Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide.

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S. mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S. mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S. mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S. mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S. mitis CSP signal can modulate QS response in S. pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S. mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S. mitis as it inhabits its natural niche.

Human Serum Supplementation Promotes Streptococcus mitis Growth and Induces Specific Transcriptomic Responses.

Streptococcus mitis is a normal member of the human oral microbiota and a leading opportunistic pathogen causing infective endocarditis (IE). Despite the complex interactions between S. mitis and the human host, understanding of S. mitis physiology and its mechanisms of adaptation to host-associated environments is inadequate, especially compared with other IE bacterial pathogens. This study reports the growth-promoting effects of human serum on S. mitis and other pathogenic streptococci, including S. oralis, S. pneumoniae, and S. agalactiae. Using transcriptomic analyses, we identified that, with the addition of human serum, S. mitis downregulates uptake systems for metal ions and sugars, fatty acid biosynthetic genes, and genes involved in stress response and other processes related with growth and replication. S. mitis upregulates uptake systems for amino acids and short peptides in response to human serum. Zinc availability and environmental signals sensed by the induced short peptide binding proteins were not sufficient to confer the growth-promoting effects. More investigation is required to establish the mechanism for growth promotion. Overall, our study contributes to the fundamental understanding of S. mitis physiology under host-associated conditions. IMPORTANCE S. mitis is exposed to human serum components during commensalism in the human mouth and bloodstream pathogenesis. However, the physiological effects of serum components on this bacterium remain unclear. Using transcriptomic analyses, S. mitis biological processes that respond to the presence of human serum were revealed, improving the fundamental understanding of S. mitis physiology in human host conditions.

The c-di-AMP signaling system influences stress tolerance and biofilm formation of Streptococcus mitis.

Streptococcus mitis is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization, S. mitis must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic-di-AMP (c-di-AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic-di-AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in S. mitis, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c-di-AMP. In this study, we utilized S. mitis deletion mutants of cdaA, pde1, and pde2 to analyze the role of c-di-AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δpde1 mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of cdaA increases auto-aggregation of S. mitis but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of pde1 or pde2 reduced the tolerance to ciprofloxacin, and UV radiation and the Δpde1 mutant was more susceptible to Triton X-100, indicating a role for c-di-AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δpde2 mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c-di-AMP signaling in cellular processes important for colonization of the mouth.

Commensal Streptococcus mitis produces two different lipoteichoic acids of type I and type IV.

The opportunistic pathogen Streptococcus mitis possesses, like other members of the Mitis group of viridans streptococci, phosphorylcholine (P-Cho)-containing teichoic acids (TAs) in its cell wall. Bioinformatic analyses predicted the presence of TAs that are almost identical with those identified in the pathogen Streptococcus pneumoniae, but a detailed analysis of S. mitis lipoteichoic acid (LTA) was not performed to date. Here, we determined the structures of LTA from two S. mitis strains, the high-level beta-lactam and multiple antibiotic resistant strain B6 and the penicillin-sensitive strain NCTC10712. In agreement with bioinformatic predictions, we found that the structure of one LTA (type IV) was like pneumococcal LTA, except the exchange of a glucose moiety with a galactose within the repeating units. Further genome comparisons suggested that the majority of S. mitis strains should contain the same type IV LTA as S. pneumoniae, providing a more complete understanding of the biosynthesis of these P-Cho-containing TAs in members of the Mitis group of streptococci. Remarkably, we observed besides type IV LTA, an additional polymer belonging to LTA type I in both investigated S. mitis strains. This LTA consists of ß-galactofuranosyl-(1,3)-diacylglycerol as glycolipid anchor and a poly-glycerol-phosphate chain at the O-6 position of the furanosidic galactose. Hence, these bacteria are capable of synthesizing two different LTA polymers, most likely produced by distinct biosynthesis pathways. Our bioinformatics analysis revealed the prevalence of the LTA synthase LtaS, most probably responsible for the second LTA version (type I), among S. mitis and Streptococcus pseudopneumoniae strains.

Streptococcus pneumoniae, S. mitis, and S. oralis Produce a Phosphatidylglycerol-Dependent, ltaS-Independent Glycerophosphate-Linked Glycolipid.

