Neisseria gonorrhoeae Population Genomics: Use of the Gonococcal Core Genome to Improve Surveillance of Antimicrobial Resistance.

BACKGROUND: Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a globally prevalent sexually transmitted infection. The dynamics of gonococcal population biology have been poorly defined due to a lack of resolution in strain typing methods. METHODS: In this study, we assess how the core genome can be used to improve our understanding of gonococcal population structure compared with current typing schemes. RESULTS: A total of 1668 loci were identified as core to the gonococcal genome. These were organized into a core genome multilocus sequence typing scheme (N gonorrhoeae cgMLST v1.0). A clustering algorithm using a threshold of 400 allelic differences between isolates resolved gonococci into discrete and stable core genome groups, some of which persisted for multiple decades. These groups were associated with antimicrobial genotypes and non-overlapping NG-STAR and NG-MAST sequence types. The MLST-STs were more widely distributed among core genome groups. CONCLUSIONS: Clustering with cgMLST identified globally distributed, persistent, gonococcal lineages improving understanding of the population biology of gonococci and revealing its population structure. These findings have implications for the emergence of antimicrobial resistance in gonococci and how this is associated with lineages, some of which are more predisposed to developing antimicrobial resistance than others.

Neisseria gonorrhoeae-Induced Inflammatory Pyroptosis in Human Macrophages is Dependent on Intracellular Gonococci and Lipooligosaccharide.

Neisseria gonorrhoeae, the human obligate pathogen responsible for the sexually transmitted disease gonorrhea, has evolved several mechanisms to evade the host immune response. One such mechanism is the modulation of host cell death pathways. In this study, we defined cell death pathways induced by N gonorrhoeae in human monocyte-derived macrophages (MDMs). In a dose-dependent manner, N gonorrhoeae stimulation of MDMs resulted in caspase 1 and 4-dependent cell deaths, indicative of canonical and noncanonical pyroptosis, respectively. Internalization of bacteria or stimulation with lipooligosaccharide (LOS) specifically induced pyroptosis in MDMs and increased secretion of IL-1ß. Collectively, our results demonstrate that N gonorrhoeae induces inflammatory pyroptosis in human macrophages due in part to intracellular LOS. We propose that this in turn may exacerbate inflammatory outcomes observed during mucosal infection.

Distinct roles for dietary lipids and Porphyromonas gingivalis infection on atherosclerosis progression and the gut microbiota.

Mounting evidence in humans supports an etiological role for the microbiota in inflammatory atherosclerosis. Atherosclerosis is a progressive disease characterized by accumulation of inflammatory cells and lipids in vascular tissue. While retention of lipoprotein into the sub-endothelial vascular layer is believed to be the initiating stimulus leading to the development of atherosclerosis, activation of multiple pathways related to vascular inflammation and endothelial dysfunction sustain the process by stimulating recruitment of leukocytes and immune cells into the sub-endothelial layer. The Gram-negative oral pathogen Porphyromonas gingivalis has been associated with the development and acceleration of atherosclerosis in humans and these observations have been validated in animal models. It has been proposed that common mechanisms of immune signaling link stimulation by lipids and pathogens to vascular inflammation. Despite the common outcome of P. gingivalis and lipid feeding on atherosclerosis progression, we established that these pro-atherogenic stimuli induced distinct gene signatures in the ApoE-/- mouse model of atherosclerosis. In this study, we further defined the distinct roles of dietary lipids and P. gingivalis infection on atherosclerosis progression and the gut microbiota. We demonstrate that diet-induced lipid lowering resulted in less atherosclerotic plaque in ApoE-/- mice compared to ApoE-/- mice continuously fed a Western diet. However, the effect of diet-induced lipid lowering on plaque accumulation was blunted by P. gingivalis infection. Using principal component analysis and hierarchical clustering, we demonstrate that dietary intervention as well as P. gingivalis infection result in distinct bacterial communities in fecal and cecal samples of ApoE-/- mice as compared to ApoE-/- mice continuously fed either a Western diet or a normal chow diet. Collectively, we identified distinct microbiota changes accompanying atherosclerotic plaque, suggesting a future avenue for investigation on the impact of the gut microbiota, diet, and P. gingivalis infection on atherosclerosis.

Microbiota, Immune Subversion, and Chronic Inflammation.

