The gingival epithelium plays a key role in protecting the supporting structures of the teeth from bacteria and their products. In ex vivo experiments, we recently showed that the cytolethal distending toxin (Cdt) from the periodontal pathogen Aggregatibacter actinomycetemcomitans causes extensive damage to gingival tissue. Morphological changes included detachment of the keratinized outer layer, distention of spinous and basal cells in the oral epithelium, disruption of rete pegs, and apparent dissolution of cell junctions. Adherens junctions (zonula adherens) are essential for maintaining barrier function and integrity of gingival epithelium. Therefore, immunohistochemical and RT-PCR analyses of human gingival explants (HGX) and human gingival epithelial cells (HGEC) were utilized for a closer examination of the effects of the Cdt on E-cadherin, the key membrane component of adherens junctions. Although there was some variability among tissue donors, exposure of gingival tissue or isolated epithelial cells to the toxin generally resulted in a pronounced increase in the expression and cytosolic distribution of E-cadherin, accompanied by an increase in levels of the intracellular scaffolding proteins β-catenin and β-actin. These results indicate that the Cdt induced substantial remodeling of adherens junctions, with a potential impact on the barrier function of gingival epithelium.
Abbreviations: cytolethal distending toxin (Cdt), 4′,6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), transepithelial resistance (TER).
adherens junctions; Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; E-cadherin; gingival epithelial cells; gingival explants
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
Actinobacillus actinomycetemcomitans; DNA fingerprinting; polymerase chain reaction; restriction fragment length polymorphism; DNA probe; Southern blot
Background and Objective
Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT.
Material and Methods
Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed.
Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits.
Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.
Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; enzyme-linked immunosorbent assay (ELISA); immunoglobulin G; serotype
Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of “corncobs”, a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.
Streptococcus crista; Fusobacterium nucleatum; corncob; transposon; transformation; dental plaque
Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.
juvenile periodontitis; epidemiology; virulence; restriction fragment-length polymorphism; DNA probes; Actinobaccillus actinomycetemcomitans
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive; five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting, 11 strains were assigned to RFLP group II (disease–associated); 4 to RFLP type XIII (carrier status-associated); and the others to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.
Actinobacillus actinomycetemcomitans; invasion; KB epithelial cell; restriction fragment-length polymorphism; leukotoxin
The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 ± 19 nm long with longer, less abundant fibrils 403 ± 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 ± 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 ± 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.
corncobs; fibrils; SrpA; Streptococcus cristatus; tufts
Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species. Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 106 target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples.
Porphyromonas gingivalis; Bacteroides forsythus; DNA probes; non-isotopic labeling
The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models.
Abbreviations: cytolethal distending toxin (Cdt), connective tissue (CT), 4′,6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE)
Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; gingival epithelial cells; gingival fibroblasts; gingival explants
Resistance to treatment and the appearance of secondary tumors in head and neck squamous cell carcinomas (HNSCC) have been attributed to the presence of cells with stem-cell-like properties in the basal layer of the epithelium at the site of the lesion. In this study, we tested the hypothesis that these putative cancer stem cells (CSC) in HNSCC could be specifically targeted and inhibited. We found that 9 of 10 head and neck tumor biopsies contained a subpopulation of cells that expressed CD133, an unusual surface-exposed membrane-spanning glycoprotein associated with CSC. A genetically modified cytolethal distending toxin (Cdt), from the periodontal pathogen Aggregatibacter actinomycetemcomitans, was conjugated to an anti-human CD133 monoclonal antibody (MAb). The Cdt-MAb complex preferentially inhibited the proliferation of CD133+ cells in cultures of established cell lines derived from HNSCC. Inhibition of the CD133+ cells was rate- and dose-dependent. Saturation kinetics indicated that the response to the Cdt-MAb complex was specific. Healthy primary gingival epithelial cells that are native targets of the wild-type Cdt were not affected. Analysis of these data provides a foundation for the future development of new therapies to target CSC in the early treatment of HNSCC. Abbreviations: Cdt, cytolethal distending toxin; CSC, cancer stem cells; HNSCC, head and neck squamous cell carcinoma; MAb, monoclonal antibody.
cancer stem cells; CD133; cytolethal distending toxin; squamous cell carcinoma
Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls.
Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells.
Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20–75%).
Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.