Lipoteichoic acid (LTA) is a Gram-positive bacterial cell surface polymer that participates in host-microbe interactions. It was previously reported that the major human pathogen Streptococcus pneumoniae and the closely related oral commensals S. mitis and S. oralis produce type IV LTAs. Herein, using liquid chromatography/mass spectrometry-based lipidomic analysis, we found that in addition to type IV LTA biosynthetic precursors, S. mitis, S. oralis, and S. pneumoniae also produce glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a biosynthetic precursor of type I LTA. cdsA and pgsA mutants produce DHDAG but lack (Gro-P)-DHDAG, indicating that the Gro-P moiety is derived from phosphatidylglycerol (PG), whose biosynthesis requires these genes. S. mitis, but not S. pneumoniae or S. oralis, encodes an ortholog of the PG-dependent type I LTA synthase, ltaS By heterologous expression analyses, we confirmed that S. mitisltaS confers poly(Gro-P) synthesis in both Escherichia coli and Staphylococcus aureus and that S. mitisltaS can rescue the growth defect of an S. aureusltaS mutant. However, we do not detect a poly(Gro-P) polymer in S. mitis using an anti-type I LTA antibody. Moreover, Gro-P-linked DHDAG is still synthesized by an S. mitisltaS mutant, demonstrating that S. mitis LtaS does not catalyze Gro-P transfer to DHDAG. Finally, an S. mitisltaS mutant has increased sensitivity to human serum, demonstrating that ltaS confers a beneficial but currently undefined function in S. mitis Overall, our results demonstrate that S. mitis, S. pneumoniae, and S. oralis produce a Gro-P-linked glycolipid via a PG-dependent, ltaS-independent mechanism.IMPORTANCE The cell wall is a critical structural component of bacterial cells that confers important physiological functions. For pathogens, it is a site of host-pathogen interactions. In this work, we analyze the glycolipids synthesized by the mitis group streptococcal species, S. pneumoniae, S. oralis, and S. mitis We find that all produce the glycolipid, glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a precursor for the cell wall polymer type I lipoteichoic acid in other bacteria. We investigate whether the known enzyme for type I LTA synthesis, LtaS, plays a role in synthesizing this molecule in S. mitis Our results indicate that a novel mechanism is responsible. Our results are significant because they identify a novel feature of S. pneumoniae, S. oralis, and S. mitis glycolipid biology.

Differential influence of Streptococcus mitis on host response to metals in reconstructed human skin and oral mucosa.

BACKGROUND: Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties. OBJECTIVE: Determine how commensal Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG). METHODS: RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis for 24 hours. Histology, cytokine secretion, and Toll-like receptors (TLRs) expression were assessed. RESULTS: S. mitis increased interleukin (IL)-6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co-application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis-induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG. CONCLUSION: Co-exposure of S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals.

Phosphatidylcholine Biosynthesis in Mitis Group Streptococci via Host Metabolite Scavenging.

The mitis group streptococci include the major human pathogen Streptococcus pneumoniae and the opportunistic pathogens Streptococcus mitis and Streptococcus oralis, which are human oral cavity colonizers and agents of bacteremia and infective endocarditis in immunocompromised patients. Bacterial membrane lipids play crucial roles in microbe-host interactions; for many pathogens, however, the composition of the membrane is poorly understood. In this study, we characterized the lipidomes of selected species of mitis group streptococci and investigated the mechanistic basis for biosynthesis of the phospholipid phosphatidylcholine (PC). PC is a major lipid in eukaryotic cellular membranes, but it is considered to be comparatively rare in bacterial taxa. Using liquid chromatography-mass spectrometry in conjunction with stable isotope tracing, we determined that mitis group streptococci synthesize PC via a rare host-metabolite-scavenging pathway, the glycerophosphocholine (GPC) pathway, which is largely uncharacterized in bacteria. Our work demonstrates that mitis group streptococci, including S. pneumoniae, remodel their membranes in response to the major human metabolites GPC and lysophosphatidylcholine.IMPORTANCE We lack fundamental information about the composition of the cellular membrane even for the best-studied pathogens of critical significance for human health. The mitis group streptococci are closely linked to humans in health and disease, but their membrane biology is poorly understood. Here, we demonstrate that these streptococci scavenge major human metabolites and use them to synthesize the membrane phospholipid PC. Our work is significant because it identifies a mechanism by which the major human pathogen S. pneumoniae and the primary human oral colonizers S. mitis and S. oralis remodel their membranes in response to host metabolites.

Daptomycin Dose-Ranging Evaluation with Single-Dose versus Multidose Ceftriaxone Combinations against Streptococcus mitis/oralis in an Ex Vivo Simulated Endocarditis Vegetation Model.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of ß-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-ß-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

High-resolution profiles of the Streptococcus mitis CSP signaling pathway reveal core and strain-specific regulated genes.