Several host-adapted pathogens and commensals have evolved mechanisms to evade the host innate immune system inducing a state of low-grade inflammation. Epidemiological studies have also documented the association of a subset of these microorganisms with chronic inflammatory disorders. In this review, we summarize recent studies demonstrating the role of the microbiota in chronic inflammatory diseases and discuss how specific microorganisms subvert or inhibit protective signaling normally induced by toll-like receptors (TLRs). We highlight our work on the oral pathogen Porphyromonas gingivalis and discuss the role of microbial modulation of lipid A structures in evasion of TLR4 signaling and resulting systemic immunopathology associated with atherosclerosis. P. gingivalis intrinsically expresses underacylated lipid A moieties and can modify the phosphorylation of lipid A, leading to altered TLR4 signaling. Using P. gingivalis mutant strains expressing distinct lipid A moieties, we demonstrated that expression of antagonist lipid A was associated with P. gingivalis-mediated systemic inflammation and immunopathology, whereas strains expressing agonist lipid A exhibited modest systemic inflammation. Likewise, mice deficient in TLR4 were more susceptible to vascular inflammation after oral infection with P. gingivalis wild-type strain compared to mice possessing functional TLR4. Collectively, our studies support a role for P. gingivalis-mediated dysregulation of innate and adaptive responses resulting in immunopathology and systemic inflammation. We propose that anti-TLR4 interventions must be designed with caution, given the balance between the protective and destructive roles of TLR signaling in response to microbiota and associated immunopathologies.

Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses.

The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses.

Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network.

UNLABELLED: The Neisseria gonorrhoeae ferric uptake regulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of an N. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in an N. gonorrhoeae wild-type or fur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in the fur strain, an effect that was reversed in a fur-complemented strain. Fur was shown to bind to the promoter region of the arsR gene downstream of a predicted σ(70) promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to the norB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcal arsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type and arsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that the N. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins. IMPORTANCE: Gene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic bacteria, successful infection. Bacterial DNA binding proteins are a common mechanism utilized by pathogens to control gene expression under various environmental conditions. Here, we show that the DNA binding protein Fur, expressed by the human pathogen Neisseria gonorrhoeae, controls the expression of a large repertoire of genes and extends this regulon by controlling expression of additional DNA binding proteins. One of these proteins, an ArsR-like regulator, was required for N. gonorrhoeae survival within host cells. These results show that the Fur regulon extends to additional regulatory proteins, which together contribute to gonococcal mechanisms of pathogenesis.

A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

Fur-mediated global regulatory circuits in pathogenic Neisseria species.

The ferric uptake regulator (Fur) protein has been shown to function as a repressor of transcription in a number of diverse microorganisms. However, recent studies have established that Fur can function at a global level as both an activator and a repressor of transcription through both direct and indirect mechanisms. Fur-mediated indirect activation occurs via the repression of additional repressor proteins, or small regulatory RNAs, thereby activating transcription of a previously silent gene. Fur mediates direct activation through binding of Fur to the promoter regions of genes. Whereas the repressive mechanism of Fur has been thoroughly investigated, emerging studies on direct and indirect Fur-mediated activation mechanisms have revealed novel global regulatory circuits.

Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

Macrophage-elicited osteoclastogenesis in response to bacterial stimulation requires Toll-like receptor 2-dependent tumor necrosis factor-alpha production.

The receptor activator of NF-kappaB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-alpha)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-alpha and occurred independently of RANKL, interleukin-1beta (IL-1beta), and IL-6. CS fluids from P. gingivalis-stimulated TLR2(-/-) macrophages failed to express TNF-alpha, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4(-/-) macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-alpha production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-alpha-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.

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.

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells.

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.

Identification of amino acid residues involved in heme binding and hemoprotein utilization in the Porphyromonas gingivalis heme receptor HmuR.

We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in Porphyromonas gingivalis. To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of P. gingivalis A7436 hmuR mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both P. gingivalis and Escherichia coli. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of P. gingivalis when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the P. gingivalis HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.

Pathogen-accelerated atherosclerosis occurs early after exposure and can be prevented via immunization.

Here we report on early inflammatory events associated with Porphyromonas gingivalis-accelerated atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice. Animals challenged with P. gingivalis presented with increased macrophage infiltration, innate immune marker expression, and atheroma without elevated systemic inflammatory mediators. This early local inflammatory response was prevented in mice immunized with P. gingivalis. We conclude that localized up-regulation of innate immune markers early after infection, rather than systemic inflammation, contributes to pathogen-accelerated atherosclerosis.

