Murine Lesion Model Research Articles

Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

Proteinase-adhesin complexes of Porphyromonas gingivalis wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpA(cat), the Lys-specific proteinase Kgp(cat) and adhesin domains RgpA A1, RgpA A2, RgpA A3, Kgp A1 and Kgp A2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpA A4, indicated that this band contained C-terminally truncated Kgp A1 (which has an identical N-terminus to RgpA A4) as well as the HagA A1* adhesin. Analysis of the Triton X-114 extracted complexes from the P. gingivalis isogenic mutants kgp (RgpA complex) and rgpA (Kgp complex) suggested that the Kgp complex consisted of Kgp(cat), Kgp A1 and Kgp A2/HagA A2 and that the RgpA complex consisted of RgpA(cat), RgpA A1, HagA A1*, RgpA A2 and RgpA A3. Each of the complexes was found to have equilibrium binding constants (K(D)) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower K(D) values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.

Vaccination with recombinant adhesins from the RgpA–Kgp proteinase–adhesin complex protects against Porphyromonas gingivalis infection

Porphyromonas gingivalis is a pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. A major virulence factor for P. gingivalis is the RgpA–Kgp proteinase–adhesin complex. We have prepared the following recombinant proteins corresponding to domains/regions of the RgpA–Kgp complex; rRgpA cat, rRgpA A1, rRgpA A2, rRgpA A4, rRgpA A1(784–1009), rRgpA A1(911–1009), rKgp A1 and rKgp A1(759–989). The ability of each recombinant protein to attenuate P. gingivalis infection, when used as a vaccine, in the murine lesion model was determined. All of the recombinant adhesin domains were found to significantly attenuate P. gingivalis infection with the most effective being rKgp A1 and rKgp A1(759–989) followed by rRgpA A1, rRgpA A1(784–1009) and rRgpA A1(911–1009). The predominant antibody isotype was IgG1. The A1 adhesins, which gave the best protection, contain specific motifs implicated in binding to host tissue. Immunisation with rRgpA cat had no effect on P. gingivalis infection. As well as detecting the Kgp A1 adhesin in a Western blot, the rKgp A1 and rKgp A1(759–989) antisera were also immunoreactive with the A1 and A3 adhesins of RgpA and HagA. Flow cytometric analysis indicated that rKgp A1 and rRgpA A1 antisera recognised native antigen on P. gingivalis whole cells. Furthermore, the rKgp A1 and rKgp A1(759–989) antisera exhibited a similar immunoreactive profile with outer membrane preparations of all P. gingivalis serotypes and clinical isolates tested. The recombinant A1 adhesin therefore has potential in the development of a P. gingivalis vaccine.

Characterization of two outer membrane protein antigens of Porphyromonas gingivalis that are protective in a murine lesion model.

Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis. The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis. Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked. Analysis of 14 laboratory and clinical isolates of P. gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro. Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation. While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P. gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease.

CPG70 Is a Novel Basic Metallocarboxypeptidase with C-terminal Polycystic Kidney Disease Domains from Porphyromonas gingivalis

In a search for a basic carboxypeptidase that might work in concert with the major virulence factors, the Arg- and Lys-specific cysteine endoproteinases of Porphyromonas gingivalis, a novel 69.8-kDa metallocarboxypeptidase CPG70 was purified to apparent homogeneity from the culture fluid of P. gingivalis HG66. Carboxypeptidase activity was measured by matrix-assisted laser desorption ionization-mass spectrometry using peptide substrates derived from a tryptic digest of hemoglobin. CPG70 exhibited activity with peptides containing C-terminal Lys and Arg residues. The k(cat)/K(m) values for the hydrolysis of the synthetic dipeptides FA-Ala-Lys and FA-Ala-Arg by CPG70 were 99 and 56 mm(-1)s(-1), respectively. The enzyme activity was strongly inhibited by the Arg analog (2-guanidinoethylmercapto)succinic acid and 1,10-phenanthroline. High resolution inductively coupled plasma-mass spectrometry demonstrated that 1 mol of CPG70 was associated with 0.6 mol of zinc, 0.2 mol of nickel, and 0.2 mol of copper. A search of the P. gingivalis W83 genomic data base (TIGR) with the N-terminal amino acid sequence determined for CPG70 revealed that the enzyme is an N- and C-terminally truncated form of a predicted 91.5-kDa protein (PG0232). Analysis of the deduced amino acid sequence of the full-length protein revealed an N-terminal signal sequence followed by a pro-segment, a metallocarboxypeptidase catalytic domain, three tandem polycystic kidney disease domains, and an 88-residue C-terminal segment. The catalytic domain exhibited the highest sequence identity with the duck metallocarboxypeptidase D domain II. Insertional inactivation of the gene encoding CPG70 resulted in a P. gingivalis isogenic mutant that was avirulent in the murine lesion model under the conditions tested.

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence.

Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5alpha. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the mid-exponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5alpha transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45% less Rgp and 30% less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.

Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model.

Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively. The contribution to pathogenicity of each of the proteinase genes of P. gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain. Whole-cell Lys-x proteolytic activity of the RgpA(-) and RgpB(-) mutants was not significantly different from that of wild-type W50, whereas the Kgp(-) mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA(-) and RgpB(-) isogenic mutants did not die but developed lesions. Mice challenged with the Kgp(-) isogenic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp(-) mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 10(9) viable-cell dose. All the mice challenged with the RgpA(-) mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB(-) mutant at this dose. Wild-type phenotype was restored to the RgpB(-) mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene. There was no difference between the pNJR12::rgpB-complemented RgpB(-) mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model. These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P. gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp >> RgpB > or = RgpA.

RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model.

Porphyromonas gingivalis, a gram-negative bacterium, has been linked to the onset and progression of periodontitis, a chronic inflammatory disease of the supporting tissues of the teeth. A major virulence factor of P. gingivalis is an extracellular complex of Arg- and Lys-specific proteinases and adhesins designated the RgpA-Kgp complex (formerly the PrtR-PrtK complex). In this study we show that the RgpA-Kgp complex, when used as an immunogen with incomplete Freund adjuvant (IFA), protects against challenge with invasive and noninvasive strains of P. gingivalis in the murine lesion model. We identified a variety of peptide vaccine candidates from the RgpA and Kgp polyprotein sequences that involved the putative active site histidine of both proteinases and five repeat motifs in the adhesin domains of both polyproteins implicated in aggregation and binding to host substrates, designated adhesin-binding motif (ABM) peptides. These peptides were synthesized using standard, solid-phase protocols for 9-fluorenylmethoxy carbonyl chemistry with S-acetylmercaptoacetic acid (SAMA) as the N-terminal residue. The SAMA-peptides were then conjugated to diphtheria toxoid and used with IFA to immunize BALB/c mice. Both active-site peptides and three of the five ABM peptides gave protection (P < 0.005) against challenge with P. gingivalis in the murine lesion model. The three ABM peptide sequences that conferred protection exist within a 100-residue span in the RgpA44 and Kgp39 adhesins of the RgpA-Kgp complex. Protective anti-RgpA-Kgp complex mouse antisera recognized the RgpA27, Kgp39, and RgpA44 adhesins in an immunoblot. Epitope mapping of the RgpA27 adhesin using the protective anti-RgpA-Kgp antisera identified a major protective epitope that mapped immediately N terminal to one of the protective ABM peptides in the 100-residue span in RgpA44 and Kgp39. This identified protective epitope contains clusters of basic residues spatially surrounded by hydrophobic amino acids, a finding which is characteristic of a heparin binding motif.

Porphyromonas gingivalisvirulence in a murine lesion model: effects of immune alterations

This study utilized various mouse strains with documented alterations in immune system components to assess their contribution to modify the virulence ofPorphyromonas gingivalis. P. gingivalisW50 was cultivated on blood agar plates, harvested and used to challenge mice by subcutaneous injection on the dorsolateral surface of the back. Soft tissue lesion development was estimated by measuring the area of the spreading lesion formed by this microorganism over a period of 15 days. Challenge of various normal inbred and outbred mouse strains including: BALB/cN, BALB/cJ, BALB/c nu/+, ICR, B10.A(4R), B10.MBR, A/J, C57BL/6J, CBA/CaH, C.B-17/Icv Tacf DF and C3H/HeN with 2×1010bacteria showed similar lesion size among these strains (400 mm2). Genetically deficient mouse strains [C.B-17/Icr Tac (SCID); DBA/2 (C5 deficient); BALB/c nu/nu (T cell deficient); CBA/CaHN-XID/J (B cell deficient) and C3H/HeJ (LPS hyporesponsive)] demonstrated a lesion size which was similar to normal animals. C57BL/6J-BgJ (NK cell deficient) mice exhibited a significantly more severe lesion than the other strains tested. Following healing of the lesions, we initiated a secondary infection of the surviving animals to estimate the acquisition of protective immunity following recovery from the primary infection. Normal mice demonstrated a delayed onset and decrease in lesion size of 15 to 30% compared with the primary infection. In contrast, each of the immunodeficient strains appeared unable to develop immune protection to the secondary challenge. The findings suggest that protection against primary infections withP. gingivalisare mediated by innate immune mechanisms (PMN. NK cells). Additionally, it appears that T-cell-dependent humoral responses are critical to developing immunity to subsequentP. gingivalisinfection.

Mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in a murine lesion model: potential synergistic effects on virulence

These studies determined the characteristics of tissue destruction in a murine abscess model elicited by mixed infection with the periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis. The interbacterial effects of this synergism, the kinetics of the relationship of the bacterial interaction, and the characteristics of the bacteria required for the tissue destruction were studied. Infection of mice with P. gingivalis and F. nucleatum strains elicited lesions of various sizes as a function of infective dose. Primary infection with F. nucleatum plus P. gingivalis at various ratios (i.e., <1:1) resulted in a significantly greater lesion size (P < 0.001) compared with that resulting from primary infection with P. gingivalis alone. At F. nucleatum/P. gingivalis ratios of > or = 1:1, spreading lesion formation and progression were significantly (P < 0.001) decreased, suggesting that bacterial interaction (i.e., coaggregation) may have inhibited the spread of the P. gingivalis infection to a site distant from the initial injection. Infection with F. nucleatum and P. gingivalis simultaneously (at different sites) or F. nucleatum administered within 4 h prior to or 1 h following P. gingivalis infection significantly enhanced the ability of P. gingivalis to form large phlegmonous lesions. Chemical inhibition of the P. gingivalis trypsin-like protease activity or the use of a trypsin-negative P. gingivalis strain abrogated tissue destruction either alone or in combination with F. nucleatum. Therefore, it was possible to examine aspects of virulence of these pathogens in a murine lesion model by either altering bacterial ratios, manipulating the time of infection, or targeting vital bacterial virulence factors.