Microbial Lipid A Remodeling Controls Cross-Presentation Efficiency and CD8 T Cell Priming by Modulating Dendritic Cell Function.

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis, an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-ß) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis-associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders.

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1ß (IL-1ß) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1ß production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, in Porphyromonas gingivalis.

Removal of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. In Porphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes the P. gingivalis lipid A deacylase enzyme. This gene, PGN_1123 in P. gingivalis 33277, is highly conserved within P. gingivalis, and putative orthologs are phylogenetically restricted to the Bacteroidetes phylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-type P. gingivalis Heterologous expression of PGN_1123 in Bacteroides thetaiotaomicron promoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCE Periodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population. Porphyromonas gingivalis is a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection. P. gingivalis is particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

Cardiolipins Act as a Selective Barrier to Toll-Like Receptor 4 Activation in the Intestine.

UNLABELLED: Intestinal homeostasis mechanisms must protect the host intestinal tissue from endogenous lipopolysaccharides (LPSs) produced by the intestinal microbiota. In this report, we demonstrate that murine intestinal fecal lipids effectively block Toll-like receptor 4 (TLR4) responses to naturally occurring Bacteroidetes sp. LPS. Cardiolipin (CL) represents a significant proportion of the total intestinal and fecal lipids and, furthermore, potently antagonizes TLR4 activation by reducing LPS binding at the lipopolysaccharide binding protein (LBP), CD14, and MD-2 steps of the TLR4 signaling pathway. It is further demonstrated that intestinal lipids and CL are less effective at neutralizing more potent Enterobacteriaceae-type LPS, which is enriched in feces obtained from mice with dextran sodium sulfate (DSS)-treated inflammatory bowel disease. The selective inhibition of naturally occurring LPS structures by intestinal lipids may represent a novel homeostasis mechanism that blocks LPS activation in response to symbiotic but not dysbiotic microbial communities. IMPORTANCE: The guts of animals harbor a variety of Gram-negative bacteria associated with both states of intestinal health and states of disease. Environmental factors, such as dietary habits, can drive the microbial composition of the host animal's intestinal bacterial community toward a more pathogenic state. Both beneficial and harmful Gram-negative bacteria are capable of eliciting potentially damaging inflammatory responses from the host intestinal tissues via a lipopolysaccharide (LPS)-dependent pathway. Physical mucosal barriers and antibodies produced by the intestinal immune system protect against the undesired inflammatory effects of LPS, although it is unknown why some bacteria are more effective at overcoming the protective barriers than others. This report describes the discovery of a lipid-type protective barrier in the intestine that reduces the deleterious effects of LPSs from beneficial bacteria but is less effective in dampening the inflammatory effects of LPSs from harmful bacteria, providing a novel mechanistic insight into inflammatory intestinal disorders.

Distinct lipid a moieties contribute to pathogen-induced site-specific vascular inflammation.

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE(-/-) mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE(-/-) mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

A novel class of lipoprotein lipase-sensitive molecules mediates Toll-like receptor 2 activation by Porphyromonas gingivalis.

Infection by the chronic periodontitis-associated pathogen Porphyromonas gingivalis activates a Toll-like receptor 2 (TLR2) response that triggers inflammation in the host but also promotes bacterial persistence. Our aim was to define ligands on the surfaces of intact P. gingivalis cells that determine its ability to activate TLR2. Molecules previously reported as TLR2 agonists include lipopolysaccharide (LPS), fimbriae, the lipoprotein PG1828, and phosphoceramides. We demonstrate that these molecules do not comprise the major factors responsible for stimulating TLR2 by whole bacterial cells. First, P. gingivalis mutants devoid of the reported protein agonists, PG1828 and fimbriae, activate TLR2 as strongly as the wild type. Second, two-phase extraction of whole bacteria resulted in a preponderance of TLR2 agonist activity partitioning to the hydrophilic phase, demonstrating that phosphoceramides are not a major TLR2 ligand. Third, analysis of LPS revealed that TLR2 activation is independent of lipid A structural variants. Instead, activation of TLR2 and TLR2/TLR1 by LPS is in large part due to copurifying molecules that are sensitive to the action of the enzyme lipoprotein lipase. Strikingly, intact P. gingivalis bacterial cells treated with lipoprotein lipase were attenuated in their ability to activate TLR2. We propose that a novel class of molecules comprised by lipoproteins constitutes the major determinants that confer to P. gingivalis the ability to stimulate TLR2 signaling.