Gingipain-specific IgG in the sera of patients with periodontal disease is necessary for opsonophagocytosis of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is a primary etiologic agent of generalized aggressive periodontitis (GAgP), and gingipains, a group of cysteine proteinases, are critical virulence factors expressed by this organism. GAgP patients develop specific antibodies to gingipains; however, the function of these antibodies in the clearance of P. gingivalis infection is poorly understood. METHODS: In this study, we defined the levels of gingipain-specific antibodies in GAgP patient sera and examined the ability of gingipain-specific antibodies to facilitate opsonophagocytosis of P. gingivalis by human polymorphonuclear leukocytes (PMNs) using a fluorescent phagocytosis assay. RESULTS: GAgP patient sera possessed elevated levels of P. gingivalis-, arginine-gingipain (Rgp)A-, RgpB-, and lysine-gingipain (Kgp)-specific IgG (Kgp > RgpA > P. gingivalis > RgpB). Adsorption of GAgP sera with P. gingivalis whole organisms, RgpA, RgpB, and Kgp conjugated to sepharose beads reduced opsonophagocytosis of P. gingivalis by PMNs. CONCLUSIONS: Our studies demonstrate that GAgP patient sera possess elevated levels of P. gingivalis- and gingipain-specific IgG. Furthermore, we show that gingipain antibodies promote uptake of P. gingivalis by PMNs, and our data suggest that gingipain-specific antibodies may be important for the control of P. gingivalis infections.

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.

Coordinate expression of the Porphyromonas gingivalis lysine-specific gingipain proteinase, Kgp, arginine-specific gingipain proteinase, RgpA, and the heme/hemoglobin receptor, HmuR.

Heme utilization in Porphyromonas gingivalis requires the participation of an outer membrane hemin/hemoglobin receptor, HmuR, the lysine-specific gingipain proteinase (Kgp) and arginine-specific gingipain proteinase (Rgp). In this study, the expression of hmuR , kgp and rgpA genes in response to growth with different heme sources was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunoassay. Coordinate regulation of hmuR , kgp and rgpA gene expression was evaluated through utilization of P. gingivalis hmuR and kgp mutants or by selective inactivation of proteinases with Kgp- and Rgp-specific inhibitors. We observed that expression of the kgp and rgpA genes was not tightly regulated by heme, but rather by the growth phase. In contrast, expression of the hmuR gene was negatively regulated by heme, while growth of P. gingivalis with human serum resulted in increased hmuR expression. A P. gingivalis kgp isogenic mutant demonstrated significantly increased hmuR gene expression, and inactivation of Kgp and Rgp activity by specific inhibitors up-regulated hmuR gene transcription. Moreover, inactivation of Kgp up-regulated rgpA transcription. Finally, a P. gingivalis hmuR mutant exhibited repressed kgp gene expression and lysine-specific proteinase activity. Collectively, these results indicate that kgp , rgpA and hmuR gene transcription is coordinately regulated and may facilitate greater efficiency of heme utilization in P. gingivalis .

Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Infectious diseases have emerged as potential risk factors for cardiovascular disease (CVD). Epidemiological studies support a connection between periodontal disease, a chronic inflammatory disease of the supporting tissues of the teeth, and CVD. METHODS AND RESULTS: To directly test the connection between periodontal disease and atherosclerosis, apoE-/- mice were orally challenged with the periodontal disease pathogen Porphyromonas gingivalis or an invasion-impaired P gingivalis fimbriae-deficient mutant (FimA-). Both wild-type P gingivalis and the FimA- mutant were detected in blood and aortic arch tissue of apoE-/- mice by PCR after challenge. ApoE-/- mice challenged with wild-type P gingivalis presented with increased atherosclerotic plaque and expressed the innate immune response markers Toll-like receptor (TLR)-2 and TLR-4 in aortic tissue. Despite detection of the FimA- mutant in the blood and in aortic arch tissue, apoE-/- mice challenged with the FimA- mutant did not present with periodontal disease, upregulation of TLRs, or accelerated atherosclerosis. Furthermore, we demonstrate that immunization to control P gingivalis-elicited periodontal disease concomitantly prevents P gingivalis-accelerated atherosclerosis. CONCLUSIONS: We conclude that invasive P gingivalis accelerates atherosclerosis.