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.

Quorum sensing (QS) is a process by which bacteria communicate using secreted chemical signaling molecules called autoinducers (AIs). By this process, many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density. In this study, the periodontal pathogen Eikenella corrodens was observed to secrete type 2 signaling molecules. An ortholog of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the E. corrodens genome. A V. harveyi bioassay showed luxS functionality in E. corrodens and the ability of luxS to complement the luxS-negative phenotype of Escherichia coli DH5alpha. AI activity was detected in the supernatant, and the maximum expression of AI-2 was observed during the late exponential phase. To determine the potential role of luxS in the colonization processes, an E. corrodens luxS mutant was constructed and tested for its capacity to form an in vitro biofilm on a polystyrene surface. The biofilm forming efficiency of the luxS mutant was approximately 1.3-fold greater than that of the wild type. These data suggest that a LuxS-dependent signal plays a role in the biofilm formation by E. corrodens.

Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids(aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.

A lectin-like substance (LS), that was isolated from Eikenella corrodens (Ec) 1073, migrated as proteins of about 300 and 45 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In this study, we cloned the gene encoding the 45-kDa protein and predicted its structure and function. Based on the N-terminal 23-amino acid (aa) sequence of this protein, we cloned the region for its N-terminus. We cloned the entire gene by means of gene walking using polymerase chain reaction and Southern hybridization. The nucleotide sequences of cloned fragments revealed an open reading frame encoding a polypeptide of 330 aa (M(r), 35748). This ORF displayed high homology to those of porins of Neisseria species. Using the T7-expression system, the 45-kDa protein was produced in E. coli. Our results suggested that the 45-kDa protein of Ec 1073 is a component of the EcLS complex, and that it is the major outer membrane protein.

Eikenella corrodens is a Gram-negative microaerophilic rod which is gaining recognition as an important human pathogen. We have previously reported the cloning and expression in Escherichia coli of a 3.6 kb Eik. corrodens genomic DNA fragment which encodes a 31.5 kDa haemagglutinin. Maxicell analysis revealed that this fragment also encodes two proteins of approximately 14 kDa. Nucleotide sequencing of the 2.2 kb fragment upstream of the haemagglutinin gene revealed two open reading frames with strong homology to genes encoding pilin subunit proteins of the type 4 or N-methylphenylalanine class. The two pilin genes, ecpA and ecpB, are complete and are expressed in E. coli. Southern analysis of ten additional Eik. corrodens strains revealed that all possess fragments homologous to ecpA. These data represent the first molecular evidence for pili in E. corrodens.

Eikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections. From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins. By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct. Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31.5 kDa and two of approximately 14 kDa each. Expression of each of the proteins appears to be under the control of an Eikenella promoter(s). Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa. Expression of both of these proteins in E. coli requires the lac promoter in the vector. By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31.5 kDa polypeptide identified as the haemagglutinin. Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2. The nucleotide sequences of both haemagglutinin genes were determined and are presented. Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins. These are the first genes of this genus to be cloned and sequenced.

Eikenella corrodens normally inhabits the human respiratory and gastrointestinal tracts but is frequently the cause of abscesses at various sites. Using the N-terminal portion of the Moraxella nonliquefaciens pilin gene as a hybridization probe, we cloned two tandemly located pilin genes of E. corrodens 31745, ecpC and ecpD, and expressed the two pilin genes separately in Escherichia coli. A comparison of the predicted amino acid sequences of E. corrodens 31745 EcpC and EcpD revealed considerable divergence between the sequences of these two pilins and even less similarity to EcpA and EcpB of E. corrodens type strain ATCC 23834. EcpC from E. corrodens 31745 displayed high degrees of homology to the pilins of Neisseria gonorrhoeae and Pseudomonas aeruginosa. EcpD from E. corrodens 31745 showed the highest homologies with the pilin of one of the three P. aeruginosa classes, whereas EcpA and EcpB of strain ATCC 23834 most closely resemble Moraxella bovis pilins. These findings raise interesting questions about potential genetic transfer between different bacterial species, as opposed to convergent evolution.

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.