Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) of Porphyromonas gingivalis

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the beta-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a usable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.     

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine-specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K(d)=1.31+/-0.26x10(-7) M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule.     

Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.     

A domain of the Leptospira LigB contributes to high affinity binding of fibronectin

Adhesion of pathogenic Leptospira spp. to mammalian cells is mediated by their adhesins interacting with host cell receptors. In a previous study, we have identified two potential fibronectin (Fn) binding sites in central variable region (LigBCen) and C-terminal variable region (LigBCtv) of LigB, an adhesin of pathogenic Leptospira spp. In this study, we have further localized the Fn-binding site on LigBCen and found a domain of LigB (LigBCen2) (amino acids 1014-1165) strongly bound to Fn. LigBCen2 bound to a 70kDa domain of Fn including N-terminal domain (NTD) and gelatin binding domain (GBD), but with a higher binding affinity to NTD (K(d)=272nM) than to GBD (K(d)=1200nM). Except Fn, LigBCen2 also bound laminin and fibrinogen. LigBCen2 could bind MDCK cells, and blocked the binding of Leptospira on MDCK cells by 45%. These results suggest that LigBCen2 contributed to high affinity binding on NTD or GBD of Fn, laminin, and fibrinogen and mediated Leptospira binding on host cells.     

Epidermal cell migration on laminin-coated substrates

Summary Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for the migrating wound epithelium. Laminin, a protein that has been implicated as an epithelial-specific adhesin, was a moderately good migration substrate. Type-IV collagen, fibrinogen and fibronectin, however, were significantly better. Fetuin, myoglobin, and casein all proved to be very poor substrates, allowing practically no migration. The inability of fetuin, myoglobin, and casein to support migration is further evidence that the considerable migration that occurs on collagen (Donaldson et al. 1982) fibrinogen and fibronectin (Donaldson and Mahan 1983) and the moderate migration on laminin, is a relatively specific response to these proteins and is therefore of special significance. The fact that laminin is a poorer migration substrate than collagen, fibrinogen or fibronectin suggests that the absence of cell surface laminin that has been associated with epithelial movement in several studies (Stanley et al. 1981; Clark et al. 1982; Madri and Stenn 1982; Gulati et al. 1983) may promote motility by allowing epithelial cells to interact directly with other extracellular macromolecules.     

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.     

Identification of Porphyromonas gingivalis components that mediate its interactions with fibronectin.

Porphyromonas (Bacteroides) gingivalis W12 binds and degrades human plasma fibronectin. In the presence of the protease inhibitor N-alpha-p-tosyl-L-lysyl chloromethyl ketone, P. gingivalis cells accumulated substantial amounts of 125I-fibronectin as a function of incubation time. Fibronectin binding was specific, reversible, and saturable. The Kd for the reaction was estimated to be on the order of 100 nM, and there was an average of 3.5 x 10(3) fibronectin binding sites per cell. Unlabeled fibronectin inhibited the binding of 125I-fibronectin to bacteria; however, fibrinogen was an even more efficient inhibitor of 125I-fibronectin binding. Unrelated proteins were without effect on fibronectin binding. A fibronectin-binding component (Mr, 150,000) was identified in sodium dodecyl sulfate-solubilized P. gingivalis. Fibronectin was degraded into discrete peptides by P. gingivalis W12. The degradation of fibronectin was inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone. Two P. gingivalis components (Mrs, 120,000 and 150,000) degraded fibronectin in substrate-containing gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a previous study (M. S. Lantz, R. D. Allen, T. A. Vail, L. M. Switalski, and M. Hook, J. Bacteriol. 173:495-504, 1991), we found that the same strain of P. gingivalis bound and subsequently degraded human fibrinogen via apparently distinct cell surface components of molecular sizes similar to those of components now implicated in the binding and degradation of fibronectin. These results raise the possibility that the two ligands are recognized and modified by the same components on P. gingivalis W12. In support of this hypothesis, unlabeled fibrinogen effectively inhibited the binding of 125I-fibronectin to bacteria and blocked 125I-fibronectin binding to a P. gingivalis ligand-binding component (Mr, 150,000 immobilized on a nitrocellulose membrane.     

Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.     

Activation of human prothrombin by arginine-specific cysteine proteinases (Gingipains R) from porphyromonas gingivalis

The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that alpha-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k(cat)/K(m) = 1.2 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534-S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis.     

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.     

The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding

Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.     

IsdA of Staphylococcus aureus is a broad spectrum, iron-regulated adhesin

As an important facet of host-pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad-spectrum ligand-binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron-regulated proteins of S. aureus and in other Gram-positive organisms. The isdA gene is only expressed in iron-limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron-limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.     

Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal Pathogen Porphyromonas gingivalis

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 microM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 microM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.     

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.     

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.     

Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic rod, has been closely associated with the initiation and progression of periodontal disease. This organism has been shown to produce a large number of proteolytic enzymes which can degrade a variety of tissue proteins, and these are considered to be major virulence factors. One of the proteinases produced by this organism, referred to as gingipain-1, has been purified to homogeneity from P. gingivalis culture medium by a combination of gel filtration and ion-exchange chromatography. The enzyme was found to have a molecular mass near 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for cysteine for activation and Ca2+ for stabilization. Amino-terminal sequence analysis indicated that gingipain belongs to a new, so far unknown, subfamily of cysteine proteinases. Three unusual features of this proteinase are: (a) the stimulation of amidolytic activity by glycine-containing dipeptides; (b) a narrow specificity which is limited to peptide bonds containing arginine residues; and (c) resistance to inhibition by proteinase inhibitors in human plasma.     

An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss

Porphyromonas gingivalis, a pathogen associated with periodontitis, bound to fibrinogen, fibronectin, hemoglobin, and collagen type V with a similar profile to that of its major virulence factor, the cell surface RgpA-Kgp proteinase-adhesin complex. Using peptide-specific, purified Abs in competitive inhibition ELISAs and epitope mapping assays, we have identified potential adhesin binding motifs (ABMs) of the RgpA-Kgp complex responsible for binding to host proteins. The RgpA-Kgp complex and synthetic ABM and proteinase active site peptides conjugated to diphtheria toxoid, when used as vaccines, protected against P. gingivalis-induced periodontal bone loss in the murine periodontitis model. The most efficacious peptide and protein vaccines were found to induce a high-titer IgG1 Ab response. Furthermore, mice protected in the lesion and periodontitis models had a predominant P. gingivalis-specific IL-4 response, whereas mice with disease had a predominant IFN-gamma response. The peptide-specific Abs directed to the ABM2 sequence (EGLATATTFEEDGVA) protected against periodontal bone loss and inhibited binding of the RgpA-Kgp complex to fibrinogen, fibronectin, and collagen type V. Furthermore, the peptide-specific Abs directed to the ABM3 sequence (GTPNPNPNPNPNPNPGT) protected against periodontal bone loss and inhibited binding to hemoglobin. However, the most protective Abs were those directed to the active sites of the RgpA and Kgp proteinases. The results suggest that when the RgpA-Kgp complex, or functional binding motif or active site peptides are used as a vaccine, they induce a Th2 response that blocks function of the RgpA-Kgp complex and protects against periodontal bone loss.     

The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein

Interaction of the basement-membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]-labelled type IV collagen to O75X-positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N-terminal 7S domain of type IV collagen, and the binding of [125I]-labelled type IV collagen to O75X-positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase-H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein-protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.