Thao T. To

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 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.

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.

Adjuvant Activity of Naturally Occurring Monophosphoryl Lipopolysaccharide Preparations from Mucosa-Associated Bacteria

ABSTRACT Natural heterogeneity in the structure of the lipid A portion of lipopolysaccharide (LPS) produces differential effects on the innate immune response. Gram-negative bacterial species produce LPS structures that differ from the classic endotoxic LPS structures. These differences include hypoacylation and hypophosphorylation of the diglucosamine backbone, both differences known to decrease LPS toxicity. The effect of decreased toxicity on the adjuvant properties of many of these LPS structures has not been fully explored. Here we demonstrate that two naturally produced forms of monophosphorylated LPS, from the mucosa-associated bacteria Bacteroides thetaiotaomicron and Prevotella intermedia, function as immunological adjuvants for antigen-specific immune responses. Each form of mucosal LPS increased vaccination-initiated antigen-specific antibody titers in both quantity and quality when given simultaneously with vaccine antigen preparations. Interestingly, adjuvant effects on initial T cell clonal expansion were selective for CD4 T cells. No significant increase in CD8 T cell expansion was detected. MyD88/Toll-like receptor 4 (TLR4) and TRIF/TLR4 signaling pathways showed equally decreased signaling with the LPS forms studied here as with endotoxic LPS or detoxified monophosphorylated lipid A (MPLA). Natural monophosphorylated LPS from mucosa-associated bacteria functions as a weak but effective adjuvant for specific immune responses, with preferential effects on antibody and CD4 T cell responses over CD8 T cell responses.

Subgingival Plaque in Periodontal Health Antagonizes at Toll-Like Receptor 4 and Inhibits E-Selectin Expression on Endothelial Cells

ABSTRACT The ability of the subgingival microbial community to induce an inappropriate inflammatory response ultimately results in the destruction of bone and gingival tissue. In this study, subgingival plaque samples from both healthy and diseased sites in the same individual were obtained from adults with chronic periodontitis and screened for their ability to either activate Toll-like receptor 2 (TLR2) or TLR4 and to antagonize TLR4-specific activation by agonist, Fusobacterium nucleatum LPS. Subgingival plaque from diseased sites strongly activated TLR4, whereas matched plaque samples obtained from healthy sites were significantly more variable, with some samples displaying strong TLR4 antagonism, while others were strong TLR4 agonists when combined with F. nucleatum LPS. Similar results were observed when TLR4 dependent E-selectin expression by endothelial cells was determined. These results are the first to demonstrate TLR4 antagonism from human plaque samples and demonstrate that healthy but not diseased sites display a wide variation in TLR4 agonist and antagonist behavior. The results have identified a novel characteristic of clinically healthy sites and warrant further study on the contribution of TLR4 antagonism in the progression of a healthy periodontal site to a diseased one.

Draft Genome Sequence of Actinomyces odontolyticus subsp. actinosynbacter Strain XH001, the Basibiont of an Oral TM7 Epibiont

ABSTRACT Here, we present the draft genome sequence of Actinomyces odontolyticus subsp. actinosynbacter strain XH001, isolated from the human oral cavity. Uniquely, it was discovered as a host bacterium to the ultrasmall epibiont TM7x, which is the first cultivated member of “Candidatus Saccharibacteria” (formerly candidate phylum TM7).

Evidence of independent acquisition and adaption of ultra-small bacteria to human hosts across the highly diverse yet reduced genomes of the phylum Saccharibacteria

