A novel type of two-component regulatory system affecting gingipains in Porphyromonas gingivalis

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two-component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene-disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine- and lysine-specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wildtype strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly-discovered two-component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.     

A novel dnaK operon from Porphyromonas gingivalis

The nucleotide sequence of the dnaK operon cloned from Porphyromonas gingivalis revealed that the operon does not contain homologues of either dnaJ or grpE. However, there were two genes which encode small heat shock proteins immediately downstream from the dnaK and they were transcribed together with dnaK as one unit. The ATPase activity of the P. gingivalis DnaK was synergistically stimulated up to 40-fold in the simultaneous presence of Escherichia coli DnaJ and GrpE. These results suggest that the DnaK homologue of P. gingivalis, with its unique genetic structure and evolutionary features, works as a member of the DnaK chaperone system.     

Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis. In this paper, we report the characterization of a protein, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by P. gingivalis. IhtB was localized to the cell surface of P. gingivalis by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose. The growth of heme-limited, but not heme-replete, P. gingivalis cells was inhibited by preincubation with IhtB peptide-specific antisera. The ihtB gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases. Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity. These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by P. gingivalis.     

A novel Porphyromonas gingivalis FeoB plays a role in manganese accumulation

FeoB is an atypical transporter that has been shown to exclusively mediate ferrous ion transport in some bacteria. Unusually the genome of the periodontal pathogen Porphyromonas gingivalis has two genes (feoB1 and feoB2) encoding FeoB homologs, both of which are expressed in bicistronic operons. Kinetic analysis of ferrous ion transport by P. gingivalis W50 revealed the presence of a single, high affinity system with a K(t) of 0.31 microM. FeoB1 was found to be solely responsible for this transport as energized cells of the isogenic FeoB1 mutant (W50FB1) did not transport radiolabeled iron, while the isogenic FeoB2 mutant (W50FB2) transported radiolabeled iron at a rate similar to wild type. This was reflected in the iron content of W50FB1 grown in iron excess conditions which was approximately half that of the wild type and W50FB2. The W50FB1 mutant had increased sensitivity to both oxygen and hydrogen peroxide and was avirulent in an animal model of infection whereas W50FB2 exhibited the same virulence as the wild type. Analysis of manganous ion uptake using inductively coupled plasma-mass spectrometry revealed a greater than 3-fold decrease in intracellular manganese accumulation in W50FB2 which was also unable to grow in manganese-limited media. The protein co-expressed with FeoB2 appears to be a novel FeoA-MntR fusion protein that exhibits homology to a manganese-responsive, DNA-binding metalloregulatory protein. These results indicate that FeoB2 is not involved in iron transport but plays a novel role in manganese transport.     

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane β-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.     

A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.     

Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis

The Gram-negative periodontal pathogen Porphyromonas gingivalis synthesizes several classes of novel phosphorylated complex lipids, including the recently characterized phosphorylated dihydroceramides. These sphingolipids promote the interleukin-1 (IL-1)-mediated secretion of inflammatory mediators from fibroblasts, including prostaglandin E2 and 6-keto prostaglandin F2alpha, and alter gingival fibroblast morphology in culture. This report demonstrates that one additional class of phosphorylated complex lipids of P. gingivalis promotes IL-1-mediated secretory responses and morphological changes in cultured fibroblasts. Structural characterization identified the new phospholipid class as 1,2-diacyl phosphatidylethanolamine, which substituted predominantly with isobranched C15:0 and C13:0 fatty acids. The isobranched fatty acids, rather than unbranched fatty acids, and the phosphoethanolamine head group were identified as the essential structural elements required for the promotion of IL-1-mediated secretory responses. These structural components are also observed in specific phosphorylated sphingolipids of P. gingivalis and likely contribute to the biological activity of these substances, in addition to the phosphatidylethanolamine lipids described in this report.     

Identification of a Porphyromonas gingivalis novel protein sov required for the secretion of gingipains

Gingipains are extracellular proteases important for the virulence of Porphyromonas gingivalis; however, the mechanism for the secretion of gingipains is poorly understood. In this report, we found that insertion mutants for PG0809 (83K1 and 83K2) were defective in black pigmentation and hemolysis. We cloned and sequenced PG0809 and found that PG0809 contains two additional nucleotides that are not deposited in the W83 genome database. The revised sequence reveals an in-frame fusion of PG0810 and PG0809 and is designated the sov gene. We constructed a sov deletion mutant (83K3) and showed that 83K3 was defective in the activities of black pigmentation, hemolysis, and hemagglutination. Furthermore, in 83K3, the activities of gingipains were severely reduced whereas those of other secreted proteases DPPIV, DPP-7, and PtpA were not affected. Immunoblot analysis using anti-RgpB antiserum showed that Arg-gingipains were poorly secreted in an outer membrane or into an extracellular portion but accumulated within the cells of 83K3, suggesting the secretion of gingipains is defected in 83K3. Taken together, our findings indicated that Sov is a novel protein required for the secretion of gingipains and suggested that the secretion system for gingipains is different from the conserved secretion systems.     