BACKGROUND: In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321. RESULTS: All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation. CONCLUSIONS: Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.

Diversity of Mosaic pbp2x Families in Penicillin-Resistant Streptococcus pneumoniae from Iran and Romania.

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for ß-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

The epithelial cell response to health and disease associated oral biofilm models.

BACKGROUND AND OBJECTIVE: Different bacteria differentially stimulate epithelial cells. Biofilm composition and viability are likely to influence the epithelial response. In vitro model systems are commonly used to investigate periodontitis-associated bacteria and their interactions with the host; therefore, understanding factors that influence biofilm-cell interactions is essential. The present study aimed to develop in vitro monospecies and multispecies biofilms and investigate the epithelial response to these biofilms. MATERIAL AND METHODS: Bacterial biofilms were cultured in vitro and then either live or methanol-fixed biofilms were co-cultured with epithelial cells. Changes in epithelial cell viability, gene expression and cytokine content of culture supernatants were evaluated. RESULTS: Bacterial viability was better preserved within mixed-species biofilm culture than within single-species biofilm culture. Both mixed- and single-species biofilms stimulated increased expression of mRNA for interleukin 8 (IL8), C-X-C motif chemokine ligand 3 (CXCL3), C-X-C motif chemokine ligand 1 (CXCL1), interleukin 1 (IL1), interleukin 6 (IL6), colony-stimulating factor 2 (CSF2) and tumour necrosis factor (TNF), and the response was greatest in response to mixed-species biofilms. Following co-culture, cytokines detected in the supernatants included IL-8, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, with the greatest release of cytokines found following co-culture with methanol-fixed, mixed-species biofilms. CONCLUSIONS: These data show that epithelial cells generate a distinct cytokine gene- and protein-expression signature in response to live or fixed, single- or multispecies biofilms.

Zinc oxide and titanium dioxide nanoparticles induce oxidative stress, inhibit growth, and attenuate biofilm formation activity of Streptococcus mitis.

Streptococcus mitis from the oral cavity causes endocarditis and other systemic infections. Rising resistance against traditional antibiotics amongst oral bacteria further aggravates the problem. Therefore, antimicrobial and antibiofilm activities of zinc oxide and titanium dioxide nanoparticles (NPs) synthesized and characterized during this study against S. mitis ATCC 6249 and Ora-20 were evaluated in search of alternative antimicrobial agents. ZnO and TiO2-NPs exhibited an average size of 35 and 13 nm, respectively. The IC50 values of ZnO and TiO2-NPs against S. mitis ATCC 6249 were 37 and 77 µg ml(-1), respectively, while the IC50 values against S. mitis Ora-20 isolate were 31 and 53 µg ml(-1), respectively. Live and dead staining, biofilm formation on the surface of polystyrene plates, and extracellular polysaccharide production show the same pattern. Exposure to these nanoparticles also shows an increase (26-83 %) in super oxide dismutase (SOD) activity. Three genes, namely bapA1, sodA, and gtfB like genes from these bacteria were identified and sequenced for quantitative real-time PCR analysis. An increase in sodA gene (1.4- to 2.4-folds) levels and a decrease in gtfB gene (0.5- to 0.9-folds) levels in both bacteria following exposure to ZnO and TiO2-NPs were observed. Results presented in this study verify that ZnO-NPs and TiO2-NPs can control the growth and biofilm formation activities of these strains at very low concentration and hence can be used as alternative antimicrobial agents for oral hygiene.

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.

Involvement of mitochondrial signalling pathway in HGFs/S. mitis coculture response to TEGDMA treatment.

Although triethylene glycol dimethacrylate (TEGDMA), a resin monomer widely used in dental practice, has been shown to have cytotoxic effects on eukaryotic cells, little is known about how the oral environment influences the cytotoxicity of this biomaterial. The aim of this study was to evaluate eukaryotic cell reaction to TEGDMA in terms of the production of reactive oxygen species (ROS), the expression of Bax, the disturbance of mitochondrial membrane potential (MMP), and the occurrence of apoptosis in an in vitro coculture model of human gingival fibroblasts (HGFs) and Streptococcus mitis strain in presence of saliva. We found that S. mitis and saliva reduced the production of ROS (from 2.2 to 1.8 fold), the occurrence of apoptosis (from 11.3 to 4.7%), and the decrease of MMP (from 0.75 to 0.9 fold) induced by TEGDMA treatment. Addition of N-acetylcysteine, a well known antioxidant, improved cell viability in all experimental conditions. The results obtained in this study suggest that the presence of S. mitis and saliva in the periodontal environment could protect cells against TEGDMA toxicity. These results, shedding more light on the biological and molecular events that occur in conjuction with TEGDMA treatment in vitro in a coculture model that mimics the environment of the oral cavity, confirm the key role played by oral bacteria and saliva in preventing toxic events that can occur in vivo in HGFs.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

Profiles of ILY, VLY and Sm-hPAF interaction with human CD59.