Temperature-dependent modulation of Porphyromonas gingivalis lipid A structure and interaction with the innate host defenses.

Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalis lipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalis grown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by ß-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

The lipid A phosphate position determines differential host Toll-like receptor 4 responses to phylogenetically related symbiotic and pathogenic bacteria.

The human symbiont Bacteroides thetaiotaomicron promotes intestinal function and health, whereas the phylogenetically related pathogen Porphyromonas gingivalis is associated with the chronic oral inflammatory disease periodontitis. Although both B. thetaiotaomicron and P. gingivalis synthesize lipopolysaccharides (LPS) consisting of penta-acylated, monophosphorylated lipid A in addition to immunologically silent, nonphosphorylated lipid A, they elicit strikingly distinct Toll-like receptor 4 (TLR4) responses. We show that the phosphate position of penta-acylated, monophosphorylated lipid A is a key feature for determining the differential TLR4 responses elicited by these evolutionarily related bacteria. B. thetaiotaomicron produces TLR4-stimulatory lipid A bearing a 1-phosphate, in contrast to P. gingivalis, which produces TLR4-evasive lipid A bearing a 4'-phosphate. Confirming these observations, recombinant Escherichia coli LPS containing penta-acylated, 1-phosphorylated lipid A is more TLR4 stimulatory than LPS containing 4'-phosphorylated lipid A. The specific capacity of a Gram-negative bacterium to alert or evade the host innate immune defense system through TLR4-dependent signaling is currently recognized as a critical aspect defining the relationship between the host and the bacterium. We propose that the distinct lipid A phosphate positions observed for the B. thetaiotaomicron and P. gingivalis LPS contributes to the manifestation of these bacteria as commensal or pathogen within the human host.

Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1- and 4'-phosphatase activities.

Signal transduction following binding of lipopolysaccharide (LPS) to Toll-like receptor 4 (TLR4) is an essential aspect of host innate immune responses to infection by Gram-negative pathogens. Here, we describe a novel molecular mechanism used by a prevalent human bacterial pathogen to evade and subvert the human innate immune system. We show that the oral pathogen, Porphyromonas gingivalis, uses endogenous lipid A 1- and 4'-phosphatase activities to modify its LPS, creating immunologically silent, non-phosphorylated lipid A. This unique lipid A provides a highly effective mechanism employed by this bacterium to evade TLR4 sensing and to resist killing by cationic antimicrobial peptides. In addition, lipid A 1-phosphatase activity is suppressed by haemin, an important nutrient in the oral cavity. Specifically, P. gingivalis grown in the presence of high haemin produces lipid A that acts as a potent TLR4 antagonist. These results suggest that haemin-dependent regulation of lipid A 1-dephosphorylation can shift P. gingivalis lipid A activity from TLR4 evasive to TLR4 suppressive, potentially altering critical interactions between this bacterium, the local microbial community and the host innate immune system.

The structurally similar, penta-acylated lipopolysaccharides of Porphyromonas gingivalis and Bacteroides elicit strikingly different innate immune responses.

Lipid A structural modifications can substantially impact the host's inflammatory response to bacterial LPS. Bacteroides fragilis, an opportunistic pathogen associated with life-threatening sepsis and intra-abdominal abscess formation, and Bacteroides thetaiotaomicron, a symbiont pivotal for proper host intestinal tissue development, both produce an immunostimulatory LPS comprised of penta-acylated lipid A. Under defined conditions, Porphyromonas gingivalis, an oral pathogen associated with periodontitis, also produces an LPS bearing a penta-acylated lipid A. However, this LPS preparation is 100-1000 times less potent than Bacteroides LPS in stimulating endothelial cells. We analyzed Bacteroides and P. gingivalis lipid A structures using MALDI-TOF MS and gas chromatography to determine the structural basis for this phenomenon. Even though both Bacteroides and P. gingivalis lipid A molecules are penta-acylated and mono-phosphorylated, subtle differences in mass and fatty acid content could account for the observed difference in LPS potency. This fatty acid heterogeneity is also responsible for the peak "clusters" observed in the mass spectra and obfuscates the correlation between LPS structure and immunostimulatory ability. Further, we show the difference in potency between Bacteroides and P. gingivalis LPS is TLR4-dependent. Altogether, the data suggest subtle changes in lipid A structure may profoundly impact the host's innate immune response.

Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4'-phosphatase, PGN_0524.

AIM: To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B. METHODOLOGY: A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)). RESULTS: P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate. CONCLUSION: The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Antagonistic lipopolysaccharides block E. coli lipopolysaccharide function at human TLR4 via interaction with the human MD-2 lipopolysaccharide binding site.

Lipopolysaccharides containing underacylated lipid A structures exhibit reduced abilities to activate the human (h) Toll-like receptor 4 (TLR4) signalling pathway and function as potent antagonists against lipopolysaccharides bearing canonical lipid A structures. Expression of underacylated lipopolysaccharides has emerged as a novel mechanism utilized by microbial pathogens to modulate host innate immune responses. Notably, antagonistic lipopolysaccharides are prime therapeutic candidates for combating Gram negative bacterial sepsis. Penta-acylated msbB and tetra-acylated Porphyromonas gingivalis lipopolysaccharides functionally antagonize hexa-acylated Escherichia coli lipopolysaccharide-dependent activation of hTLR4 through the coreceptor, hMD-2. Here, the molecular mechanism by which these antagonistic lipopolysaccharides act at hMD-2 is examined. We present evidence that both msbB and P. gingivalis lipopolysaccharides are capable of direct binding to hMD-2. These antagonistic lipopolysaccharides can utilize at least two distinct mechanisms to block E. coli lipopolysaccharide-dependent activation of hTLR4. The main mechanism consists of direct competition between the antagonistic lipopolysaccharides and E. coli lipopolysaccharide for the same binding site on hMD-2, while the secondary mechanism involves the ability of antagonistic lipopolysaccharide-hMD-2 complexes to inhibit E. coli lipopolysaccharide-hMD-2 complexes function at hTLR4. It is also shown that both hTLR4 and hMD-2 contribute to the species-specific recognition of msbB and P. gingivalis lipopolysaccharides as antagonists at the hTLR4 complex.

Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli.

The ability of lipid A structural variants to elicit unique endothelial cell gene expression was examined by measuring global gene expression profiles in human umbilical cord vein endothelial cells (HUVEC) using Affymetrix full genome chips. Two lipid A structural variants obtained from Porphyromonas gingivalis designated PgLPS(1435/1449) and PgLPS(1690) as well as LPS obtained from Escherichia coli wild type and an E. coli msbB mutant (missing myristic acid in the lipid A) were examined. Each of these lipid A structures has been shown to interact with TLR4; however, PgLPS(1435/1449) and E. coli msbB LPS have been shown to be TLR4 antagonists while PgLPS(1690) and wild-type E. coli LPS are TLR4 agonists. It was found that PgLPS(1435/1449) and PgLPS(1690) as well as E. coli msbB LPS activated a subset of those genes significantly transcribed in response to E. coli wild-type LPS. Furthermore, the subset of genes expressed in response to the different lipid A structural forms were those most significantly activated by wild-type E. coli LPS demonstrating a hierarchy in TLR4-dependent endothelial cell gene activation. A unique gene expression profile for the weak TLR4 agonist PgLPS(1690) was observed and represents a TLR4 hierarchy in endothelial cell gene activation.

Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity: differential activities of tetra- and penta-acylated lipid A structures on E-selectin expression and TLR4 recognition.

Porphyromonas gingivalis is a gram-negative bacterium strongly associated with periodontitis, a chronic inflammatory disease of the tissue surrounding the tooth root surface. Lipopolysaccharide (LPS) obtained from P. gingivalis is unusual in that it has been shown to display an unusual amount of lipid A heterogeneity containing both tetra- and penta-acylated lipid A structures. In this report, it is shown that penta-acylated lipid A structures facilitate E-selectin expression whereas tetra-acylated lipid A structures do not. Furthermore, it is shown that tetra-acylated lipid A structures are potent antagonists for E-selectin expression. Both tetra- and penta-acylated lipid A structures interact with TLR4 although experiments utilizing human, mouse and human/mouse chimeric TLR4 proteins demonstrated that they interact differentially with the TLR4 signalling complexes. The presence of two different structural types of lipid A in P. gingivalis LPS, with opposing effects on the E-selectin response suggests that this organism is able to modulate innate host responses by alterations in the relative amount of these lipid A structures.