Aggregatibacter (Actinobacillus) actinomycetemcomitans; antibody; cytolethal distending toxin; localized aggressive periodontitis
A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium. The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A. actinomycetemcomitans from Brazil, Kenya, Japan and Sweden. Thirty-nine of 40 cell lysates caused distension of Chinese hamster ovary cells. At least one of the cdt genes was detected in all strains examined. The three cdt genes were detected, by PCR, in 34 DNA samples. DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity. Restriction analysis was performed on every amplicon obtained. PCR-RFLP patterns revealed that the three cdt genes were conserved. These data provided evidence that the cdt genes are found and expressed in the majority of the A. actinomycetemcomitans isolates. Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found.
Actinobacillus actinomycetemcomitans; cdt genes; cytolethal distending toxin; periodontitis
The periodontal pathogen Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT), a complex multicomponent toxin that arrests the growth of many types of eukaryotic cell. The kinetics of the effects of CDT-containing extracts, from an invasive strain of this bacterium, were examined on epithelial-like cells routinely used in invasion studies. Both KB and HEp-2 cells were exquisitely sensitive to the effects of the CDT with TD50 of 30 and 300 pg of total bacterial protein, respectively. Initial cell morphology changes were relatively rapid, occurring within the first 13 h of exposure. CDT-treated KB cells increased in size to 4–5 times the size of untreated controls. Cytotoxicity was irreversible when attached cells were incubated, for a minimum of 120 min, with nanogram quantities of CDT-containing extract. As cultures aged, the cells became more resistant to the effects of the CDT-containing extracts. These findings have important implications for understanding the ability of A. actinomycetemcomitans to invade and multiply in epithelial cells.
Actinobacillus actinomycetemcomitans; cytolethal distending toxin; invasion; KB cells; Hep-2 cells
Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans.
A new member of the lraI family of putative adhesin genes was cloned, from Streptococcus crista CC5A, and sequenced. The gene, scbA appears to be part of an ABC transport operon and encodes a putative peptide of 34.7 kDa. The protein contains a signal sequence with residues 17 to 21 (L-A-A-C-S) matching the consensus sequence for the prolipoprotein cleavage site of signal peptidase II. ScbA is 57 to 93% identical, at the amino acid level, with the five previous sequenced members of the LraI family. Surprisingly, ScbA does not exhibit adhesion properties characteristic of the other LraI proteins. Strain CC5A bound poorly to saliva-coated hydroxyapatite and did not coaggregate with Actinomyces naeslundii PK606. An scbA insertion-duplication mutation that abolished expression (of ScbA was created. There was no difference in fibrin binding between this mutant and wild-type CC5A. Since it is possible that ScbA could play a role in corncob formation between S. crista and Fusobacterium nucleatum, this property was examined. The mutant strain retained the ability to form corncobs. On the basis of the lack of adhesin properties it appears that ScbA is an atypical member of the LraI family.
Corncobs, which are distinct morphological units formed by the ordered coaggregation of a filamentous microorganism and streptococci, can be made in vitro by using oral strains of Fusobacterium nucleatum and Streptococcus sanguis. Previous studies have shown that strains of F. nucleatum contain one of at least two different types of corncob receptor. The objective of this study was to isolate the receptor from F. nucleatum ATCC 10953 as the first step in the elucidation of the molecular basis of corncob formation. The cell envelope fraction from this bacterium was treated with trypsin, delipidated with chloroform-methanol, and subjected to ion-exchange chromatography. A single polypeptide (apparent Mr, 39,500), which was eluted from the column with 0.5 M sodium chloride and extracted with dodecyltrimethylammonium bromide to remove contaminating lipopolysaccharide, inhibited corncob formation between strain ATCC 10953 and S. sanguis CC5A. Similarly derived cell fractions from either F. nucleatum FDC 364 or Fusobacterium necrophorum failed to effect coaggregation in the inhibition assay. Amino acid analysis of the polypeptide showed a moderately hydrophobic character (polarity index, 41) and 11% basic residues. Antiserum made against the purified polypeptide agglutinated F. nucleatum ATCC 10953, neutralized the ability of this bacterium to form corncobs, and agglutinated whole cells of S. sanguis CC5A that were precoated with the receptor polypeptide. The identification and isolation of this receptor should greatly enhance our ability to define some of the complex intergeneric coaggregation mechanisms that are thought to occur in the human oral cavity.
