Pamela H. Braham

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.

Hemin-Dependent Modulation of the Lipid A Structure of Porphyromonas gingivalis Lipopolysaccharide

ABSTRACT Porphyromonas gingivalis is a periopathogen strongly associated with the development of adult-type periodontitis. Both the virulence characteristics of periopathogens and host-related factors are believed to contribute to periodontitis. P. gingivalis lipopolysaccharide (LPS) displays a significant amount of lipid A structural heterogeneity, containing both penta- and tetra-acylated lipid A structures. However, little is known concerning how the lipid A structural content of P. gingivalis is regulated. Alterations in the lipid A content may facilitate the ability of P. gingivalis to modulate the innate host response to this bacterium. In this report, it is shown that the concentration of hemin in the growth medium significantly modulates the lipopolysaccharide lipid A structural content of P. gingivalis. Hemin is a key microenvironmental component of gingival cervicular fluid which is believed to vary depending upon the state of vascular ulceration. At low hemin concentrations, one major penta-acylated lipid A structure was found, whereas at high concentrations of hemin, multiple tetra- and penta-acylated lipid A structures were observed. Hemin concentrations, not iron acquisition, were responsible for the alterations in the lipid A structural content. The modifications of the lipid A structural content were independent of the LPS extraction procedure and occurred in a variety of laboratory strains as well as a freshly obtained clinical isolate. The known hemin binding proteins Kgp and HmuR contributed to the lipid A modulation sensing mechanism. To the best of our knowledge, this is the first report that hemin, a clinically relevant microenvironmental component for P. gingivalis, can modulate the lipid A structure found in a bacterium. Since tetra- and penta-acylated P. gingivalis lipid A structures have opposing effects on Toll-like receptor 4 activation, the alteration of the lipid A structural content may have significant effects on the host response to this bacterium.

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.

Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4＇-Phosphatase, PGN_0524

AimTo elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.MethodologyA 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 μg·mL−1).ResultsP. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 μg·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 μg·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 wild‐type 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.ConclusionThe 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 TLR4 sensing.

Antigenic variation and cross-reactivity in Bacteroides forsythus clinical isolates detected by western blot

Bacteroides forsythus is one of the etiologic agents of destructive periodontal diseases. Determining which antigenic components of the bacterium are recognized in the immune response of periodontitis patients is an important step in assessing strategies for vaccine development. The aim of this study was to identify the major strain-variable and cross-reactive antigens of B. forsythus clinical isolates recognized by serum IgG from patients with early-onset rapidly progressive periodontitis. Ten patient sera with measurable IgG against antigenic components of the species were identified by Western blot. Positive sera were tested by checkerboard ELISA to identify those most responsive to strain-variable antigens in nine clinical isolates and ATCC strain 43037. Correlation analysis of the ELISA data suggested that different subsets of isolates were preferentially recognized by different sera. Western blots revealed that certain sera also recognized major shared components across all the isolates, but preferential recognition of different isolate subsets by different patients was clearly confirmed. To determine if the variable antigens recognized were nonprotein, proteinase K-digested isolates were compared to undigested controls by Western blot. The main strain-variable antigens were proteinase resistant, while proteins at 200 and 210 kDa were identified as the major shared components. Two-dimensional SDS-PAGE revealed that these proteins are the quantitatively dominant heat-modifiable components of the cell envelope. Even though variable antigens are prominent in the immune response of patients, a cross-protective vaccine based on the shared envelope proteins of B. forsythus seems feasible in light of these observations.