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion.

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex.

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.

Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4.

The innate host response to lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that P. gingivalis LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at m/z 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2(-/-) and TLR4(-/-) mice also responded to P. gingivalis LPS in a manor consistent with the HEK results, demonstrating that P. gingivalis LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that P. gingivalis LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in P. gingivalis LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.

Differential and opposing regulation of PAI-1 promoter activity by estrogen receptor alpha and estrogen receptor beta in endothelial cells.

To investigate the molecular mechanisms involved in the estrogen-dependent control of plasminogen activator inhibitor-1 (PAI-1) gene expression in vascular cells, we compared the transactivation properties of estrogen receptors (ERalpha and ERbeta) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ERalpha increased PAI-1 promoter activity in BAECs by an estrogen-dependent mechanism, whereas ERbeta suppressed PAI-1 promoter activity by an estrogen-independent mechanism. The suppressive activity of ERbeta was dominant over the inductive activity of ERalpha. Mutation of a putative estrogen response element (ERE) located at position -427 in the proximal promoter abolished the ERalpha action without influencing the suppressive effects of ERbeta. Mutation of either AP1-like site did not eliminate the ERalpha or ERbeta actions at the PAI-1 promoter, suggesting that other promoter elements are involved in these responses. These mutations significantly reduced the -3.4kbp PAI-1 promoter response to serum. We concluded that ERalpha and ERbeta exert differential effects on the PAI-1 promoter activity in transfected BAECs. ERalpha activated the PAI-1 promoter through a proximal ERE (-427) and possibly additional EREs located within the PAI-1 promoter, whereas ERbeta suppressed the promoter construct via an unidentified mechanism. This is the first demonstration of the differential regulation of a vascular gene promoter by ERalpha and ERbeta.

Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells.

E. coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells. In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E. coli LPS-dependent activation of human endothelial cells. P. gingivalis LPS at 1 micro g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 micro g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1beta) stimulation. P. gingivalis LPS (1 micro g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1beta-dependent activation of NF-kappaB in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88. Surprisingly, P. gingivalis LPS weakly but significantly activated NF-kappaB in HMEC-1 cells in the absence of E. coli LPS, and the P. gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4. Pretreatment of HUVECs with P. gingivalis LPS did not influence the ability of E. coli LPS to stimulate E-selectin mRNA expression. Taken together, these data provide the first evidence that P. gingivalis LPS-dependent antagonism of E. coli LPS in human endothelial cells likely involves the ability of P. gingivalis LPS to directly compete with E. coli LPS at the TLR4 signaling complex.

Ligand-specific control of src-suppressed C kinase substrate gene expression.

The src-suppressed C-kinase substrate, SSeCKS, is now recognized as a key regulator of cell signaling and cytoskeletal dynamics. However, few ligands that control SSeCKS expression have been identified. We report that platelet-derived growth factor-BB (PDGF-BB), lysophosphatidic acid (LPA), and eicosapentaenoic acid (EPA) potently modulate SSeCKS gene expression in cultured smooth muscle (RASM) cells relative to other bioactive ligands tested. In addition, EPA-dependent regulation of SSeCKS expression correlates with distinct changes in cell morphology and adhesion in RASM cells. Independent evidence that ligand-specific control of SSeCKS expression links to the regulation of cell adhesion and morphology was obtained using ras-transformed fibroblasts, KNRK. Sodium butyrate (NaB) upregulates SSeCKS mRNA and protein expression corresponding to increased cell-spreading and adhesion. In addition, ectopic expression of recombinant SSeCKS recapitulates attributes of NaB-induced morphogenesis in KNRK cells. The data provide novel evidence that SSeCKS functions in PDGF-BB-, LPA-, EPA-, and NaB-mediated cell signaling.