Structural Identification of Lipid-α: A Glycosyl Lipid Involved in Oligo- And Polysaccharides Metabolism in Streptococcus agalactiae (Group B Streptococcus).

Streptococcus agalactiae (group B Streptococcus, GBS) is a gram-positive bacterium that is an asymptomatic colonizer commonly found in the gastrointestinal and genitourinary tract of healthy adults. GBS is also the most common cause of life-threatening bacterial infections in newborns and is emerging as a pathogen in immunocompromised and diabetic adults. The GBS cell wall and covalently linked capsular polysaccharides (CPS) are vital to the protection of the bacterial cell and act as virulence factors. GBS-CPS have been successfully used to produce conjugate vaccines for all currently identified GBS serotypes. However, the mechanisms of biosynthesis and assembly of CPS and the other cell wall components remain poorly defined due to their complex surface structures. In this biosynthetic study of the GBS cell wall-CPS complex, glycolipids with varying lengths of glycosyl-chains were discovered. Among those, one of the smallest glycolipids (named GBS Lipid-α) was structurally characterized. Lipid-α is involved in GBS saccharide metabolism and presumably acts as a glycosyl acceptor to elongate the glycosyl chain. GBS Lipid-α was determined to be a 3-monosaccharide 1,2 acyl glycerol with a molecular mass in the range of m/z = 724-808. GBS Lipid-α is highly heterogenic with various acyl groups and glycosyl moieties. This knowledge will pave the way for future studies to elucidate the entire metabolic pathway and genes involved. The Lipid-α pathway may also exist in other bacterial species and has the potential to be a biomarker for future drug development.

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis.

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1ß (IL-1ß), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.

Immunization with cell-free-generated vaccine protects from Porphyromonas gingivalis-induced alveolar bone loss.

INTRODUCTION: Periodontal diseases (PD) are complex oral inflammatory diseases initiated by keystone bacteria such as Porphyromonas gingivalis. A vaccine for PD is desirable as clinical treatment involves protracted maintenance strategies aimed to retain dentition. Although prior immunization approaches targeting P. gingivalis have reported variable success in limiting facets of disease such as oral bone loss, it remains that a vaccine for this disease may be attainable. AIM: To investigate cell-free protein synthesis (CFPS) as a platform to produce vaccinable targets suitable for efficacy testing in a P. gingivalis-induced murine oral bone loss model. MATERIALS AND METHODS: Recombinantly generated P. gingivalis minor fimbriae protein (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were combined in equivalent doses in adjuvants and injected intramuscularly to immunize mice. Serum levels of protein-specific antibody were measured by ELISA, and oral bone levels were defined by morphometrics. RESULTS: Recombinantly generated P. gingivalis proteins possessed high fidelity to predicted size and elicited protein-specific IgG following immunization. Importantly, immunization with the vaccine cocktail protected from P. gingivalis elicited oral bone loss. CONCLUSION: These data verify the utility of the CFPS technology to synthesize proteins that have the capacity to serve as novel vaccines.

Cell Wall Hydrolytic Enzymes Enhance Antimicrobial Drug Activity Against Mycobacterium.

Cell wall hydrolases are enzymes that cleave bacterial cell walls by hydrolyzing specific bonds within peptidoglycan and other portions of the envelope. Two major sources of hydrolases in nature are from hosts and microbes. This study specifically investigated whether cell wall hydrolytic enzymes could be employed as exogenous reagents to augment the efficacy of antimicrobial agents against mycobacteria. Mycobacterium smegmatis cultures were treated with ten conventional antibiotics and six anti-tuberculosis drugs-alone or in combination with cell wall hydrolases. Culture turbidity, colony-forming units (CFUs), vital staining, and oxygen consumption were all monitored. The majority of antimicrobial agents tested alone only had minimal inhibitory effects on bacterial growth. However, the combination of cell wall hydrolases and most of the antimicrobial agents tested, revealed a synergistic effect that resulted in significant enhancement of bactericidal activity. Vital staining showed increased cellular damage when M. smegmatis and Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) were treated with both drug and lysozyme. Respiration analysis revealed stress responses when cells were treated with lysozyme and drugs individually, and an acute increase in oxygen consumption when treated with both drug and lysozyme. Similar trends were also observed for the other three enzymes (hydrolase-30, RipA-His6 and RpfE-His6) evaluated. These findings demonstrated that cell wall hydrolytic enzymes, as a group of biological agents, have the capability to improve the potency of many current antimicrobial drugs and render ineffective antibiotics effective in killing mycobacteria. This combinatorial approach may represent an important strategy to eliminate drug-resistant bacteria.

Macrophage-specific TLR2 signaling mediates pathogen-induced TNF-dependent inflammatory oral bone loss.

Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf(-/-)) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.

Distinct gene signatures in aortic tissue from ApoE-/- mice exposed to pathogens or Western diet.

BACKGROUND: Atherosclerosis is a progressive disease characterized by inflammation and accumulation of lipids in vascular tissue. Porphyromonas gingivalis (Pg) and Chlamydia pneumoniae (Cp) are associated with inflammatory atherosclerosis in humans. Similar to endogenous mediators arising from excessive dietary lipids, these Gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. In this study, we identified gene expression profiles that characterize P. gingivalis, C. pneumoniae, and Western diet (WD) at acute and chronic time points in aortas of Apolipoprotein E (ApoE-/-) mice. RESULTS: At the chronic time point, we observed that P. gingivalis was associated with a high number of unique differentially expressed genes compared to C. pneumoniae or WD. For the top 500 differentially expressed genes unique to each group, we observed a high percentage (76%) that exhibited decreased expression in P. gingivalis-treated mice in contrast to a high percentage (96%) that exhibited increased expression in WD mice. C. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. Gene Set Enrichment Analysis (GSEA) revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and PPAR pathways in response to P. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and PPAR pathways in response to C. pneumoniae; WD was associated with increased expression of immune and inflammatory pathways. DAVID analysis of gene clusters identified by two-way ANOVA at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in ApoE-/- mice; these changes were enhanced in P. gingivalis-treated mice but attenuated in C. pneumoniae-treated mice. Notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli. CONCLUSIONS: Despite the common outcome of P. gingivalis, C. pneumoniae, and WD on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified. Our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways.

Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.

Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via TLRs link stimulation by multiple pathogens to atherosclerosis. However, how pathogen-specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood. In this study, atherosclerosis-prone apolipoprotein-E null (ApoE(-/-)) and TLR4-deficient (ApoE(-/-)TLR4(-/-)) mice were orally infected with the periodontal pathogen Porphyromonas gingivalis. ApoE(-/-)TLR4(-/-) mice were markedly more susceptible to atherosclerosis after oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE(-/-)TLR4(-/-) mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of Th1 immunity and regulatory T cell infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell IL-12 and IL-10, which are prototypic Th1 and regulatory T cell polarizing cytokines. We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.

A Porphyromonas gingivalis hypothetical protein controlled by the type I-C CRISPR-Cas system is a novel adhesin important in virulence.

The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.

Characterization of a Bacterial Kinase That Phosphorylates Dihydrosphingosine to Form dhS1P.

Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation. IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.

A Porphyromonas gingivalis Capsule-Conjugate Vaccine Protects From Experimental Oral Bone Loss.

Periodontal diseases are chronic inflammatory diseases of the periodontium that result in progressive destruction of the soft and hard tissues supporting the teeth, and it is the most common cause of tooth loss among adults. In the US alone, over 100 million individuals are estimated to have periodontal disease. Subgingival bacteria initiate and sustain inflammation, and, although several bacteria have been associated with periodontitis, Porphyromonas gingivalis has emerged as the key etiological organism significantly contributing to the disease. Currently, intensive clinical maintenance strategies are deployed to mitigate the further progression of disease in afflicted individuals; however, these treatments often fail to stop disease progression, and, as such, the development of an effective vaccine for periodontal disease is highly desirable. We generated a conjugate vaccine, comprising of the purified capsular polysaccharide of P. gingivalis conjugated to eCRM®, a proprietary and enhanced version of the CRM197 carrier protein with predetermined conjugation sites (Pg-CV). Mice immunized with alum adjuvanted Pg-CV developed robust serum levels of whole organism-specific IgG in comparison to animals immunized with unconjugated capsular polysaccharide alone. Using the murine oral bone loss model, we observed that mice immunized with the capsule-conjugate vaccine were significantly protected from the effects of P. gingivalis-elicited oral bone loss. Employing a preclinical model of infection-elicited oral bone loss, our data support that a conjugate vaccine incorporating capsular polysaccharide antigen is effective in reducing the main clinical endpoint of periodontal disease-oral bone destruction. Further development of a P. gingivalis capsule-based conjugate vaccine for preventing periodontal diseases is supported.

Immune response of macrophages from young and aged mice to the oral pathogenic bacterium Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1ß, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.

Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalis In vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalis IMPORTANCE Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.

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.

Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis.

