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.

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.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND: The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS: Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS: AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS: The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

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.

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.

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.

A simple, semiselective medium for anaerobic isolation of anginosus group streptococci from patients with chronic lung disease.

The anaerobic isolation of anginosus group streptococci (AGS) from respiratory specimens containing diverse microbiota using a semiselective blood agar medium incorporating nalidixic acid and sulfamethazine (NAS) is described. AGS were detected in 60% of tested sputa from patients with cystic fibrosis, chronic obstructive pulmonary disease, and bronchiectasis. This demonstrates NAS to be a diagnostic tool for detecting AGS within the complex microbial communities associated with chronic lung disorders.

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models.

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1ß, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1ß and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.

Synthesis, Drug Release, and Antibacterial Properties of Novel Dendritic CHX-SrCl2 and CHX-ZnCl2 Particles.

This work demonstrated for the first time the synthesis of novel chlorhexidine particles containing strontium and zinc, to provide an effective, affordable, and safe intervention in the treatment of recurrent infections found in Medicine and Dentistry. The CHX-SrCl2 and CHX-ZnCl2 particles were synthesized by co-precipitation of chlorhexidine diacetate (CHXD) and zinc chloride or strontium chloride, where particle size was manipulated by controlling processing time and temperature. The CHX-ZnCl2 and CHX-SrCl2 particles were characterized using SEM, FTIR, and XRD. UV-Vis using artificial saliva (pH 4 and pH 7) was used to measure the drug release and ICP-OES ion release. The antibacterial properties were examined against P. gingivalis, A. actinomycetemcomitans, and F. nucleatum subsp. Polymorphum, and cytotoxicity was evaluated using mouse fibroblast L929 cells. The novel particles were as safe as commercial CHXD, with antibacterial activity against a range of oral pathogens. UV-Vis results run in artificial saliva (pH 4 and pH 7) indicated a higher release rate in acidic rather than neutral conditions. The CHX-ZnCl2 particles provided the functionality of a smart Zinc and CHX release, with respect to environmental pH, allowing responsive antibacterial applications in the field of medicine and dentistry.

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.

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.

Controlled release of chlorhexidine from a HEMA-UDMA resin using a magnetic field.

OBJECTIVES: To functionalize novel chlorhexidine (CHX) particles with iron oxide (Fe3O4) nanoparticles and control their release kinetics in a dental resin using an external magnetic field. METHODS: Fe3O4 nanoparticles were synthesized and incorporated into spherical CHX particles and the powder was freeze dried. Resin disc specimens were produced using a UDMA-HEMA resin mixed with freeze dried spherical Fe3O4-CHX particles (5wt.%), which were placed into a Teflon mould (10mm diameter×1mm depth) and covered with a Mylar strip. A MACS magnet was left in contact for 0min (Group 1), 5min (Group 2) or 10min (Group 3) and the resin discs subsequently light cured (Bluedent LED pen, Bulgaria) for 60s per side. The resin discs were immersed in deionized water at various time points up to 650h. UV-Vis absorbance was used to determine the CHX content. CHX released for each time point was determined. The functionalized CHX particles and resin discs were characterized using TEM, TGA, EDX and SEM. RESULTS: Fe3O4 nanoparticles (20nm) incorporated into the spherical CHX particles led to a mean (SD) particle size reduction from 17.15 (1.99)µm to 10.39 (2.61)µm. The presence of Fe3O4 nanoparticles in the spherical CHX particles was confirmed with SEM, EDX, and TGA. SEM of Group 1 resin discs (no magnetic exposure) showed functionalized CHX spheres were homogeneously distributed within the resin discs. For resin discs which had magnetic exposure (5 or 10min) the particles started to cluster nearer the surface (Group 2: 43.7%, Group 3: 57.3%), to a depth of 94µm. UV-Vis absorbance revealed Group 1 resin discs had a cumulative CHX release of 4.4% compared to 5.9% for Group 2 and 7.4% for Group 3 resin discs, which had magnetic exposure (5, 10min). SIGNIFICANCE: Fe3O4 nanoparticle functionalized CHX spheres demonstrated a magnetic field responsive property. A magnetic field responsive release of CHX may be useful in clinical situations where the drug can be directed to give a tailored release at the site of infection.

Correction: Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

[This corrects the article DOI: 10.1371/journal.pone.0173741.].

Modulation of behaviour and virulence of a high alginate expressing Pseudomonas aeruginosa strain from cystic fibrosis by oral commensal bacterium Streptococcus anginosus.

