Stanley C. Holt

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5′-phosphate-dependent protein acting as a haemolytic enzyme

Cystalysin is a Cβ–Sγ lyase from the oral pathogen Treponema denticola catabolyzing L‐cysteine to produce pyruvate, ammonia and H2S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5′‐phosphate (PLP)‐dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 Å resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S‐containing amino acid substrates yielding H2S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin–L‐aminoethoxyvinylglycine (AVG) complex revealed a ‘dead end’ ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin–AVG complex may provide the chemical basis for rational drug design.

Iron-regulated outer membrane proteins in the periodontopathic bacterium, Bacteroides gingivalis

Hemin has been implicated in the pathogenesis of the oral pathogen, Bacteroides gingivalis. In order to elucidate the role of hemin (iron) in the growth and expression of outer membrane proteins, B. gingivalis strain W50 was grown with and without hemin to induce iron-limitation. Cells grew slower under iron stress and growth was completely inhibited in the absence of added hemin. The outer membrane protein profiles of B. gingivalis grown under iron-replete and iron-restricted conditions were studied by extrinsic radiolabelling with [125I] and polyacrylamide gel-electrophoresis. The induction of 10 surface proteins, with apparent molecular weights of 26, 29, 50, 56, 58, 60, 62, 71, 77, and 80 Kd, was observed in B. gingivalis grown under iron-restricted conditions. These proteins were repressed under iron-replete conditions. We postulate the involvement of the iron-regulated proteins in hemin uptake and virulence in B. gingivalis.

Periodontal diseases: to protect or not to protect is the question?

For decades, investigations have identified local and systemic humoral immune responses to microorganisms comprising the supra- and subgingival biofilms in the oral cavity. Inflammation and tissue destruction in the periodontium are accompanied by alterations in the quantity, quality, and specificity of antibody. The conundrum in this scenario is the existence of a substantial plasma cell infiltrate at sites of periodontal lesions and a seemingly robust antibody response in the oral cavity and the serum, apparently coincident with progressing disease. Consequently, much effort has been expended to elucidate the critical characteristics of protective humoral responses and to develop strategies for enhancing these unique features. We and others have conducted studies attempting to distinguish disease susceptibility associated with: i) variations in response levels-significantly increased to some species with disease, minimal response to others; ii) functional comparisons of antibody-subclass differences, genetic regulation, and maturation of responses; iii) microbial and antigenic specificity of the antibody-focus on specific pathogens and identification of selected antigens as targets for immunoprotection; and, iv) kinetics of responses during disease and therapeutic interventions-linking immune changes with infection and as a measure of treatment success. This report summarizes varied research designs and results, to provide a profile of antibody in health, gingivitis, and periodontitis. These profiles may be used to provide a framework focusing on the humoral response to commensal microorganisms and likely pathogens, as they emerge in the biofilm-etiologic for or in response to disease processes. Models for antibody as a diagnostic adjunct and for predicting protective antibody responses are suggested. These concepts are likely relevant for considering vaccine approaches to periodontitis.

Oral bone loss is increased in ovariectomized rats

Alveolar bone loss associated with periodontal disease occurs frequently in postmenopausal females, the same group that is predisposed to osteoporosis. To determine if the estrogen-deficient state enhances oral bone loss, we studied ovariectomized rats administered the potent bone-resorbing cytokine interleukin-1 or the periodontal pathogen Campylobacter rectus lipopolysaccharide (LPS). Distal root canals of first mandibular molars were instrumented with endodontic files, and bone resorbing factors were deposited and sealed into the root canal. Radiographs of periapical bone loss were evaluated using computer assisted image analysis to determine lesion size. Both interleukin-1 and C. rectus LPS caused a significant increase in lesion area in both ovariectomized and normal rats when compared with controls and a significant increase in ovariectomized animals compared to nonovariectomized animals receiving LPS. Using this endodontic model, we have demonstrated that estrogen deficiency results in increased oral bone loss in rats.

Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis : identification of a novel 26 kDa hemin-repressible surface protein

Porphyromonas gingivalis is capable of in vitro growth when iron sources are either complexed to hemin or host iron transport proteins, or exist in an inorganic form. This study examined the effect of these iron sources on outer membrane protein (OMP) expression in P. gingivalis W50. Hemin (iron) starved P. gingivalis was transferred into growth medium containing hemin, hemoglobin, hemin-saturated human serum albumin, hemin-free human serum albumin, transferrin, lactoferrin, or inorganic iron. Surface proteins were identified by 125I-labeling and resolved by SDS-PAGE and autoradiography. When grown under hemin starved conditions, P. gingivalis W50 and related strains expressed a major 26 kDa OMP, as revealed by 125I-autoradiography. Autoradiographic analysis demonstrated the absence of this 26 kDa OMP from the P. gingivalis surface in hemin-containing environments. Growth of P. gingivalis W50 in the presence of host iron transport proteins (hemin-free) or inorganic iron resulted in surface expression of a 26 kDa OMP. The presence of protoporphyrin IX or substitution of hemin-associated iron with zinc, resulted in continued surface expression of the 26 kDa OMP, indicating that repressibility of this OMP required the coordination of iron to the protoporphyrin IX molecule (i.e. hemin). A survey of 125I-labeled OMPs from several hemin starved P. gingivalis and related strains, demonstrated that a hemin-repressible 26 kDa OMP occurred only in P. gingivalis. We report here a newly described 26 kDa hemin-regulated surface protein occurring in several strains of P. gingivalis which is expressed on the cell surface in hemin starved conditions and is lost from the cell surface in response to an environment containing iron coordinated specifically to protoporphyrin IX (i.e. hemin).

Cloning and Expression of Two Novel Hemin Binding Protein Genes from Treponema denticola

ABSTRACT Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed,T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of thehbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in severalT. denticola ATCC strains and clinical isolates, but not inTreponema pectinovorum, Treponema socranskii, orEscherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, calledhbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. ThehbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature‐induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid‐pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E2 (PGE2) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)‐6 occurred in the experimental animals by the time of delivery. IL‐8, MCP‐1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE2, LBP, IL‐8 and MCP‐1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL‐6, IL‐8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE2, macrophage chemotactic protein (MCP)‐1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature‐induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals.

Cystalysin, a 46-kDa L-Cysteine Desulfhydrase from Treponema denticola: Biochemical and Biophysical Characterization

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin‐binding or ‐associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent‐solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29‐34 kDa exhibited association with human apo‐ and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin‐associated proteins from T. pallidum and T. denticola in competitive‐binding assays. However, the T. denticola proteins dissociated from a lacto‐ferrin‐affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29‐34 kDa band of T. pallidum suggesting that the polypeptide was lipid‐modified. Each of the lactoferrin‐binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin‐binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.

Coaggregation between bacterial species

Bacterial coaggregation, or interbacterial adherence, is one mechanism involved in the development of bacterial biofilms that are found on surfaces in nature. Assays used to measure coaggregation rely on the interaction of bacterial cells in suspension or attachment of one species to a second species that has been fixed to a solid substrate. Both semiquantitative and quantitative assays are described. These methods have also been used to determine the nature of the adherence and molecules involved in mediating the interaction, to characterize potential inhibitors, to isolate the bacterial adhesins and receptors, and to isolate adherence-deficient mutant strains. Each of the assay systems offers different advantages, with significant variations in sensitivity. Selection of a particular assay system should depend on the goals of the study to be performed.