Outer membrane proteome of Prevotella intermedia 17: identification of thioredoxin and iron-repressible hemin uptake loci

Although hemin is an indispensable nutrient for the oral pathogen Prevotella intermedia, not much is known regarding the molecular mechanisms of hemin acquisition. The availability of the genomic sequence of the bacterium allowed us to apply proteomic approaches to identify proteins that may be mediating the hemin acquisition process. As hemin acquisition mechanisms have been shown to be induced in iron-depleted conditions, we applied proteomic approaches to detect those proteins whose expressions were affected by iron. We analyzed 40 protein spots and identified 19 such proteins. Interestingly, two proteins drastically upregulated in iron-depleted conditions, PIN0009 and PINA0611, are homologs of hemin uptake receptors in other bacteria. PIN0009 is predicted to be an outer membrane lipoprotein. It is encoded by a gene that is the first of a seven-gene genomic locus encoding proteins of a novel hemin acquisition system. The second protein, PINA0611, is a homolog of numerous TonB-dependent outer membrane receptors including outer membrane iron uptake receptors of various Gram-negative bacteria. There was also another protein, regulated by iron, that was previously demonstrated to bind hemoglobin in P. intermedia. Finally, we identified a thioredoxin-like protein that has a novel outer membrane location.     

Hemolytic and hemagglutinating activities of Prevotella intermedia and Prevotella nigrescens

A total of 91 isolates of Prevotella intermedia or Prevotella nigrescens from subgingival sites were identified by PCR using primers specific for sequences of 16S rRNA. The hemolytic and hemagglutinating activities of the P. intermedia isolates exhibited significantly higher levels compared to those of the P. nigrescens isolates by quantitative analysis. The hemagglutinin gene (phg) was found in 23 of 26 P. intermedia isolates (88.5%), whereas it was found in only two of 44 isolates (4.5%) of P. nigrescens. The high hemolytic and hemagglutinating activities of P. intermedia may be involved in the pathogenicity of P. intermedia in the progression of periodontal disease.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens

Abstract Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens . These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.     

Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia

The hemolysin from Prevotella intermedia was partially purified from culture supernatant and then characterized. The hemolysin produced a clear beta-hemolytic zone on a blood agar plate. Hemolytic activity was 2.5-fold greater in culture supernatant compared to that cell-associated. The isolation and purification procedure involved ammonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The activity of this hemolysin was stimulated by reductants such as cysteine, dithiothreitol, glutathione etc., and was lost upon oxidation. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Ca(2+), Mg(2+) and EDTA did not affect the activity. The optimal pH of this hemolysin was 7.5.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

The influence of molecular oxygen exposure on the biology of Prevotella intermedia, with emphasis on its antibiotic susceptibility

AIM: This study focuses on investigating the molecular and physiological characteristics of Prevotella intermedia after molecular oxygen exposure (MOE) and the effect on drug susceptibility patterns. METHODS AND RESULTS: Samples of P. intermedia were used as parent strains: ATCC25611 and four clinical isolates. Strains adapted to oxidative stress by MOS were obtained by the enrichment technique. Drug susceptibility was evaluated by minimal inhibitory concentrations (MIC) using agar dilution. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to evaluate the genetic diversity of all strains and physiological analyses were made by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of crude, cell-free extracts. The genetic profile showed that lineages with altered MIC values were selected after MOE. Overall, we found significant decrease in drug susceptibility for the aero-strains against all tested antimicrobials (amoxicillin, amoxicillin+clavulanic acid, clindamycin, chloramphenicol, ertapenen and metronidazole). We also observed markedly different protein expression patterns between the parent and selected aero-strains. CONCLUSIONS: MOE induces changes in the genetic profile and protein expression patterns of P. intermedia that may also be linked to its drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of MOE on anaerobic bacterial physiology and behaviour may influence antimicrobial susceptibility patterns with potential consequences to antimicrobial chemotherapy.     

A beta-lactamase belonging to group 2e from oral clinical isolates of Prevotella intermedia

A beta-lactamase in oral clinical isolates of Prevotella intermedia that hydrolyzed cefuroxime and cephalothin with rates of 600 and 53.3 respectively, relative to that for cephaloridine (100), was characterized as a 2e-cephalosporinase. Inhibition was observed by clavulanic acid (IC50 0.72 microM), tazobactam (IC50 0.21 microM) and sulbactam (IC50 0.07 microM) and was not inhibited by cloxacillin, EDTA, NaCl or p-CMB. The pI and pH optima were 4.7 and 5.4, respectively.     

Degradation of human hemoglobin by Prevotella intermedia

In this study, the ability of Prevotella intermedia, an obligate anaerobic rod, to degrade human hemoglobin was determined by SDS-PAGE and the degradation was quantified by scanning densitometry. Both bacterial cells and culture supernatants degraded hemoglobin. The hemoglobin degradation by P. intermedia was time-dependent, heat sensitive, pH related and was not influenced by iron restriction. Inhibition studies demonstrated that a cysteine protease might be involved in hemoglobin degradation and this protease might require metal ions for its activity and it might be thiol-requiring and trypsin-inducible. The results indicate that P. intermedia is capable to release heme from hemoglobin, hence provide a source of iron for its proliferation.     

Incidence of Prevotella intermedia and Prevotella nigrescens in Periodontal Health and Disease

The incidence of black-pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n = 45), and in 47.1% of healthy sites (n = 34) and 87.8% of active sites (n = 41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase-lipase, acid-phosphatase and α-fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P < 0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.     

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens

Abstract The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via a high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species ( P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis , is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had orginated from healthy or diseased oral sites.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Long-range charge transport through double-stranded DNA mediated by manganese or iron porphyrins

Guanine oxidation by electron transfer results in the formation of a guanine radical cation, which is at the origin of long-range charge transport through double-stranded DNA. It is possible to observe guanine lesions at a long distance from the oxidative reagent covalently bound to DNA owing to the migration of the positive hole in the DNA pi-stacks. This phenomenon of long-range hole transport is classically studied in the literature with photosensitizers used as one-electron oxidants. It is shown in the present work that the process of long-range charge transport and the concomitant formation of guanine lesions at a long distance can be observed also in the case of two-electron oxidants. This is the signature of the formation of a transient guanine radical cation in the course of the two-electron abstraction process and consequently evidence of the separated one plus one electron abstraction steps. Long-range charge transport is likely to be a universal mechanism for any two-electron oxidant acting by electron abstraction provided that the second electron abstraction is slower than hole transfer.     

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species

Abstract Dioxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10⁴–10⁵ bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80–90 samples within two days with low material costs.     

Lipase and acidic phosphatase from the psychrotrophic bacterium Pseudomonas fluorescens: Two enzymes whose synthesis is regulated by the growth temperature

Abstract The characterization of a protease inhibitor produced by a strain of Prevotella intermedia is reported in the present study. The protease inhibitor was secreted into the culture medium during the exponential growth phase and was also present in significant amounts in the periplasmic space. The inhibitor was active against a wide variety of proteolytic enzymes, including the trypsin-like activity of Porphyromonas gingivalis . The inhibitory compound, a heat labile protein, was isolated by non-denaturing preparative polyacrylamide gel electrophoresis, using concentrated culture supernatant as starting material. The exact functions and the ecological roles of the protease inhibitor produced by P. intermedia remain to be determined.