Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens.

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious ß-proteobacterium Eikenella corrodens grown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N'-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2 without the bc1 complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitive cbb' cytochrome c oxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system of E. corrodens ATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation.

BACKGROUND: Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. METHODS: Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. RESULTS: Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 µM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. CONCLUSIONS: TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

The periodontopathogenic bacterium Eikenella corrodens produces an autoinducer-2-inactivating enzyme.

Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development.

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Characterization and expression of adjacent proline iminopeptidase and aspartase genes from Eikenella corrodens.

Two adjacent genes involved in nitrogen metabolism from Eikenella corrodens, with a potential role in pathogenesis, were studied. Proline iminopeptidase (Pip) activity, which may be essential for energy production and protection against host immune mechanisms, is exhibited by E. corrodens. Analysis of Pip-expressing clones revealed an ORF of 939 bases with a predicted amino acid sequence identity of 67% to the Pip of Neisseria gonorrhoea. 200 bp downstream from pip, an ORF of 1395 bases, encoding a protein with 87% identity to a putative aspartase from the Neisseria meningitidis genome sequence, was identified. Enzymatic function was confirmed with a complemented Escherichia coli aspartase deficient mutant. The E. corrodens aspartase was found to be 77% identical to the Haemophilus influenzae aspartase sequence, which was originally identified on the basis of its ability to bind plasminogen. However, the E. corrodens aspartase had no such activity. Southern hybridization indicated both genes to be single copy and conserved within the genomes of a diverse panel of E. corrodens isolates from health and disease.

Antibody-based diagnostic for 'refractory' periodontitis.

OBJECTIVE: About 10-15% of US adults are 'refractory' to therapy for chronic periodontitis. Recently, studies suggest that these patients have elevated lysine decarboxylase activity in the sulcular microbiota. The aim of this study was to determine whether an elevated IgG antibody response to lysine decarboxylase, alone or with antibody to other bacterial antigens and baseline clinical measurements, would predict 'refractory' patients with high accuracy. METHODS: Chronic periodontitis patients were treated using scaling and root planing (SRP) followed by maintenance SRP and 3-monthly re-examinations. If there was a loss of mean full mouth attachment or more than three sites appeared with > 2.5 mm new loss within a year, the subjects were re-treated (modified Widman flap surgery and systemically administered tetracycline). If attachment loss as above recurred, the subjects were 'refractory'. Baseline clinical measurements and specific antibody responses were used in a logistic regression model to predict 'refractory' subjects. RESULTS: Antibody to a peptide portion of lysine decarboxylase (HKL-Ab) and baseline bleeding on probing (BOP) prevalence measurements predicted attachment loss 3 months after initial therapy [pIAL = loss (0) or gain (1)]. IgG antibody contents to a purified antigen from Actinomyces spp. (A-Ab) and streptococcal d-alanyl glycerol lipoteichoic acid (S-Ab) were related in 'refractory' patients (R2 = 0.37, p < 0.01). From the regression equation, the relationship between the antibodies was defined as linear (pLA/S-Ab = 0) or non-linear pLA/S-Ab = 1). Using pLA/S-Ab, pIAL and age, a logistic regression equation was derived from 48 of the patients. Of 59 subjects, 37 had 2-4 mm attachment loss and were assigned as 'refractory' or successfully treated with 86% accuracy. CONCLUSION: HKL-Ab facilitated an accurate prediction of therapeutic outcome in subjects with moderate periodontitis.

The characterization of a (nutritionally important) proline iminopeptidase from Eikenella corrodens.

Eikenella corrodens generates energy primarily through the oxidative deamination of specific amino acids, a process that is coupled to dissimilatory nitrate reduction to nitrite. Cell yields resulting from chemostat-growth of the organism in simple, chemically defined media containing varying amounts of proline confirm that this amino acid is a likely source of energy for E. corrodens in the oral environment. The importance of proline in ATP generation by the organism is reflected in molar growth yields, which showed that biomass production per mole of this amino acid was significantly higher than that for other amino acids. The organism was found to express, constitutively, the enzyme proline iminopeptidase, which releases proline from the N-terminus of small peptides. The enzyme was partially purified and characterized and found to exist in the cytoplasm as a 35 kDa monomer. Inhibition studies showed that the enzyme, although classified as a serine protease, also appears to require thiol groups for activity, a finding which is consistent with previous reports. The enzyme obeyed Michaelis-Menten kinetics and was found to have a Km value of 0.223 mM for the substrate proline-p-nitroanilide.

Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease.

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library of E. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. epsilon-Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

Catalase-positive Eikenella corrodens and Eikenella-like isolates of human and canine origin.

