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Polyphosphate (polyP) is a food additive with antimicrobial activity. Here we evaluated the effects of sodium tripolyphosphate (polyP3, Na 5 P 3 O 10 ) on four major oral bacterial species, in both single- and mixed-culture. PolyP3 inhibited three opportunistic pathogenic species: Fusobacterium nucleatum, Prevotella intermedia , and Porphyromonas gingivalis . On the contrary, a commensal bacterium Streptococcus gordonii was relatively less susceptible to polyP3 than the pathogens. When all bacterial species were co-cultured, polyP3 (≥ 0.09%) significantly reduced their total growth and biofilm formation, among which the three pathogenic bacteria were selectively inhibited. Collectively, polyP3 may be an alternative antibacterial agent to control oral pathogenic bacteria., Polyphosphate (polyP) is a food additive with antimicrobial activity. Here we evaluated the effects of sodium tripolyphosphate (polyP3, Na 5 P 3 O 10 ) on four major oral bacterial species, in both single- and mixed-culture. PolyP3 inhibited three opportunistic pathogenic species: Fusobacterium nucleatum, Prevotella intermedia , and Porphyromonas gingivalis . On the contrary, a commensal bacterium Streptococcus gordonii was relatively less susceptible to polyP3 than the pathogens. When all bacterial species were co-cultured, polyP3 (≥ 0.09%) significantly reduced their total growth and biofilm formation, among which the three pathogenic bacteria were selectively inhibited. Collectively, polyP3 may be an alternative antibacterial agent to control oral pathogenic bacteria.

Background: Polyphosphate (polyP) has bactericidal activity against a gram-negative periodontopathogen Porphyromonas gingivalis, a black-pigmented gram-negative anaerobic rod. However, current knowledge about the mode of action of polyP against P. gingivalis is incomplete. To elucidate the mechanisms of antibacterial action of polyP against P. gingivalis, we performed the full-genome gene expression microarrays, and gene ontology (GO) and protein-protein interaction network analysis of differentially expressed genes (DEGs)., Results: We successfully identified 349 up-regulated genes and 357 down-regulated genes (>1.5-fold, P < 0.05) in P. gingivalis W83 treated with polyP75 (sodium polyphosphate, Na(n+2)P(n)O3(n+1); n = 75). Real-time PCR confirmed the up- and down-regulation of some selected genes. GO analysis of the DEGs identified distinct biological themes. Using 202 DEGs belonging to the biological themes, we generated the protein-protein interaction network based on a database of known and predicted protein interactions. The network analysis identified biological meaningful clusters related to hemin acquisition, energy metabolism, cell envelope and cell division, ribosomal proteins, and transposon function., Conclusions: polyP probably exerts its antibacterial effect through inhibition of hemin acquisition by the bacterium, resulting in severe perturbation of energy metabolism, cell envelope biosynthesis and cell division, and elevated transposition. Further studies will be needed to elucidate the exact mechanism by which polyP induces up-regulation of the genes related to ribosomal proteins. Our results will shed new light on the study of the antibacterial mechanism of polyP against other related bacteria belonging to the black-pigmented Bacteroides species.

Strains of Porphyromonas gingivalis, a periodontopathic bacterium, are classified into six genotypic variants based on nucleotide sequence differences in the fimA gene encoding FimA. A PCR assay using primer sets specific for each genotype has demonstrated that the most predominant fimA genotype in periodontitis patients is type II, which is now commonly referred to as the periodontitis-associated fimA genotype of P. gingivalis. However, the potential for false type II fimA positives caused by cross-hybridization of type II fimA-specific primers with type Ib fimA has complicated the genotyping. A previous study developed new primers that specifically amplified only the DNA fragment of type II fimA. The aim of the present study was to assess the prevalence of P. gingivalis fimA genotypes in Korean adults and to reconfirm the relationship between type II fimA and periodontitis using the new primers. Among 412 Korean adults, P. gingivalis was detected in 97.5 % of patients and 57.8 % of healthy subjects. Type II fimA was the most widely distributed type among healthy and periodontitis subjects. Organisms with types II, Ib and IV fimA had a significant frequency of occurrence in periodontitis subjects. Statistical analysis, however, revealed that a more significant correlation was found between periodontitis and the occurrence of type Ib fimA.

