Enterococcus faecalis lipoteichoic acid regulates macrophages autophagy via PI3K/Akt/mTOR pathway.

Enterococcus faecalis (E. faecalis) infection is considered an important etiological factor for the development of persistent apical periodontitis (PAP), but the exact mechanisms of autophagy between E. faecalis and immune cells remain unknown. In this study, we elucidated how E. faecalis lipoteichoic acid (LTA) is associated with macrophages autophagy. We found that E. faecalis LTA apparently activated macrophage autophagy with significant increase of autophagosomes and autophagy relative protein. Meanwhile, we noticed significantly decreasing expression of p-Akt and p-mTOR. However, these effect were absent in macrophages knockdown of Beclin1. In summary, these findings suggested E. faecalis LTA may increased macrophages autophagy via inhibiting PI3K/Akt/mTOR pathway and this process was Beclin1 dependent.

Phthalate Esters in Indoor Window Films in a Northeastern Chinese Urban Center: Film Growth and Implications for Human Exposure.

Indoor window film samples were collected in buildings during 2014-2015 for the determination of six phthalate diesters (PAEs). Linear regression analysis suggested that the film mass was positively and significantly correlated with the duration of film growth (from 7 to 77 days). PAEs were detected in all window film samples (n = 64). For all the samples with growth days ranged from 7 to 77 days, the median concentrations of total six PAEs (∑6PAEs) in winter and summer window film samples were 9900 ng/m(2) film (2000 µg/g film) and 4700 ng/m(2) film (650 µg/g film), respectively. Among PAEs analyzed, di-2-ethyl-hexyl phthalate (DEHP) was the major compound (71 ± 9.7%), followed by di-n-butyl phthalate (DBP; 20 ± 7.4%) and diisobutyl phthalate (DiBP; 5.1 ± 2.2%). Positive correlations among PAEs suggested their common sources in the window film samples. Room temperature and relative humidity were negatively and significantly correlated with PAEs concentations (in ng/m(2)). Poor ventilation in cold winter in Noreastern China significantly influenced the concentrations of PAEs in window film which suggested higher inhalation exposure dose in winter. The median hazard quotient (HQ) values from PAEs exposure were below 1, suggesting that the intake of PAEs via three exposure pathways was considered as acceptable.

Gap2 promotes the formation of a stable protein complex required for mature Fap1 biogenesis.

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins conserved in streptococci and staphylococci. Fap1, a SRRP identified in Streptococcus parasanguinis, is the major constituent of bacterial fimbriae and is required for adhesion and biofilm formation. An 11-gene cluster is required for Fap1 glycosylation and secretion; however, the exact mechanism of Fap1 biogenesis remains a mystery. Two glycosylation-associated proteins within this cluster--Gap1 and Gap3--function together in Fap1 biogenesis. Here we report the role of the third glycosylation-associated protein, Gap2. A gap2 mutant exhibited the same phenotype as the gap1 and gap3 mutants in terms of Fap1 biogenesis, fimbrial assembly, and bacterial adhesion, suggesting that the three proteins interact. Indeed, all three proteins interacted with each other independently and together to form a stable protein complex. Mechanistically, Gap2 protected Gap3 from degradation by ClpP protease, and Gap2 required the presence of Gap1 for expression at the wild-type level. Gap2 augmented the function of Gap1 in stabilizing Gap3; this function was conserved in Gap homologs from Streptococcus agalactiae. Our studies demonstrate that the three Gap proteins work in concert in Fap1 biogenesis and reveal a new function of Gap2. This insight will help us elucidate the molecular mechanism of SRRP biogenesis in this bacterium and in pathogenic species.

Structural insights into serine-rich fimbriae from Gram-positive bacteria.

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

Stable surface expression of a gene for Helicobacter pylori toxic porin protein with pBAD expression system.

Helicobacter pylori (H. pylori) infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among H. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express HopE protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

A conserved C-terminal 13-amino-acid motif of Gap1 is required for Gap1 function and necessary for the biogenesis of a serine-rich glycoprotein of Streptococcus parasanguinis.

Adhesion of Streptococcus parasanguinis to saliva-coated hydroxyapatite (SHA), an in vitro tooth model, is mediated by long peritrichous fimbriae. Fap1, a fimbria-associated serine-rich glycoprotein, is required for fimbrial assembly. Biogenesis of Fap1 is controlled by an 11-gene cluster that contains gly, nss, galT1 and -2, secY2, gap1 to -3, secA2, and gtf1 and -2. We had previously isolated a collection of nine nonadherent mutants using random chemical mutagenesis approaches. These mutants fail to adhere to the in vitro tooth model and to form fimbriae. In this report, we further characterized these randomly selected nonadherent mutants and classified them into three distinct groups. Two groups of genes were previously implicated in Fap1 biogenesis. One group has a mutation in a glycosyltransferase gene, gtf1, that is essential for the first step of Fap1 glycosylation, whereas the other group has defects in the fap1 structural gene. The third group mutant produces an incompletely glycosylated Fap1 and exhibits a mutant phenotype similar to that of a glycosylation-associated protein 1 (Gap1) mutant. Analysis of this new mutant revealed that a conserved C-terminal 13-amino-acid motif was missing in Gap1. Site-directed mutagenesis of a highly conserved amino acid tryptophan within this motif recapitulated the deletion phenotype, demonstrating the importance of the Gap1 C-terminal motif for Fap1 biogenesis. Furthermore, the C-terminal mutation does not affect Gap1-Gap3 protein-protein interaction, which has been shown to mediate Fap1 glycosylation, suggesting the C-terminal motif has a distinct function related to Fap1 biogenesis.