Cystic fibrosis (CF) airways harbour complex and dynamic polymicrobial communities that include many oral bacteria. Despite increased knowledge of CF airway microbiomes the interaction between established CF pathogens and other resident microbes and resulting impact on disease progression is poorly understood. Previous studies have demonstrated that oral commensal streptococci of the Anginosus group (AGS) can establish chronic pulmonary infections and become numerically dominant in CF sputa indicating that they play an important role in CF microbiome dynamics. In this study a strain of Pseudomonas aeruginosa (DWW2) of the mucoid alginate overproducing phenotype associated with chronic CF airway infection and a strain of the oral commensal AGS species Streptococcus anginosus (3a) from CF sputum were investigated for their ability to co-exist and their responses to biofilm co-culture. Bacteria in biofilms were quantified, pyocyanin expression by DWW2 was measured and the effect of AGS strain 3a on reversion of DWW2 to a non-mucoidal phenotype investigated. The virulence of DWW2, 3a and colony variant phenotypes of DWW2 in mono- and co-culture were compared in a Galleria mellonella infection model. Co-culture biofilms were formed in normoxic, hypercapnic (10% CO2) and anoxic atmospheres with the streptococcus increasing in number in co-culture, indicating that these bacteria would be able to co-exist and thrive within the heterogeneous microenvironments of the CF airway. The streptococcus caused increased pyocyanin expression by DWW2 and colony variants by stimulating reversion of the mucoid phenotype to the high pyocyanin expressing non-mucoid phenotype. The latter was highly virulent in the infection model with greater virulence when in co-culture with the streptococcus. The results of this study demonstrate that the oral commensal S. anginosus benefits from interaction with P. aeruginosa of the CF associated mucoid phenotype and modulates the behaviour of the pseudomonad in ways that may be clinically relevant.

Environment and colonisation sequence are key parameters driving cooperation and competition between Pseudomonas aeruginosa cystic fibrosis strains and oral commensal streptococci.

Cystic fibrosis (CF) patient airways harbour diverse microbial consortia that, in addition to the recognized principal pathogen Pseudomonas aeruginosa, include other bacteria commonly regarded as commensals. The latter include the oral (viridans) streptococci, which recent evidence indicates play an active role during infection of this environmentally diverse niche. As the interactions between inhabitants of the CF airway can potentially alter disease progression, it is important to identify key cooperators/competitors and environmental influences if therapeutic intervention is to be improved and pulmonary decline arrested. Importantly, we recently showed that virulence of the P. aeruginosa Liverpool Epidemic Strain (LES) could be potentiated by the Anginosus-group of streptococci (AGS). In the present study we explored the relationships between other viridans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus sanguinis) and the LES and observed that co-culture outcome was dependent upon inoculation sequence and environment. All four streptococcal species were shown to potentiate LES virulence factor production in co-culture biofilms. However, in the case of S. oralis interactions were environmentally determined; in air cooperation within a high cell density co-culture biofilm occurred together with stimulation of LES virulence factor production, while in an atmosphere containing added CO2 this species became a competitor antagonising LES growth through hydrogen peroxide (H2O2) production, significantly altering biofilm population dynamics and appearance. Streptococcus mitis, S. gordonii and S. sanguinis were also capable of H2O2 mediated inhibition of P. aeruginosa growth, but this was only visible when inoculated as a primary coloniser prior to introduction of the LES. Therefore, these observations, which are made in conditions relevant to the biology of CF disease pathogenesis, show that the pathogenic and colonisation potential of P. aeruginosa isolates can be modulated positively and negatively by the presence of oral commensal streptococci.

ARDRA and RAPD analyses of human and animal isolates of Streptococcus gallolyticus.

A total of 23 Streptococcus gallolyticus strains, consisting of 12 strains from feces of healthy animals and 11 from clinical cases of human or cow mastitis milk, were examined genealogically. Four strains of S. bovis "biotype II/1" and 3 strains of S. equinus, the closely related organisms to S. gallolyticus, were also analyzed for outgroup comparison. Neither the amplified ribosomal DNA restriction analysis (ARDRA) nor the randomly amplified polymorphic DNA (RAPD) analysis that had been designed to recognize S. gallolyticus strains virulent in pigeons could differentiate clinical strains from the others of S. gallolyticus. No correspondence between the DNA profile in either analysis and the host animal species was detected.

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 ß-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 ß-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of ß-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.

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.