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. Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with chronic periodontitis, an inflammatory disease that results in tooth loss (1). P. gingivalis is typically found in diseased subgingival sites (2). Mouse models of P. gingivalis infection have demonstrated its capacity to elicit periodontitis, as measured by bone loss (3-5). A recent study revealed a key role played by P. gingivalis infection in altering the composition, and increasing the abundance, of oral commensal bacteria in conventionally grown mice (4). The interaction between an increasing bacterial load and the host innate immune system triggers a proinflammatory response, which is considered a major factor in causing periodontitis.The Toll-like receptor (TLR) family of innate immune receptors plays a pivotal role in host surveillance and in initiating an immediate immune response that is designed to neutralize microbial threats to the host (6). P. gingivalis infection triggers activation of TLR2, which leads to production of proinflammatory cytokines. One impact of these cytokines is a damaging effect on alveolar bone, resulting in bone loss, as demonstrated by studies using TLR2 Ϫ/Ϫ mice (3, 7). Paradoxically, the robust TLR2 proinflammatory response does not clear P. gingivalis infection in wild-type mice. Instead, the infection is cleared more efficiently in TLR2 Ϫ/Ϫ mice and by macrophages from TLR2 Ϫ/Ϫ mice (3, 5, 7), indicating that TLR2 stimulation confers enhanced persistence to this chronic pathogen. A lack of bacterial clearance illustra...

Porphyromonas gingivalis Outer Membrane Vesicles Induce Selective Tumor Necrosis Factor Tolerance in a Toll-Like Receptor 4- and mTOR-Dependent Manner

“…Other reports, however, describe P. gingivalis LPS preparations as a highly heterogeneous mixture of different types of lipid A species with differential levels of stimulatory potency in their effects on TLR2 and TLR4 (10,14,49). In addition, a few studies demonstrated that TLR2 activity can be attributed to P. gingivalis lipoproteins (50)(51)(52). However, since methods that are more refined than the SDS-PAGE performed in this study (see Fig.…”

Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

“…Previous investigations into the effect of P. gingivalis LPS on nonpolarized macrophages have shown that the induced immune responses is varied and that many cytokines were only transiently expressed compared to Escherichia coli LPS and other Gram-negative pathogens (7)(8)(9). Furthermore, P. gingivalis LPS is atypical in that it is structurally different from the canonical enterobacterial LPS and has been reported to stimulate both TLR4 and TLR2 (10)(11)(12). The stimulation of TLR4 has been linked to penta-acylated lipid A structures (13)(14)(15); however, the molecular entity for stimulation of TLR2, even in highly purified LPS samples, has not yet been identified (12).…”

“…Furthermore, P. gingivalis LPS is atypical in that it is structurally different from the canonical enterobacterial LPS and has been reported to stimulate both TLR4 and TLR2 (10)(11)(12). The stimulation of TLR4 has been linked to penta-acylated lipid A structures (13)(14)(15); however, the molecular entity for stimulation of TLR2, even in highly purified LPS samples, has not yet been identified (12). It has been suggested that TLR2 stimulation is due to the presence of novel lipoprotein contaminants that copurify with the P. gingivalis LPS (12).…”

Importance of heterogeneity in Porhyromonas gingivalis lipopolysaccharide lipid A in tissue specific inflammatory signalling

“…In another study, multiple lipid A species interacted functionally with both TLR2 and TLR4 [15]. However, as already mentioned, the TLR2 agonist activity in P. gingivalis is most likely due to a lipoprotein, and treatment of LPS with lipoproteinase as free LPS substantially attenuated the TLR2 engaging activity [17]. These findings explain previous observations that P. gingivalis LPS can act both as a TLR4 agonist and antagonist, with an end result of varying cytokine secretion profiles [5,11,12].…”

“…Although TLR2 activation by P. gingivalis LPS is possible, the difference lies in the form of LPS presented to the host. For example, the protein-free LPS is unable to activate TLR2, whereas the bound LPS on the live bacterium can mediate TLR2 activation via a novel class of lipoprotein lipase-sensitive molecules highlighting the importance of active infection [17]. In essence, and according to Lasica et al [18], the tightly associated or covalently attached protein to LPS on live P. gingivalis accounts for the TLR2 activation.…”