LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens.

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.

Molecular strategies for fimbrial expression and assembly.

Fimbriae or pili are long, filamentous, multimeric macromolecules found on the bacterial cell surface. Bacteria express a diverse array of fimbriae or pili that are involved in bacterial adherence and invasion. Fimbriae can be categorized based on their modes of expression and assembly. Type I fimbriae and P pili are distributed peritrichously and translocated to the cell surface by a chaperone/usher pathway. Type 4 pili are located at the pole of the cell and assembled via the type II secretion system. Curli fimbriae are coiled surface structures assembled by an extracellular nucleation/precipitation pathway. Fimbriae of oral gram-negative and gram-positive bacteria have not been well-studied as compared with the fimbriae of enteric pathogens. Oral pathogens, such as Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis, possess fimbriae that have been implicated in bacterial adhesion and invasion. These fimbriae are potential virulence factors in oral infectious processes. A. actinomycetemcomitans and E. corrodens have Type 4-like fimbriae, whereas P. gingivalis displays a unique type of fimbriae. To date, fimbriae of the oral primary colonizers, Actinomyces naeslundii and Streptococcus parasanguis, represent the only fimbriae characterized for any gram-positive bacteria. The putative major fimbrial subunits, FimA and FimP of A. naeslundii and Fap1 of S. parasanguis, contain a signal sequence and cell-wall-sorting signal. The presence of extensive dipeptide repeats in Fap1 makes it unique among fimbrial molecules. Based on experimental data, a nucleation/precipitation pathway is proposed for fimbrial biogenesis of both S. parasanguis and A. naeslundii, although we cannot rule out an alternative covalent linkage model. The model systems described in this review served as a framework for hypotheses for how the known molecular factors of fimbriae on oral bacteria may be expressed and assembled.

Eikenella corrodens phase variation involves a posttranslational event in pilus formation.

The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagA resembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.

Interleukin-6 (IL-6) and IL-8 are induced in human oral epithelial cells in response to exposure to periodontopathic Eikenella corrodens.

Periodontitis is the inflammatory response in periodontal tissues elicited by bacterial colonization in periodontal pockets. In this response, pocket epithelial cells are the first cells to come into contact with bacteria. To elucidate this mechanism, we determined the adherence of the periodontopathic bacterium Eikenella corrodens 1073, which has a GalNAc-sensitive lectin-like adhesin (EcLS), to a human oral epithelial carcinoma cell line (KB) and the induction of proinflammatory cytokine production in the cells following exposure to this bacterium in vitro. In the adherence assay, EcLS played a role as the adhesin of this bacterium in adherence to KB cells. In a reverse transcriptase PCR, significant interleukin-8 (IL-8) and IL-6 mRNA levels were induced in response to exposure to this bacterium. In an enzyme-linked immunosorbent assay after an 8-h bacterial exposure, the IL-8 and IL-6 protein levels were 13.5- and 8.3-fold higher than those in the nonexposed controls, respectively. These protein responses were time dependent. Interestingly, when E. corrodens was separated from KB cells by cell culture inserts, a slight stimulation of the IL-6 and IL-8 mRNA and secreted protein levels was seen. These results imply that the direct contact of E. corrodens 1073 with oral epithelial cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion. We suggest that E. corrodens induces the epithelial cells to secrete proinflammatory cytokines which serve as an early signaling system to host immune and inflammatory cells in underlying connective tissues.

Purification and characterization of Eikenella corrodens type IV pilin.

Eikenella corrodens is a gram-negative human pathogen associated with periodontal diseases and soft-tissue infections. Pilin was purified by association-dissociation and fast protein liquid chromatography; it had an apparent molecular mass of about 14.8 kDa and an N-terminal amino acid sequence reflective of type IV pilins. Antibodies to the purified protein reacted with pili on whole cells. This is the first report of purification of type IV pili/pilin from this organism. Other type IV pili are important virulence factors; we are currently investigating the biological role of pili in E. corrodens.

