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.

Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the ␤-defensin family, human ␤-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-␣) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-␣ and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower (ϳ10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-␣ as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-␣ that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli and F. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Summary 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 4Ј-phosphatase activities to modify its LPS, creating immunologically silent, nonphosphorylated 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.

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msb B gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msb B mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msb B mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF ␣ production by adherent monocytes. The cloned msb B gene was able to functionally complement the msb B mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msb B mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells. ( J. Clin. Invest. 1996. 97:359-365.)

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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 g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1␤) stimulation. P. gingivalis LPS (1 g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1␤-dependent activation of NF-B 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-B 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.The role that Porphyromonas gingivalis plays in the development of periodontal disease likely involves its ability to invade the gingiva and modulate innate host inflammatory responses via proteinases and lipopolysaccharide (LPS) (28,32,46,47). Previous studies have demonstrated that P. gingivalis disrupts the ability of gingival epithelial cells to produce interleukin-8 (IL-8) (8). These data suggest that such "chemokine paralysis" suppresses the host's ability to recruit and localize neutrophils to gingival sites of the infection via an IL-8 gradient (48). Gingival fibroblasts are likely to figure prominently in inflammatory responses to P. gingivalis. For example, P. gingivalis LPS has been shown to stimulate the production of a variety of cytokines, including IL-1, IL-6, and IL-8, in gingival fibroblasts, and it is chronic and excessive cytokine production that is believed to participate in tissue destruction during the course of periodontal disease (49). On the other hand, monocytes and human endothelial cells exhibit a low responsiveness to P. gingivalis LPS compared to E. coli LPS (7, 9, 30). In addition, in vivo studies demonstrated the low biological activity of P. gingivalis LPS in stimulating cytokine and adhesion molecule expression in mice (38).Another key property of P. gingivalis LPS is that it not only fails to stimulate E-selectin expression or p38 mitogen-activated protein kinase activation in human umbilical endothelial cells (HUVEC), but can potently antagonize the ability of E. coli LPS...

Summary Streptococcus sanguinis colonizes teeth and is an important cause of infective endocarditis. Our prior work showed that the lipoprotein SsaB is critical for S. sanguinis virulence for endocarditis and belongs to the LraI family of conserved metal transporters. In this study, we demonstrated that an ssaB mutant accumulates less manganese and iron than its parent. A mutant lacking the manganese-dependent superoxide dismutase, SodA, was significantly less virulent than wild-type in a rabbit model of endocarditis, but significantly more virulent than the ssaB mutant. Neither the ssaB nor the sodA mutation affected sensitivity to phagocytic killing or efficiency of heart valve colonization. Animal virulence results for all strains could be reproduced by growing bacteria in serum under physiological levels of O2. SodA activity was reduced, but not eliminated in the ssaB mutant in serum and in rabbits. Growth of the ssaB mutant in serum was restored upon addition of Mn2+ or removal of O2. Antioxidant supplementation experiments suggested that superoxide and hydroxyl radicals were together responsible for the ssaB mutant’s growth defect. We conclude that manganese accumulation mediated by the SsaB transport system imparts virulence by enabling cell growth in oxygen through SodA-dependent and independent mechanisms.