Identification of critical residues in Gap3 of Streptococcus parasanguinis involved in Fap1 glycosylation, fimbrial formation and in vitro adhesion.

BACKGROUND: Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation. RESULTS: Deletion and site-directed mutagenesis were employed to dissect the regions within Gap3 that were important for its function in Fap1 glycosylation and biogenesis. A deletion of 6 consecutive amino acids, PDLPIL, eliminated the production of the mature 200 kDa Fap1 protein and gave rise instead to a 470 kDa Fap1 intermediate that was only partially glycosylated. Site-directed mutagenesis of the 6 amino acids revealed that only three of these amino acids were required. Mutants in these amino acids (L64R, P65R and L67T) produced the premature 470 kDa Fap1 intermediate. Mutants in the remaining amino acids produced the mature form of Fap1. Cell surface expression of the Fap1 precursor among L64R, P65R and L67T mutants was reduced to levels consistent with that of a gap3 insertional mutant. Electron micrographs showed that these 3 mutants lost their long peritrichous fimbriae. Furthermore, their in vitro adhesion ability to saliva-coated hydroxylapatite (SHA) was inhibited. CONCLUSION: Our data suggest that 3 highly conserved, hydrophobic residues L64, P65 and L67 in Gap3 are essential for Gap3 function and are important for complete glycosylation of Fap1, fimbrial formation and bacterial adhesion.

SecA2 is distinct from SecA in immunogenic specificity, subcellular distribution and requirement for membrane anchoring in Streptococcus parasanguis.

A secA2 gene is present in the genomes of a wide variety of Gram-positive bacteria. In Streptococcus parasanguis, a primary colonizer of the tooth surface, secA2 is involved in the secretion of a small group of proteins including the fimbrial adhesin, Fap1. Although the substrate specificity is different, SecA2 is predicted to be similar to SecA in structure and function based on the homology between these two proteins. In this study, polyclonal antibodies against SecA2 and SecA did not cross-react with each other, indicating that these two proteins possessed distinct immunogenic epitopes. Fractionation analysis demonstrated that SecA2 was not evenly distributed between the cytoplasmic membrane and the cytoplasm as was noted for SecA. SecA2 was associated with the membrane in the wild type and in secA2 mutants with different regions deleted. The subcellular distribution of SecA2 was not dependent on secY2, suggesting that the membrane association is not through SecY2. These data suggested that SecA2 is distinct from SecA in many respects such as substrate specificity, immunogenic specificity, subcellular distribution and requirement for membrane anchoring.

Correlation between Enterococcus faecalis and Persistent Intraradicular Infection Compared with Primary Intraradicular Infection: A Systematic Review.

INTRODUCTION: The relationship between Enterococcus faecalis and pulpal or periradicular diseases has been studied for many years; however, whether E. faecalis is correlated with persistent intraradicular infections (teeth after failed endodontic treatments) compared with primary intraradicular infections remains controversial. The objective of this systematic review was to compare the prevalence of E. faecalis in primary and persistent intraradicular infections. METHODS: An exhaustive literature search combined with specified inclusion criteria was performed to collect all studies comparing the prevalence of E. faecalis in root canals with primary and persistent intraradicular infections. Descriptive statistics were applied first because of the high heterogeneity among studies. Subgroup analysis according to different detecting methods (culture and polymerase chain reaction) and sensitivity analysis was then applied. Meta-analysis was conducted with the help of Stata/SE 12.0 (StataCorp, College Station, TX) after excluding studies with uncertain forms of pulpal and periradicular lesions in their primary infection groups. RESULTS: The systematic review included 10 studies covering 972 teeth. Among them, 2 studies used the culture technique, 6 studies used polymerase chain reaction, and the other 2 used both techniques. The detection rate of E. faecalis by both methods was higher in persistent infections compared with untreated chronic periapical periodontitis as primary infections. The difference was statistically significant (odds ratio = 7.247; 95% confidence interval, 4.039-13.002). CONCLUSIONS: E. faecalis is more highly correlated with persistent intraradicular infections compared with untreated chronic periapical periodontitis.

