW is directly supplied to each device or part of this unit system. Experimental SAEW samples were collected from a high-speed handpiece (HS-1), an ultrasonic scaler, and a cup filler of the prototype dental unit. Control samples were taken before and after the prescribed flushing from another high-speed handpiece (HS-2) that is directly supplied with tap water in the same dental unit. The samples were analyzed for free chlorine and heterotrophic bacteria for 7 years to assess the efficacy and effects of SAEW. The substances eluted in SAEW were examined to investigate the effect of SAEW on the water line. A questionnaire survey was conducted on patients on whom dental uints supplied with SAEW were used. RESULTS: SAEW always showed a higher free chlorine concentration than tap water during the observation period of 7 years. In HS-2 supplied with tap water, the free chlorine concentration increased significantly owing to the prescribed flushing. SAEW always showed a significantly smaller number of heterotrophic bacteria than tap water. No abnormal levels values of water line components eluted into SAEW were observed. There were few negative comments from patients on whom dental units supplied with SAEW were used. CONCLUSIONS: SAEW continuously used for 7 years was effective for contamination control in the water line of dental units.

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 ß-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-ß-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.

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.

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.

Detection of penicillin-binding protein 2b gene alteration in Streptococcus mitis by polymerase chain reaction.

Three isolates of beta-lactam-resistant streptococci from the saliva of healthy adults were identified as Streptococcus mitis. Minimum inhibitory concentrations (MICs) were 2 to 4 micro g/ml for ampicillin (ABPC) and 64 to more than 128 micro g/ml for cefaclor (CCL). To determine the position of base alterations of the penicillin-binding protein 2b ( pbp2b) gene, upstream primers containing possible mutation points were designed, and used for polymerase chain reaction (PCR), together with a downstream primer. Alterations adjacent to the conserved motifs of the pbp2b gene were apparent. DNA sequencing data indicated replacements in deduced amino acid sequences in all resistant isolates: from threonine to alanine just after the serine-serine-asparagine (SSN) motif, and from alanine to glycine two residues downstream of the lysine-threonine-glycine (KTG) motif. These changes were the same as those in penicillin-resistant Streptococcus pneumoniae (PRSP), suggesting importance for the enzymatic activity of the protein. Thus, Beta-lactam susceptibility of S. mitis may be partially predicted by PCR using our primer set for pbp2b.

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

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.

Beta-lactam resistance in Streptococcus mitis isolated from saliva of healthy subjects.

The purpose of this study was to examine the percentage of Beta-lactam-resistant streptococcal carriers in healthy adults, and to investigate the relationships among minimum inhibitory concentrations (MICs) of Beta-lactams, alterations in the penicillin-binding protein genes ( pbp genes), and the affinity of penicillin-binding proteins (PBPs) for ampicillin (ABPC) in Streptococcus mitis. We also compared numbers of surviving bacteria at various ABPC concentrations in both ABPC-susceptible and -resistant S. mitis strains. The percentages of subjects carrying ABPC- and cefaclor (CCL)-resistant streptococci were 52% (27 of 52 subjects) and 100%, respectively. S. mitis, including both antibiotic-susceptible and -resistant strains, were classified into five groups according to the pbp gene mutations that resulted in alterations of the deduced amino-acid sequence in the homology boxes of PBPs. All ABPC-resistant strains showed alterations in PBP1A, 2X, and 2B, while no or only PBP2X alterations were detected in the susceptible strains. These results suggest that the accumulation of pbp gene mutations is strongly related to the MIC of ABPC for S. mitis. In the resistant strains, the affinity of PBPs for ABPC was reduced in comparison with that in the susceptible strains, and the bactericidal effect of ABPC was also reduced. Therefore, we should be aware of conditions such as infective endocarditis that are caused by Beta-lactam-nonsusceptible streptococci in the normal oral flora.

Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus(1).

Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.