BACKGROUND: The molecular features of a new member of the bacterially -derived cytolysin family were examined. In particular, the interactive mechanisms of intermedilysin (ILY), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) with human CD59 (hCD59) were analyzed. MATERIALS AND METHODS: Molecular models of VLY and Sm-hPAF were constructed based on X-ray data of ILY (protein data bank ID=1S3R), and their interactive profiles with hCD59 were examined using molecular simulation. RESULTS: Non-binding (NB) energy between ILY and hCD59 was three orders of magnitude higher than the energy between VLY and hCD59. NB energy between Sm-hPAF and hCD59 was similar to that between VLY and hCD59. CONCLUSION: A hydrogen bond (ILY Arg432-hCD59 Glu76) was observed between ILY and hCD59, and a stronger interaction was formed by flexible adjustment between them.

M-ficolin binds selectively to the capsular polysaccharides of Streptococcus pneumoniae serotypes 19B and 19C and of a Streptococcus mitis strain.

The three human ficolins (H-, L-, and M-ficolins) and mannan-binding lectin are pattern recognition molecules of the innate immune system mediating activation of the lectin pathway of the complement system. These four human proteins bind to some microorganisms and may be involved in the resolution of infections. We investigated binding selectivity by examining the binding of M-ficolin to a panel of more than 100 different streptococcal strains (Streptococcus pneumoniae and Streptococcus mitis), each expressing distinct polysaccharide structures. M-ficolin binding was observed for three strains only: strains of the pneumococcal serotypes 19B and 19C and a single S. mitis strain expressing a similar polysaccharide structure. The bound M-ficolin, in association with MASP-2, mediated the cleavage of complement factor C4. Binding to the bacteria was inhibitable by N-acetylglucosamine, indicating that the interaction with the bacterial surface takes place via the fibrinogen-like domain. The common N-acetylmannosamine residue present in the structures of the four capsular polysaccharides of group 19 is linked via a phosphodiester bond. This residue is apparently not a ligand for M-ficolin, since the lectin binds to two of the group 19 polysaccharides only. M-ficolin bound strongly to serotype 19B and 19C polysaccharides. In contrast to those of serotypes 19A and 19F, serotype 19B and 19C polysaccharides contain an extra N-acetylmannosamine residue linked via glycoside linkage only. Thus, this extra residue seems to be the M-ficolin ligand. In conclusion, we were able to demonstrate specific binding of M-ficolin to some capsular polysaccharides of the opportunistic pathogen S. pneumoniae and of the commensal bacterium S. mitis.

Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

Identification of a functional capsule locus in Streptococcus mitis.

The polysaccharide capsule of Streptococcus pneumoniae is a hallmark for virulence in humans. In its close relative Streptococcus mitis, a common human commensal, analysis of the sequenced genomes of six strains revealed the presence of a putative capsule locus in four of them. We constructed an isogenic S. mitis mutant from the type strain that lacked the 19 open reading frames in the capsule locus (Δcps mutant), using a deletion strategy similar to previous capsule functional studies in S. pneumoniae. Transmission electron microscopy and atomic force microscopy revealed a capsule-like structure in the S. mitis type strain that was absent or reduced in the Δcps mutant. Since S. mitis are predominant oral colonizers of tooth surfaces, we addressed the relevance of the capsule locus for the S. mitis overall surface properties, autoaggregation and biofilm formation. The capsule deletion resulted in a mutant with approximately two-fold increase in hydrophobicity. Binding to the Stains-all cationic dye was reduced by 40%, suggesting a reduction in the overall negative surface charge of the mutant. The mutant exhibited also increased autoaggregation in coaggregation buffer, and up to six-fold increase in biofilm levels. The results suggested that the capsule locus is associated with production of a capsule-like structure in S. mitis and indicated that the S. mitis capsule-like structure may confer surface attributes similar to those associated with the capsule in S. pneumoniae.