Effect of ZnCl2 on plaque growth and biofilm vitality.

OBJECTIVE: The aim of this study was to evaluate the effects of ZnCl(2) on plaque-growth and vitality pattern of dental biofilm and to determine the optimum zinc concentration for the inhibition of plaque formation. DESIGN: Data were collected from nine volunteers for whom a special-designed acrylic appliance was prescribed after a careful dental check up. The volunteers rinsed twice daily for 2min with ZnCl(2) of 2.5, 5, 10, 20mM as treatment and double distilled water (DDW) as control in respective assigned test weeks. The plaque index (PI) was assessed after 48h of appliance wearing. The glass discs with the adhered biofilm were removed from the splints and stained with two fluorescent dyes. The biofilm thickness (BT) and bacterial vitality of the whole biofilm as well as the mean bacterial vitality (BV) of the inner, middle and outer layers of biofilm were evaluated under confocal laser scanning microscope (CLSM). RESULTS: PI, BT and BV of biofilms treated by various concentrations of ZnCl(2) were reduced significantly when compared with the DDW group (p<0.05). PI, BT and BV of the 2.5mM ZnCl(2) group was significantly higher than groups of 5, 10, 20mM ZnCl(2) (p<0.05). The mean BV of the 3 layers (inner, middle and outer layers) showed that 2.5mM ZnCl(2) was the lowest concentration to inhibit BV in the outer layer, 5mM was the lowest concentration to extend this inhibition of BV to the middle layer, and none of the concentrations investigated in this study has shown any effect on bacteria inhibition in the inner layer. CONCLUSION: Zinc ions exhibited possible inhibitory effects on plaque formation, and have a promising potential to be used as an antibacterial agent in future dentifrices and mouthrinses.

Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5 against Streptococcus mutans.

OBJECTIVE: To investigate the killing effect of two salivary antimicrobial peptides, hLF1-11 and P-113, and identify the antibacterial mechanism of the peptides. METHODS: The antimicrobial activities of hLF1-11 and P-113 against oral Streptococci strains were determined using the broth microdilution method. The effects of hLF1-11 and P-113 on the bacterial plasma membrane were visualized by scanning electron microscopy. Cell membrane permeability was monitored using the intracellular dye calcein. The subcellular localization of hLF1-11 and P-113 in bacteria was measured by fluorescence light microscopy. An electrophoretic mobility shift assay (EMSA) was performed to evaluate the DNA binding capabilities of hLF1-11, P-113 and MUC7 12-mer. RESULTS: Both hLF1-11 and P-113 exerted potent bactericidal activities against all selected oral Streptococci. Streptococcus mutans UA 159 was the most susceptible of the oral bacterial species tested to the antimicrobial effects of the three peptides. The cell membranes of bacteria treated with hLF1-11 or P-113 were still intact after 30 min. hLF1-11 and P-113 could penetrate the bacterial cell membranes and accumulate in the cytoplasm in S. mutans. Both hLF1-11 and P-113 showed DNA binding affinity. CONCLUSIONS: Together, our results demonstrate that hLF1-11 and P-113 display antibacterial activity against dental cavity-inducing S. mutans through an intracellular mechanism that could involve DNA binding. Thus, these peptides might be attractive and valuable candidates for development into effective antimicrobial therapies to combat dental caries.

Differential roles of individual domains in selection of secretion route of a Streptococcus parasanguinis serine-rich adhesin, Fap1.

Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.

[DNA vaccine encoding Streptococcus mutans surface protein protected gnotobiotic rats from caries].

OBJECTIVE: Protein of Streptococcus mutans is considered as one of the virulence factors due to its ability to mediate the initial attachment of Streptococcus mutans to tooth surface. In this study, an anticaries DNA vaccine pCIA-P was used to immunize rats. The expression of PAc in different tissues in vivo, specific immune response and protection effects against dental caries were observed. METHODS: Plasmid pCIA-P was injected into rats by two different routs: intramuscular injection (i.m.) and targeted salivary gland immunization (TSG). Immunohistochemistry technique was used to detect the expression of PAc. Gnotobiotic rats were vaccinated with pCIA-P by three different approaches: TSG, intramuscular injection and buccal mucosal injection (i.o.). The specific immune responses were evaluated by ELISA and their anticaries effects were evaluated by Keyes caries scores. RESULTS: PAc was expressed in the sarcoplasm and sarcolemma of muscle fibers and submandibular glands, especially strongly positive in duct regions. The levels of serum specific anti-PAc IgG and salivary specific anti-PAc IgA in TSG immunization and buccal mucosal immunization group were significantly higher than those of other groups. The Keyes caries scores of those two groups were significantly lower than those of other groups. CONCLUSION: The plasmid pCIA-P could provoke specific immune responses as a novel immunogen. Mucosal immunization with pCIA-P appears to be an effective genetic immunization method against dental caries.