Stephen R. Coats

Porphyromonas gingivalis Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4

ABSTRACT 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.

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

ABSTRACT 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 μg/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 μg/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1β) stimulation. P. gingivalis LPS (1μ g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1β-dependent activation of NF-κB 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-κB 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.

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.

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.

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.

The Lipid A Phosphate Position Determines Differential Host Toll-Like Receptor 4 Responses to Phylogenetically Related Symbiotic and Pathogenic Bacteria

ABSTRACT 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.

A Novel Class of Lipoprotein Lipase-Sensitive Molecules Mediates Toll-Like Receptor 2 Activation by Porphyromonas gingivalis

ABSTRACT 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

ABSTRACT 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 β-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.

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.

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.