Iron and heme utilization in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with the initiation and progression of adult periodontal disease. Iron is utilized by this pathogen in the form of heme and has been shown to play an essential role in its growth and virulence. Recently, considerable attention has been given to the characterization of various secreted and surface-associated proteins of P. gingivalis and their contribution to virulence. In particular, the properties of proteins involved in the uptake of iron and heme have been extensively studied. Unlike other Gram-negative bacteria, P. gingivalis does not produce siderophores. Instead it employs specific outer membrane receptors, proteases (particularly gingipains), and lipoproteins to acquire iron/heme. In this review, we will focus on the diverse mechanisms of iron and heme acquisition in P. gingivalis. Specific proteins involved in iron and heme capture will be described. In addition, we will discuss new genes for iron/heme utilization identified by nucleotide sequencing of the P. gingivalis W83 genome. Putative iron- and heme-responsive gene regulation in P. gingivalis will be discussed. We will also examine the significance of heme/hemoglobin acquisition for the virulence of this pathogen.     

Tobacco-induced alterations to Porphyromonas gingivalis–host interactions

Smokers are more susceptible than non-smokers to persistent infection by Porphyromonas gingivalis , a causative agent of periodontitis. Patients who smoke exhibit increased susceptibility to periodontitis and are more likely to display severe disease and be refractory to treatment. Paradoxically, smokers demonstrate reduced clinical inflammation. We show that P. gingivalis cells exposed to cigarette smoke extract (CSE) induce a lower proinflammatory response (tumour necrosis factor-α, interleukin-6, interleukin-12 p40) from monocytes and peripheral blood mononuclear cells than do unexposed bacteria. This effect is reversed when CSE-exposed bacteria are subcultured in fresh medium without CSE. Using microarrays representative of the P. gingivalis genome, CSE-exposure resulted in differential regulation of 6.8% of P. gingivalis genes, including detoxification and oxidative stress-related genes; DNA repair genes; and multiple genes related to P. gingivalis virulence, including genes in the major fimbrial and capsular operons. Exposure to CSE also altered the expression of outer membrane proteins, most notably by inducing the virulence factors RagA and RagB, and a putative lipoprotein cotranscribed with the minor fimbrial antigen. Therefore, CSE represents an environmental stress to which P. gingivalis adapts by altering gene expression and outer membrane proteins. These changes may explain, in part, the altered virulence and host–pathogen interactions that have been documented in vivo in smokers with periodontal disease.     

Gingipains from Porphyromonas gingivalis synergistically induce the production of proinflammatory cytokines through protease-activated receptors with Toll-like receptor and NOD1/2 ligands in human monocytic cells

Gingipains (HRgpA, RgpB and Kgp) are cysteine proteinases and virulence factors of Porphyromonas gingivalis , the major causative bacterium of periodontal disease. To study synergistic effects of gingipains and signalling via Toll-like receptors (TLRs) and NOD1/2, we investigated effects of a gingipain on the secretion of proinflammatory cytokines from monocytic THP-1 cells in the presence of pathogen-associated molecular patterns (PAMPs). Gingipains stimulated interleukin (IL)-8's secretion from THP-1 cells, which was completely inhibited by proteinase inhibitors of gingipain and increased in the presence of PAMPs. Synergistic effects of gingipains and PAMPs were also seen in the secretion of IL-6 and MCP-1 and reduced to about 50% the secretion of IL-8 from THP-1 cells treated with siRNA targeting either protease-activated receptor (PAR)-1, -2 or -3. PAR agonist peptides mimicked the synergistic effects of gingipains with PAMPs. These results indicate that gingipains stimulate the secretion of cytokines from monocytic cells through the activation of PARs with synergistic effects by PAMPs. This is the first report of synergism of signalling via PARs, and TLRs or NOD1/2. The host defence system against P. gingivalis may be triggered through the activation of PARs by gingipains and augmented by PAMPs from this pathogen via TLRs or NOD1/2.     

Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.     

RIPK4 activates an IRF6-mediated proinflammatory cytokine response in keratinocytes

Keratinocytes of the oral mucosa and epidermis play key roles in host defense. In addition to functioning as a physical barrier, they also produce cytokines to elicit inflammation in response to infection or injury. We recently established that receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) function as a cell-intrinsic signaling axis to regulate keratinocyte differentiation. In this study, we have demonstrated a functional relationship between RIPK4 and IRF6 in the control of proinflammatory cytokine expression in keratinocytes. The overexpression of RIPK4 by oral keratinocytes induced the strong expression of CCL5 and CXCL11. In contrast, the expression of other cytokines (e.g. IL8 and TNF) was largely unaffected, thus demonstrating specificity in the induction of proinflammatory cytokine expression by RIPK4. CCL5 and CXCL11 expression were also induced in response to the activation of the PKC pathway, and gene silencing experiments indicated that their inducible expression was dependent on RIPK4 and IRF6. Moreover, gene reporter assays suggested that RIPK4 induces CCL5 and CXCL11 expression by stimulating the transactivation of their promoters by IRF6. Accordingly, our findings suggest that the RIPK4-IRF6 signaling axis plays a multifaceted role in barrier epithelial homeostasis through its regulation of both keratinocyte inflammation and differentiation.     

Porphyromonas gingivalis regulates the RANKL-OPG system in bone marrow stromal cells

Porphyromonas gingivalis is a Gram-negative anaerobe implicated in chronic periodontitis, a bacterial-induced inflammatory condition that causes destruction of the periodontal connective tissues and underlying alveolar bone. The receptor activator of nuclear factor-kappaB ligand (RANKL) is a cytokine that directly stimulates osteoclastogenesis and bone resorption, whereas its decoy receptor osteoprotegerin (OPG) blocks this action. This study aimed to investigate the effects of P. gingivalis culture supernatants on RANKL and OPG expression in W20-17 bone marrow stromal cells, and evaluate the involvement of its virulence factors, particularly gingipains and lipopolysaccharide. P. gingivalis up-regulated RANKL and down-regulated OPG mRNA expression and protein production. These effects were blocked by indomethacin, suggesting mediation by prostaglandins. Furthermore, P gingivalis induced the production of prostaglandin E(2). Heat-inactivation, or chemical inhibition of P. gingivalis gingipains did not affect RANKL and OPG regulation. However, lipopolysaccharide depletion by polymyxin B abolished RANKL induction, and partly rescued the suppression of OPG. In conclusion, P. gingivalis regulates the RANKL-OPG system via prostaglandin E(2) in bone marrow stromal cells, in a manner that favours osteoclastogenesis. A non-proteolytic and non-proteinaceous P. gingivalis component is involved in these events, most probably its lipopolysaccharide. This activity may contribute to the bone loss characteristic of periodontitis.     

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.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

IFN-gamma alters the response of Borrelia burgdorferi-activated endothelium to favor chronic inflammation

Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-gamma in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-gamma on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-gamma synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1beta, TNF-alpha, and LPS also cooperated with IFN-gamma to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-gamma potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-gamma, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-gamma diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-gamma thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.     

Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.     

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.     

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.     

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.