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.

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.

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.

Change during an 8-Year Period in Streptococcus Pyogenes emm Types in Pharyngeal Isolates from Children with Noninvasive Infections.

BACKGROUND: Streptococcus pyogenes, or group A streptococcus (GAS), is one of the most common bacterial pathogens in children. GAS can cause such nonserious and noninvasive diseases as pharyngitis and skin infection, as well as serious, invasive diseases like streptococcal toxic shock syndrome. One factor that makes GAS pathogenic is the type-specific M protein on its cell surface. To identify emm types and their characteristics, we previously examined GAS strains isolated from children with noninvasive infections at our hospital. The present study was conducted 8 years later, for comparison. METHODS: The 23 participants were inpatients and outpatients at Nippon Medical School Tama Nagayama Hospital during 2016 and 2017. A pharyngeal swab specimen was obtained from each child, and genes encoding M proteins were amplified by polymerase chain reaction. RESULTS: emm type analysis identified emm1 in 11 of the 23 strains and emm12 in 4. Three group G streptococcus (GGS) strains carried M-like protein genes. CONCLUSIONS: The predominant emm type was emm12 in our previous report and emm1 in this study. This study also identified 3 GGS strains among the isolates, which carried either the stg245, stg6795, or stg840 M-like protein gene. One GAS strain carried stg485, a gene associated with GGS rather than GAS.

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.

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.

[Evaluation of the GonoGen II kit for rapid identification of Neisseria gonorrhoeae using monoclonal antibody directed at gonococcal outer membrane protein 1].

The Gonogen II test for rapid identification of Neisseria gonorrhoeae (Gonococcus, GC) was evaluated. The test is based on a colorimetric reaction with monoclonal antibody to GC outer membrane protein 1. Of the 50 clinical isolates of GC, 49 isolates tested positive and only one strain tested negative. Other Neisseria. spp, H. influenzae, H. parainfluenzae, E. corrodens, M. catarrhalis, and A. baumannii showed negative test results. Non-Neisseriae. spp, such as S. aureus. P. aeruginosa, E. faecalis, and E. coli also showed negative test results. No cross-reactivity was found between GC and other Neisseriae. spp or non-Neisseriae. spp. In a mixed suspension of GC and all of non-Neisseriae. spp as mentioned above, the GonoGen II test was positive. The specificity and sensitivity of the test for the identification of GC were 98% and 100%. The minimum limit of detection of GC was > or = 1 x 10(5) cfu/mL. Decision making based on the test result is possible within 10 minutes. These findings also suggest that the test does not require pure GC. The GonoGen II test appears to be a reliable, quick and easy-to-use assay, and also to not require viable GC. Thus GonoGen II is shown to be a very useful test for the identification of GC.

Studies of recombinant streptococcal pyrogenic exotoxin B/cysteine protease (rSPE B/SCP) in the skin of guinea pigs & the release of histamine from cultured mast cells & basophilic leukocytes.

BACKGROUND & OBJECTIVES: Streptococcal pyrogenic exotoxin B/streptococcal cysteine protease (SPE B/SCP) is considered to be one of the virulence factors of Streptococcus pyogenes (S. pyogenes) which causes serious diseases such as severe invasive infections and streptococcal toxic shock syndrome (STSS). There are no reports on the histamine releasing activity of SPE B/SCP from mast cells, although several biological activities have been studied. It is not clear whether SPE B/SCP have the superantigenic activity. We studied whether SPE B/SCP plays as a pathogenic factor in streptococcal infections and STSS through a histamine releasing activity. METHODS: Human mast cells and basophils were generated from CD34 positive cells isolated from cord blood and cultured in the presence of rIL-6, stem cell factor and/or rIL-3. The capacity of increasing capillary permeability of recombinant SPE B/SCP (rSPE B/SCP) was studied by using the skin of guinea pigs. Mitogenic activity to human T-cells of rSPE B/SCP was studied by incorporation of (3)Hthymidine. The levels of histamine in the plasma of patients with STSS and controls were measured by ELISA kit. RESULTS: rSPE B/SCP induced increased capillary permeability in the skin of guinea pigs, but both SPE A and SPE C did not exhibit such activity. Histamine was released from cultured human mast cells stimulated with rSPE B/SCP. The rSPE B/SCP did not exhibit mitogenic activity to human T-cells. Three of the 7 patients with STSS showed higher levels of plasma histamine than those of normal subjects. INTERPRETATION & CONCLUSION: The results suggested that increased capillary permeability and histamine release from mast cells induced by rSPE B/SCP might be involved in STSS and/or streptococcal infection of skin and mucous membrane.

Frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae in Tokyo in 2002.

The frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae obtained in Tokyo in 2002 was studied. The frequencies of GyrA and ParC mutations in these 19 isolates were 100% (19 of 19) and 84.2% (16 of 19), respectively, and these results were 1.48-fold (100%/67.6%) and 3.58-fold (84.2%/23.5%) higher, respectively, than the frequencies reported in 1998 in 68 isolates obtained in Fukuoka during the period from 1992 to 1996. Isolates with increasing numbers of mutations were more resistant not only to levofloxacin but also to other antibiotics. The 50% and 90% minimum inhibitory concentrations (MICs) to levofloxacin during the period from 1995 to 1996 were 0.063 and 1 micro g/ml, and they increased to 4 and 8 micro g/ml, respectively, in the present study. All 19 cases of gonoccocal urethritis in the present study were cured with a single intramuscular injection of 2 g spectinomycin.

Genetic polymorphisms of CYP2C8 in Japanese population.

CYP2C8 plays important roles in metabolizing therapeutic drugs and endogenous compounds. Although genetic polymorphisms of CYP2C8 were reported, there is little information on CYP2C8 polymorphisms in the Japanese population. In the present study, we screened for previously described polymorphisms in the coding region of this gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific-PCR analyses. Eleven polymorphisms of CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), CYP2C8*4 (I264M), CYP2C8*5 (frameshift), T130N, E154D, N193K, K249R, L390S, P404A, and H411L have been comprehensively investigated in at least 200 Japanese individuals. A single subject was heterozygous for CYP2C8*5, and the allele frequency was calculated as 0.0025. The other single nucleotide polymorphisms (SNPs) were not found in the Japanese subjects in the present study. Thus, it appears that the frequencies of these alleles in Japanese are extremely low. In addition, concerning the SNPs of T130N, E154D, N193K, K249R, and H411L, it remains clear that these alleles exist as polymorphisms or represent sequence errors or cloning artifacts. Although several SNPs such as CYP2C8*2, CYP2C8*3, CYP2C8*4, and P404A have been reported to reduce the enzymatic activity, pharmacokinetic abnormalities of drugs metabolized by polymorphic CYP2C8 might be rare in Japanese.

Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells.

OBJECTIVE: To clarify the influence of moderate hypothermia on the production of proinflammatory cytokines. DESIGN: Controlled in vitro study. SETTING: Research laboratory. SUBJECTS: Peripheral blood mononuclear cells from healthy adult human subjects. INTERVENTIONS: Stimulation with 1 microg/mL lipopolysaccharide at 33 degrees C and 37 degrees C. MEASUREMENTS: Concentrations of released tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were measured chronologically by enzyme immunoassay. The number of mRNA copies of these cytokines was determined by competitive reverse transcriptase-polymerase chain reaction analysis, and nuclear factor-kappaB activations were assessed by electrophoretic mobility shift assay. MAIN RESULTS: Significant reduction of the released-tumor necrosis factor-alpha concentration was observed 1 and 2 hrs after the stimulation with lipopolysaccharide at 33 degrees C compared with 37 degrees C. The peak release of interleukin-1beta at 33 degrees C was delayed 12 hrs later than that at 37 degrees C. A delayed peak in the release of interleukin-6 also was observed at 33 degrees C. The peaks of cytokines were confirmed at the mRNA expression level by competitive reverse transcriptase-polymerase chain reaction analysis at both temperatures. The peak of the tumor necrosis factor-alpha mRNA expression level was observed at 1 hr after the stimulation at 37 degrees C and 2 hrs after the stimulation at 33 degrees C. In the interleukin-1beta mRNA expression, at 37 degrees C the first peak appeared 1 hr and the second 6 hrs after the stimulation. In contrast, at 33 degrees C, the first peak appeared 2 hrs and the second 12 hrs after the stimulation. Whereas interleukin-6 mRNA expression at 37 degrees C peaked 6 hrs after the stimulation, no definite peak was observed at 33 degrees C and the expression level was approximately half of that at 37 degrees C. The maximum intensity of nuclear factor-kappaB activation at 33 degrees C was delayed by 1.5 hrs compared with that at 37 degrees C. CONCLUSIONS: Moderate hypothermia delays the induction of proinflammatory cytokines in human peripheral blood mononuclear cells.

Cysteine protease activity and histamine release from the human mast cell line HMC-1 stimulated by recombinant streptococcal pyrogenic exotoxin B/streptococcal cysteine protease.

We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.