Toll-like receptors (TLRs) are a group of pathogen-associated molecular pattern receptors, which play an important role in innate immune signaling in response to microbial infection. It has been demonstrated that TLRs are differentially up regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. The expression of TLRs are markedly augmented in human atherosclerotic lesions and this occurs preferentially by endothelial cells and macrophages in areas infiltrated with inflammatory cells. Furthermore polymorphisms in the human gene encoding one TLR receptor (TLR4) which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with low levels of certain circulating mediators of inflammation and a decreased risk for atherosclerosis in humans. Recent advances have established a fundamental role for inflammation in mediating all stages of atherosclerosis. However, the triggers that initiate and sustain the inflammatory process have not been definitively identified. Although definitive proof of a role of infection contributing to atherogenesis is lacking, multiple investigations have demonstrated that infectious agents evoke cellular and molecular changes supportive of such a role. Evidence in humans suggesting that periodontal infection predisposes to atherosclerosis is derived from studies demonstrating that the periodontal pathogen Porphyromonas gingivalis resides in the wall of atherosclerotic vessels and seroepidemiological studies demonstrating an association between pathogen-specific IgG antibodies and atherosclerosis. Our recent work with P. gingivalis has demonstrated the effectiveness of specific intervention strategies (immunization) in the prevention of pathogen-accelerated atherosclerosis. We have also established that the inflammatory signaling pathways that P. gingivalis utilizes is dependent on the cell type and this specificity clearly influences innate immune signaling in the context of local chronic inflammation versus distant chronic inflammation. We postulate that bacterial infection mediates inflammatory responses that involve specific innate immune pathways in defined host cells. Furthermore, these inflammatory responses can be correlated with atherosclerosis and ultimately thrombotic complications.

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.

Linkage of Infection to Adverse Systemic Complications: Periodontal Disease, Toll-Like Receptors, and Other Pattern Recognition Systems.

Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that provide innate immune sensing of conserved pathogen-associated molecular patterns (PAMPs) to engage early immune recognition of bacteria, viruses, and protozoa. Furthermore, TLRs provide a conduit for initiation of non-infectious inflammation following the sensing of danger-associated molecular patterns (DAMPs) generated as a consequence of cellular injury. Due to their essential role as DAMP and PAMP sensors, TLR signaling also contributes importantly to several systemic diseases including cardiovascular disease, diabetes, and others. The overlapping participation of TLRs in the control of infection, and pathogenesis of systemic diseases, has served as a starting point for research delving into the poorly defined area of infection leading to increased risk of various systemic diseases. Although conflicting studies exist, cardiovascular disease, diabetes, cancer, rheumatoid arthritis, and obesity/metabolic dysfunction have been associated with differing degrees of strength to infectious diseases. Here we will discuss elements of these connections focusing on the contributions of TLR signaling as a consequence of bacterial exposure in the context of the oral infections leading to periodontal disease, and associations with metabolic diseases including atherosclerosis and type 2 diabetes.

Signaling events in pathogen-induced macrophage foam cell formation.

Macrophage foam cell formation is a key event in atherosclerosis. Several triggers induce low-density lipoprotein (LDL) uptake by macrophages to create foam cells, including infections with Porphyromonas gingivalis and Chlamydia pneumoniae, two pathogens that have been linked to atherosclerosis. While gene regulation during foam cell formation has been examined, comparative investigations to identify shared and specific pathogen-elicited molecular events relevant to foam cell formation are not well documented. We infected mouse bone marrow-derived macrophages with P. gingivalis or C. pneumoniae in the presence of LDL to induce foam cell formation, and examined gene expression using an atherosclerosis pathway targeted plate array. We found over 30 genes were significantly induced in response to both pathogens, including PPAR family members that are broadly important in atherosclerosis and matrix remodeling genes that may play a role in plaque development and stability. Six genes mainly involved in lipid transport were significantly downregulated. The response overall was remarkably similar and few genes were regulated in a pathogen-specific manner. Despite very divergent lifestyles, P. gingivalis and C. pneumoniae activate similar gene expression profiles during foam cell formation that may ultimately serve as targets for modulating infection-elicited foam cell burden, and progression of atherosclerosis.

Porphyromonas gingivalis-mediated signaling through TLR4 mediates persistent HIV infection of primary macrophages.

Periodontal infections contribute to HIV-associated co-morbidities in the oral cavity and provide a model to interrogate the dysregulation of macrophage function, inflammatory disease progression, and HIV replication during co-infections. We investigated the effect of Porphyromonas gingivalis on the establishment of HIV infection in monocyte-derived macrophages. HIV replication in macrophages was significantly repressed in the presence of P. gingivalis. This diminished viral replication was due partly to a decrease in the expression of integrated HIV provirus. HIV repression depended upon signaling through TLR4 as knock-down of TLR4 with siRNA rescued HIV expression. Importantly, HIV expression was reactivated upon removal of P. gingivalis. Our observations suggest that exposure of macrophages to Gram-negative bacteria influence the establishment and maintenance of HIV persistence in macrophages through a TLR4-dependent mechanism.

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.

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.