It has been reported that a large majority of periodontitis patients carry organisms with either type II or IV-fimA, while type I is the most prevalent fimA genotype among Porphyromonas gingivalis-positive healthy adults. Here we report characterization of recombinant fimbrial protein (rFimA) produced in Escherichia coli from genotype IV-fimA. In SDS-PAGE and immunoblot analysis after partial dissociation, type IV-rFimA showed a ladder-like pattern representing oligomeric/polymeric forms of native fimbrial structure. Unlike anti-type I-native fimbriae which can only recognize conformational epitopes of the respective proteins, both anti-type IV-native fimbriae and anti-type IV-rFimA antibodies recognized conformational as well as linear epitopes in type IV-fimbriae. These results suggest that the type IV-rFimA proteins retain the native fimbrial antigenicity and the antigenicity of type IV-fimbriae is different from that of type I-fimbriae., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

For more accurate PCR-based identification of Porphyromonas gingivalis harboring genotype II fimA, the most prevalent type in periodontitis patients, a new primer set was developed and evaluated. The previous type II primers hybridized to the DNA of P gingivalis strains harboring type Ib as well as type II fimA, while the new primers specifically amplified only the DNA fragment of type II fimA. In the investigation using mixed bacterial culture and 155 clinical samples from peri-implantitis patients, the new primers increased the accuracy of PCR-based detection of type II fimA by excluding false-negatives as well as false-positives.

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis., (© 2011 The Societies and Blackwell Publishing Asia Pty Ltd.)

Polyphosphate [poly(P)] has antibacterial activity against various Gram-positive bacteria. In contrast, Gram-negative bacteria are generally resistant to poly(P). Here, we describe the antibacterial characterization of poly(P) against a Gram-negative periodontopathogen, Porphyromonas gingivalis. The MICs of pyrophosphate (Na(4)P(2)O(7)) and all poly(P) (Na(n + 2)P(n)O(3n + 1); n = 3 to 75) tested for the bacterium by the agar dilution method were 0.24% and 0.06%, respectively. Orthophosphate (Na(2)HPO(4)) failed to inhibit bacterial growth. Poly-P75 was chosen for further study. In liquid medium, 0.03% poly-P75 was bactericidal against P. gingivalis irrespective of the growth phase and inoculum size, ranging from 10(5) to 10(9) cells/ml. UV-visible spectra of the pigments from P. gingivalis grown on blood agar with or without poly-P75 showed that poly-P75 reduced the formation of μ-oxo bisheme by the bacterium. Poly-P75 increased hemin accumulation on the P. gingivalis surface and decreased energy-driven uptake of hemin by the bacterium. The expression of the genes encoding hemagglutinins, gingipains, hemin uptake loci, chromosome replication, and energy production was downregulated, while that of the genes related to iron storage and oxidative stress was upregulated by poly-P75. The transmission electron microscope showed morphologically atypical cells with electron-dense granules and condensed nucleoid in the cytoplasm. Collectively, poly(P) is bactericidal against P. gingivalis, in which hemin/heme utilization is disturbed and oxidative stress is increased by poly(P).

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato (Solanum tuberosum) cells and the ctb-fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21 kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB-FimA (266-337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB-FimA fusion protein was confirmed by ELISA measurements of GM1 ganglioside receptor binding. Quantification of the CTB-FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB-FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease.

It has been reported that a large majority of periodontitis patients carry organisms with either type II or IV- fim A, while type I is the most prevalent fim A genotype among Porphyromonas gingivalis-positive healthy adults. Here we report characterization of recombinant fimbrial protein (r Fim A) produced in Escherichia coli from genotype IV- fim A. In SDS- PAGE and immunoblot analysis after partial dissociation, type IV-r Fim A showed a ladder-like pattern representing oligomeric/polymeric forms of native fimbrial structure. Unlike anti-type I-native fimbriae which can only recognize conformational epitopes of the respective proteins, both anti-type IV-native fimbriae and anti-type IV-r Fim A antibodies recognized conformational as well as linear epitopes in type IV-fimbriae. These results suggest that the type IV-r Fim A proteins retain the native fimbrial antigenicity and the antigenicity of type IV-fimbriae is different from that of type I-fimbriae. [ABSTRACT FROM AUTHOR]