Recently, we discovered that a member of the Saccharibacteria/TM7 phylum (strain TM7x) isolated from the human oral cavity, has an ultra-small cell size (200-300nm), a highly reduced genome (705 Kbp) with limited de novo biosynthetic capabilities, and a very novel lifestyle as an obligate epibiont on the surface of another bacterium 1. There has been considerable interest in uncultivated phyla, particularly those that are now classified as the proposed candidate phyla radiation (CPR) reported to include 35 or more phyla and are estimated to make up nearly 15% of the domain Bacteria. Most members of the larger CPR group share genomic properties with Saccharibacteria including reduced genomes (<1Mbp) and lack of biosynthetic capabilities, yet to date, strain TM7x represents the only member of the CPR that has been cultivated and is one of only three CPR routinely detected in the human body. Through small subunit ribosomal RNA (SSU rRNA) gene surveys, members of the Saccharibacteria phylum are reported in many environments as well as within a diversity of host species and have been shown to increase dramatically in human oral and gut diseases. With a single copy of the 16S rRNA gene resolved on a few limited genomes, their absolute abundance is most often underestimated and their potential role in disease pathogenesis is therefore underappreciated. Despite being an obligate parasite dependent on other bacteria, six groups (G1-G6) are recognized using SSU rRNA gene phylogeny in the oral cavity alone. At present, only genomes from the G1 group, which includes related and remarkably syntenic environmental and human oral associated representatives1, have been uncovered to date. In this study we systematically captured the spectrum of known diversity in this phylum by reconstructing completely novel Class level genomes belonging to groups G3, G6 and G5 through cultivation enrichment and/or metagenomic binning from humans and mammalian rumen. Additional genomes for representatives of G1 were also obtained from modern oral plaque and ancient dental calculus. Comparative analysis revealed remarkable divergence in the host-associated members across this phylum. Within the human oral cavity alone, variation in as much as 70% of the genes from nearest oral clade (AAI 50%) as well as wide GC content variation is evident in these newly captured divergent members (G3, G5 and G6) with no environmental relatives. Comparative analyses suggest independent episodes of transmission of these TM7 groups into humans and convergent evolution of several key functions during adaptation within hosts. In addition, we provide evidence from in vivo collected samples that each of these major groups are ultra-small in size and are found attached to larger cells.

Draft Genome Sequence of Low-Passage Clinical Isolate Porphyromonas gingivalis MP4-504.

ABSTRACT We present the draft genome of Porphyromonas gingivalis MP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. The traA-Q conjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277.

Draft Genome Sequence of Tannerella forsythia Clinical Isolate 9610

ABSTRACT We present here the draft genome sequence of Tannerella forsythia 9610, a clinical isolate obtained from a periodontitis patient. The genome is composed of 79 scaffolds with 82 contigs, for a length of 3,201,941 bp and a G+C of 47.3%.

The promotion of nephropathy by Porphyromonas gingivalis lipopolysaccharide via toll-like receptors

BackgroundRecently, we reported that toll-like receptor (TLR)2 and TLR4 localized on the glomerular endothelium in the glomeruli of streptozotocin (STZ)-induced type 1 diabetic mice and high fat diet feed-induced type 2 diabetic mice, and that periodontal pathogen Porphyromonas gingivalis LPS (Pg-LPS) administration lowered the survival rate of diabetic mice. The present study aims to examine the effect of TLR4 blocking on the suppression of Pg-LPS-induced diabetic nephropathy.MethodsThe survival rate and morphological/biochemical features for streptozotocin-induced diabetic mice with Pg-LPS and TLR4 blocker eritoran administration were investigated by reporter gene assay, urine and blood analysis, immunohistochemistry, and real time-PCR.Results and ConclusionsAll of the diabetic mice administered Pg-LPS were euthanized within the survival period of almost all of the diabetic mice. The blood urea nitrogen and creatinine, expression of TLR2 and TGF-b, and type 1 collagen accumulation, in the diabetic mice increased significantly with the Pg-LPS administration. In spite of the limited TLR4 activation with Pg-LPS, the TLR4 blocker eritoran decreased blood urea nitrogen and creatinine, and raised the survival rate of the Pg-LPS-administered diabetic mice slightly. The high expression levels of TLR2, TGF-b, and type 1 collagen in Pg-LPS-administered diabetic mice decreased with eritoran. Nuclear STAT3 which enhances TLR2 expression was detected in the TLR2-expressing glomeruli of diabetic mice. The TLR2 and STAT3 gene expression increased by the Pg-LPS administration but decreased with eritoran. These may suggest that Pg-LPS-induced diabetic nephropathy is mainly dependent on TLR2 signaling on glomerular endothelial cells, and that TLR4 blocker eritoran may play a role to slow the progress of diabetic nephropathy.