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gingivalis strain 381

We previously reported the existence of two different kinds of fimbriae expressed by Porphyromonas gingivalis ATCC 33277. In this study, we isolated and characterized a secondary fimbrial protein from strain FPG41, a fimA-inactivated mutant of P. gingivalis 381. FPG41 was constructed by a homologous recombination technique using a mobilizable suicide vector, and failed to express the long fimbriae (41-kDa fimbriae) that were produced on the cell surface of P. gingivalis 381. However, short fimbrial structures were observed on the cell surface of FPG41 by electron microscopy. The fimbrial protein was purified from FPG41 by DEAE-Sepharose CL-6B column chromatography. The secondary fimbrial protein was eluted at 0.15 M NaCl, and the molecular mass of this protein was approximately 53 kDa as estimated by SDS-PAGE. An antibody against the 53-kDa fimbrial protein reacted with the short fimbriae of the FPG41 and the wild-type strain. However, the 41-kDa long fimbriae of the wild-type strain and the 67-kDa fimbriae of ATCC 33277 did not react with the same antibody. Moreover, the N-terminal amino acid sequence of the 53-kDa fimbrial protein showed only 2 of 15 residues that were identical to those of the 41-kDa fimbrial protein. These results show that the properties of the 53-kDa fimbriae are different from those of the 67-kDa fimbriae of ATCC 33277 as well as those of the 41-kDa fimbriae.     

Phospholipid analogues of Porphyromonas gingivalis

Porphyromonas has lipids containing hydroxy acids and C16:0 and iso-C15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209-299 were consistent with the presence of the C13: 0, C15: 0, C16: 0 and C18: 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618-961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidentified lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue profiles obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.     

Characterization of a hemophore-like protein from Porphyromonas gingivalis

The porphyrin auxotrophic pathogen Porphyromonas gingivalis obtains the majority of essential iron and porphyrin from host hemoproteins. To achieve this, the organism expresses outer membrane gingipains containing cysteine proteinase domains linked to hemagglutinin domains. Heme mobilized in this way is taken up by P. gingivalis through a variety of potential portals where HmuY/HmuR of the hmu locus are best described. These receptors have relatively low binding affinities for heme. In this report, we describe a novel P. gingivalis protein, HusA, the product of PG2227, which rapidly bound heme with a high binding constant at equilibrium of 7 × 10(-10) M. HusA is both expressed on the outer membrane and released from the organism. Spectral analysis indicated an unusual pattern of binding where heme was ligated preferentially as a dimer. Further, the presence of dimeric heme induced protein dimer formation. Deletional inactivation of husA showed that expression of this moiety was essential for growth of P. gingivalis under conditions of heme limitation. This finding was in accord with the pronounced increase in gene expression levels for husA with progressive reduction of heme supplementation. Antibodies reactive against HusA were detected in patients with chronic periodontitis, suggesting that the protein is expressed during the course of infection by P. gingivalis. It is predicted that HusA efficiently sequesters heme from gingipains and fulfills the function of a high affinity hemophore-like protein to meet the heme requirement for growth of P. gingivalis during establishment of infection.     

PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.     

The Clostridium ramosum IgA proteinase represents a novel type of metalloendopeptidase

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.     

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ～70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.     

Binding of a histidine-rich peptide to Porphyromonas gingivalis

Porphyromonas gingivalis 381 cells were incubated with 125I-histidine-rich polypeptide (histatin) 5 in the presence or absence of unlabeled histatin 5, to evaluate the histatin-binding capacity of the cells. The binding of histatin 5 was rapid, reversible, saturable and specific. The number of histatin 5-binding sites per cell was 3,600, and the dissociation constant (Kd) was in the order of 10(-6) M. These findings suggest that histatin interacts with certain bacterial cells through specific binding sites on their surface, and will allow the development of a histatin radioreceptor assay.     

Inhibitors of benzamidine type influence the virulence properties of Porphyromonas gingivalis strains

Synthetic inhibitors of benzamidine type have been found to have inhibiting effects on arginine specific cysteine proteinases of P. gingivalis. The purpose of our study was to assess the effects of these inhibitors on the virulence properties of two P. gingivalis strains, the reference strain ATCC 33277 and JH16-1, a clinical isolate obtained from a patient with severe periodontitis. The inhibitors tested were pentamidine, benzamidine, three bis-benzamidine derivatives with a pentamidine-related structure, one bis-benzamidine derivative with another structure, and one arginine derivative as a negative control, each in the concentrations of 2 microM and 20 microM. As virulence criteria the following parameters were determined: arginine-specific amidolytic activity, growth inhibition, hemagglutination of sheep erythrocytes, adherence to KB cells and immuno-phagocytosis including intracellular killing. Pentamidine and the bis-benzamidine derivatives with pentamidine-related structure showed the most remarkable effects on reduction of amidolytic activity by 35%, growth inhibition and reduced hemagglutination. Except for the arginine derivative all other inhibitors tested enhanced the phagocytosis capacities of granulocytes. No clear influence of the inhibitors on adherence of P. gingivalis to KB cells was seen. Although in vitro effects of the synthetic inhibitors of cysteine proteinases on virulence of P. gingivalis were observed further in vitro tests concerning immunomodulatory effects should be done before these substances are used for therapy in clinically controlled studies.     

牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用

目的探讨牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2,PgHA-2）的氯化血红素结合位点多肽对牙龈卟啉单胞菌（Porphyromonasgingivalis,Pg）摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK（肽1）,DEYAVMISK（肽2,肽1中第2位氨基酸突变为谷氨酸）,ALHPDHYLI（肽3,HA-2结合位点不相关多肽）。将肽l、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2（Porphyromonas gingivalis recombinant HA-2,PgrHA-2）,收集与氯化血红素结合的PgrHA-2,SDS—PAGE电泳,分析多肽对PgrHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制PgrHA-2与氯化血红素结合,而肽2和肽3对PgrHA-2与氯化血红素的结合无抑制作用。在24、48和72h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低（P〈0．05）。结论本研究表明PgHA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。     

Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate‐affinity chromatography

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_0880, having been separated by phosphate‐affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P‐Ser), P‐Thr, and/or P‐Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis.