Specific Antibodies to
            <i>Porphyromonas gingivalis</i>
            Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection

Kuboniwa, Masae; Amano, Atsuo; Shizukuishi, Satoshi; Nakagawa, Ichirô; Hamada, Shigeyuki

doi:10.1128/iai.69.5.2972-2979.2001

Cited by 52 publications

(46 citation statements)

References 53 publications

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“…7B). Of note, proteins involved in iron acquisition in other human pathogens are under intense investigation as potential therapeutic targets (1,15,19,(27)(28)(29)35). For example, antibodies directed against lipoproteins of two ABC transporters involved in iron uptake in Streptococcus pneumoniae recently were shown to be protective in a mouse infection model (1).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

et al. 2002

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Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.Group A Streptococcus (GAS) is a gram-positive human pathogen that causes a variety of diseases, such as pharyngitis, cellulitis, bacteremia, streptococcal toxic shock syndrome, necrotizing fasciitis, and postinfection sequelae, including acute rheumatic fever, rheumatic heart disease, and glomerulonephritis (26). GAS pharyngitis results in substantial morbidity and economic loss globally, and severe invasive infections are associated with high morbidity and mortality rates.Extracellular proteins that mediate pathogen adhesion, evasion of host immune responses, tissue destruction, and nutrient uptake play vital roles in the life cycle of GAS (3, 18). GAS extracellular proteins can be assigned to two broad categories, i.e., actively secreted proteins found free in the culture supernatant and proteins that are located predominantly on the bacterial cell surface. Most actively secreted proteins have characteristic gram-positive secretion signal sequences located at the amino terminus. Cell surface proteins are attached to the cell surface in several ways. For example, lipoproteins are anchored to the bacterial cell membrane by a lipid moiety located at the amino terminus (33). In addition, many grampositive bacterial cell surface proteins, including several made by GAS, are covalently cross-linked to the cell wall through a conserved pentapeptide sequence (Leu-Pro-X-Thr-Gly [LPXTG]) that is followed by a stretch of hydrophobic residues and a short charged tail located at the carboxy terminus (9).Recently we analyzed the supernatant proteome of GAS in an initial effort to identify novel secreted proteins for pathogenesis and therapeutics research (17). However, important secreted proteins may not have been identified by our proteome analysis because of limited in vitro expression, technical difficulties associated with proteomics, or the fact that we analyzed culture supernatant proteins only. Therefore, bioinform...

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

et al. 2002

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“…Several studies have suggested that the phenotypic expression of gingipains in P. gingivalis is involved in iron utilization and virulence (20,21,24,27,29). Specifically, a defect in the gingipain Kgp results in a non-black-pigmented mutant, thus suggesting that the accumulation of iron on the cell surface has been affected.…”

Section: Discussionmentioning

confidence: 99%

Gingipain RgpB Is Excreted as a Proenzyme in the vimA -Defective Mutant Porphyromonas gingivalis FLL92

et al. 2003

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We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp-and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturationrenaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic adult periodontitis (reviewed in references 23, 25, and 43) and is associated with other systemic diseases, including atherosclerosis (reviewed in reference 11). While several virulence factors have been implicated in the pathogenicity of P. gingivalis, the high-level proteolytic abilities of this organism have been the focus of much attention and appear to play an important role in its virulence. The major proteases, called gingipains, are both extracellular and cell associated. They consist of arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]) (35). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp (28,33). In addition to being essential for growth, proteases have other functions. Several reports (reviewed in reference 22) have documented the multiple effects of proteases, including degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increases in vascular permeability and blood clotting prevention. Furthermore, these gingipains, which have been suggested...

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“…The number of bacteria in each suspension was estimated by determining the optical density at 600 nm and extrapolating from a standard curve, as described previously (20). P. gingivalis cells were added to a monolayer of HeLa cells (1 ϫ 10 5 cells/well) in a 24-well culture plate at a multiplicity of infection (MOI) of 200 and then incubated for 90 min at 37°C in the presence of 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types ofPorphyromonas gingivalisFimbriae

Nakagawa

Inaba²,

Yamamura

et al. 2006

Infect Immun

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Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by N␣-p-tosyl-L-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arggingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.

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Specific Antibodies to Porphyromonas gingivalis Lys-Gingipain by DNA Vaccination Inhibit Bacterial Binding to Hemoglobin and Protect Mice from Infection

Cited by 52 publications

References 53 publications

Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

Gingipain RgpB Is Excreted as a Proenzyme in the vimA -Defective Mutant Porphyromonas gingivalis FLL92

Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types ofPorphyromonas gingivalisFimbriae

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