Corncob formation in dental plaque was believed to be limited to strains of Bacterionema matruchotii and Streptococcus sanguis. We observed recently that strains of Fusobacterium nucleatum also interacted with S. sanguis to form corncobs. Since the fusobacteria are among the first anaerobic filaments to colonize subgingival plaque, these interactions could serve as a connecting link between the transformation of supra- to subgingival plaque. To further characterize these interactions, quantitative in vitro studies of the kinetics of corncob formation of the fusobacteria were undertaken. These studies indicated that fewer streptococci were needed to saturate F. nucleatum strain 364 compared to strain 10953. Corncob formation with both strains was enhanced with increasing pH up to pH 8, at which point autoaggregation of the streptococci occurred. Variation in ionic strength and divalent cations had little effect on the interaction, and EDTA suppressed aggregate formation only slightly. Detergents at concentrations above 0.05% also inhibited corncob formation. Electron micrographs suggested that attachment of the cocci to the fusiforms was mediated through localized tufts of fimbriae, as they are in the Bacterionema system. However, although both trypsin and heat treatment of the streptococci inhibited corncob formation with fusobacteria, the effects were not as complete as those seen in Bacterionema species. Unlike the Bacterionema model, trypsin and heat treatment of the fusobacteria resulted in inhibition of corncob formation. These results suggest that several different receptors may be involved in corncob formation.
A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status. A. actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study. Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction. All but one of these conversion subjects harbored A. actinomycetemcomitans. Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002). Six of nine conversion subjects were infected with A. actinomycetemcomitans from this group. RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects. In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects. Thus, the use of RFLP to type clinical isolates of A. actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health. These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants.
Actinobacillus actinomycetemcomitans is recognized as a primary pathogen in localized juvenile periodontitis (LJP). Restriction fragment length polymorphisms (RFLP) within a collection of subgingival plaque isolates of this bacterium were identified and characterized as the first step in understanding the pathogenesis of LJP. Over 800 isolates, from members of 18 families (LJP families) with at least one member with active LJP or a documented history of the disease and one or more siblings, less than 13 years of age, having no clinical evidence of LJP and 32 healthy control subjects, were assigned to one of 13 distinct RFLP groups (II to XIV) by using a previously characterized 4.7-kb DNA probe cloned from the reference strain FDC Y4. Isolates belonging to RFLP groups II, IV, V, and XIII predominated subgingival sites in the subjects. Members of RFLP groups II, IV, VII, VIII, X, and XI were recovered only from LJP family subjects, while group XIII and XIV variants were found exclusively in healthy controls. A synthetic oligonucleotide, homologous to the 5' end of the leukotoxin gene (lktA), and the A. actinomycetemcomitans plasmid, pVT745, were tested for their abilities to subdivide the 13 RFLP groups. The leukotoxin probe specifically identified all RFLP group II variants because of the absence of a HindIII site in the upstream noncoding region of the lkt gene complex. The plasmid probe was not as selective but may be useful for identifying clinical isolates belonging to RFLP group I. The use of these probes for the identification of genetic variants of A. actinomycetemcomitans that may be preferentially colonize diseased and healthy subjects will facilitate the study of the role of this important pathogen in periodontal diseases.
The scrY gene, part of the pUR400-borne sucrose regulon, appeared to be transcribed from its own promoter, with the transcriptional start site located 58 bp upstream from the initiation codon. An open reading frame encoding a polypeptide of 505 amino acid residues (Mr 55,408) was identified. The first 22 amino acid residues formed a leader sequence typical of those found in other procaryotic outer membrane and periplasmic proteins. A frameshift mutation in the scrY gene resulted in a dramatic decrease in sucrose transport with no effect on in vitro phosphorylation activity associated with enzyme IISer. The rate of diffusion of sucrose was 96 times greater than the rate of diffusion of lactose or maltose in liposomes containing the ScrY protein. This increase in sucrose permeability provided strong evidence that the ScrY protein functions as a sucrose porin. There was 23% amino acid sequence identity between the ScrY protein and LamB, a maltose porin from Escherichia coli.
Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans.
A major, heat-modifiable cell envelope protein was identified in Fusobacterium nucleatum FDC 364 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, designated HM-1, had apparent molecular weights of 38,500 and 50,000 when heated in sodium dodecyl sulfate at 50 and 100 degrees C, respectively. Whole cells were labeled with 125I, and the results suggested that the HM-1 protein may be exposed on the bacterial surface. The HM-1 protein was isolated in association with the peptidoglycan by extraction of whole cells or cell envelopes with 2% sodium dodecyl sulfate at 55 degrees C. Heating the peptidoglycan-HM-1 protein complex in the detergent at 100 degrees C resulted in the quantitative release of the protein. Isoelectric focusing experiments and amino acid analysis revealed that the HM-1 protein had a basic character and was moderately hydrophilic. Various strains of F. nucleatum as well as three oral fusiform isolates contained a serologically related protein. The abundance and location of the HM-1 protein in F. nucleatum suggest that it has the potential to participate in cell surface-related interactions of this bacterium.
A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease.
The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.