Ten catalase-positive isolates and one catalase-negative isolate that had been assigned to Eikenella corrodens were compared to the nomenclatural type strain regarding selected phenotypic and molecular features and chromosomal deoxyribonucleic acid (DNA) relatedness using the spectrophotometric method. Five catalase-positive human isolates were assigned to the genomic species Eikenella corrodens on the basis of high DNA relatedness levels. Three others, among them strain Chen UB 204, exhibited only moderate degrees of DNA relatedness to the type strain and with each other. Two catalase-positive isolates from dogs were closely interrelated, but yielded only low degrees of DNA binding with Eikenella corrodens and the Eikenella-like human isolates. These findings confirm that the human eikenellas comprise more than one genomic species and that the canine strains represent a distinct taxonomic entity. The differentiation of the strains investigated by conventional phenotypic features, hydrolytic enzyme reactions, and cellular carbohydrate patterns was considered.

Energy production and peptidase activity in Eikenella corrodens.

Eikenella corrodens 33EK(L), a clinical isolate, was assayed for its ability to utilise amino acids as substrates in the reduction of nitrate to nitrite. The metabolism of proline, glutamate, serine and glutamine was found to result in relatively high rates of nitrate reduction. The ability of cells to metabolise these amino acids from a variety of small peptides was also determined. E. corrodens was found to possess a relatively specific proline aminopeptidase as well as a putative carboxypeptidase activity for glutamate. Energy production in this organism appears to be via oxidative deamination of these key amino acids linked to a respiratory chain, with nitrate acting as the ultimate electron acceptor.

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens.

Eikenella corrodens isolates from periodontally healthy subjects and adult periodontitis patients were compared for their ability to produce a range of potential virulence factors. All were positive for proline aminopeptidase, thiol-dependent haemolysin and esterase activities. Low or negative activities were found against casein, phospholipid, lipid, collagen, aminophosphate, phosphate under acid or alkaline conditions, and eleven other amino acid substrates tested. In oral infections, the haemolytic activity of E. corrodens could be amplified in the reduced environment of the periodontal pocket and damage host cells. Proline aminopeptidase may act against proline residues in collagen, immunoglobulins and complement proteins.

Characterization of a beta-lactamase found in Eikenella corrodens.

Eleven strains of Eikenella corrodens with beta-lactamase activity were isolated from a patient with refractory periodontitis who had previously been treated with penicillin antibiotics. These strains were relatively resistant to benzylpenicillin, amoxicillin, and ampicillin (MICs, greater than or equal to 64 micrograms/ml); susceptible to amoxicillin-clavulanate (2:1) (MICs, less than or equal to 4 micrograms/ml); and moderately susceptible to cephalothin and cephaloridine (MICs, 0.12 to 16 micrograms/ml). The addition of 1 microgram of potassium clavulanate, a beta-lactamase inhibitor, per ml resulted in a significant increase in the susceptibilities of these strains to penicillins but not to cephalosporins. Potassium clavulanate had no effect on non-beta-lactamase-producing strains. Enzyme production was constitutive since activity was not increased when cells were cultivated in the presence of benzylpenicillin. Enzyme activity was strongly inhibited by potassium clavulanate, sulbactam, and iodine; weakly inhibited by cloxacillin, imipenem, and moxalactam; but not inhibited by aztreonam, EDTA, or p-chloromercuribenzoate. By gel infiltration, the enzyme had an estimated molecular mass of 29 kDa. Isoelectric focusing of the partially purified enzyme gave a major beta-lactamase band at pH 5.50 and a minor band at pH 5.60. Plasmids were not detected in any of the 11 beta-lactamase-positive strains. This enzyme is considered to belong to class 2a of the Bush classification scheme.

Characterization of a beta-lactamase-specifying plasmid isolated from Eikenella corrodens and its relationship to a commensal Neisseria plasmid.

A 9.4-kilobase plasmid encoding penicillin, streptomycin, and sulfonamide resistance was isolated from a beta-lactamase-producing Eikenella corrodens strain. This plasmid appears to be identical to a resistance plasmid common to saprophytic Neisseria strains.

Measurement of microbial alpha-amylases with p-nitrophenyl glycosides as the substrate complex.

The detection of alpha-amylase is commonly used in clinical microbiology laboratories to aid in differentiating Streptococcus bovis from other streptococci. It is also useful in identifying Eikenella corrodens and the gravis subspecies of Corynebacterium diphtheriae and in separating species of the genera Bacteroides, Clostridium, Actinomyces, and Bacillus. Currently, the most frequently used procedure utilizes starch as the substrate and iodine as the indicator. Starch is incorporated into a agar medium, the isolate is inoculated on the surface, and the medium is incubated for 24 to 48 h. A 15-min test containing p-nitrophenyl polyglycosides as the substrate complex was developed to yield results comparable with the agar-based starch test. The reagent was made in liquid form, 0.20 ml per tube, and could be incubated either in ambient air or at 35 degrees C. When dried, the p-nitrophenyl polyglycoside reagent could be stored at 0 degrees C for 4 weeks.

Butandioldehydrogenase in Eikenella corrodens and related bacteria.

The butandioldehydrogenase was examined in 153 strains of Eikenella, Moraxella, Acinetobacter, Agrobacterium, Haemophilus, Actinobacillus, Pasteurella, Cardiobacterium and "TM-1" of Hollis et al. This enzyme has been proved to be useful in the differentiation of this group of bacteria.