Scanning electron microscopy of Eikenella corrodens colony morphology variants.

Eikenella corrodens is a gram-negative, human pathogen which exhibits colony morphology variation. Scanning electron microscopy (SEM) was used to examine large (non-corroding) and small (corroding) colony variants from the type strain (ATCC 23834) and two clinical isolates (strains VA1 and CM1). Large colonies were large, flatter, and appeared relatively featureless compared to small colonies and had even, smooth colony margins. Small colonies were more raised from the medium surface, and often had a central raised region surrounded by flatter border. Cells on the surface of large colonies were more regularly arranged at the colony edge, and end-to-end rows of cells around the colony were seen in some strains. Cells in the center of the upper surface of small colonies were usually randomly arranged. Within cross sections of small colonies, cells were arranged randomly or perpendicular to the medium; in large colonies, cells were random or arranged horizontally. Amorphous, "slime" material was often seen covering groups of cells in large and small variants. An unusual variant, possibly a mutant, which combined features of both colony types was isolated.

Demonstration of the glycocalyces associated with three oral gram-negative bacterial species using a modern acrylic resin technique.

The complex highly hydrated chemical composition of the bacterial glycocalyx renders it difficult to preserve and visualize at the ultrastructural level. Polyanionic stains such as ruthenium red help to maintain some structural integrity, and other more modern approaches include antibody stabilization, lectins, and the addition of lysine to the primary fixative. It has been suggested that the glycocalyx of certain disease-associated organisms may play a role in the pathogenesis of some microbially based diseases such as periodontitis. New, more adequate, modern methodologies are therefore required for the further study of this structure. In the present study a cold dehydration process in conjunction with LR white acrylic resin has been employed to study the glycocalyces of three oral gram-negative bacterial species reported to be periodontopathogens. When compared with organisms processed conventionally and with ruthenium red, the organisms processed by the cold dehydration and LR white method demonstrated a fibrous matrix that was not seen in the other specimens. These results indicate that a combination of reduced dehydration temperature and cold acrylic resin embedding provides the best methodology for the visualization of the fine structure of the bacterial glycocalyx. This approach may be particularly useful in the study of organisms within specific disease-associated environments such as the periodontal pocket.

Corroding characteristics of Eikenella corrodens.

Fourteen strains of Eikenella corrodens isolated from human oral cavity were studied to determine corroding characteristics. Nine out of the 14 strains produced corroding colonies under anaerobic culture condition. One of them produced corrosion even in an aerobic culture. No morphological differences in surface structures were observed between corroding and non-corroding strains of E. corrodens by transmission electron microscopy. The morphology of corroding colonies of E. corrodens was then examined by scanning electron microscopy. The surface of the corroding-colony center was smoothly convex. A boundary line was clear between the smooth center and the surrounding corrosion region. Double or triple frill-like structures surrounded the center portion with small convexities. Spreading bacterial masses were observed in the outer portion of the colony. Morphological observations of the corroding colony edge indicated that a surface translocation termed "twitching motility" or "gliding motility" occurs in the outer portion and plays a role in its colonization of periodontal regions.

Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens.

The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.

Transmission-scanning electron microscopic observations of selected Eikenella corrodens strains.

The morphology of Eikenella corrodens 333/54-55 (ATCC 23834) and two human periodontal lesion isolates, strains 470 and 373, was examined by transmission and scanning electron microscopy. All strains exhibited a cell envelope characteristic of gram-negative bacteria. Staining with ruthenium red and alcian blue revealed a loosely organized fibrous slime layer associated with the outer surface of the outer membrane. Slime "stabilization" was achieved by incubation of cells with antisera prepared against whole cells of the Eikenella strains. The stabilized slime appeared as a thick, electron-opaque layer juxtaposed to the outer membrane. Negative staining and heavy metal shadow-casting revealed an interwoven network of fibrils approximately 4 nm in diameter. These fibrils appeared to represent subunits of a larger fibril. Scanning electron microscopy after antibody slime stabilization confirmed the presence and location of the slime layer.