Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-β. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-β in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro-and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-β.Lyme disease | endosomal receptors | type I interferons | phagocytosis

We have previously demonstrated that phagocytosed Borrelia burgdorferi induces activation programs in human peripheral blood mononuclear cells that differ qualitatively and quantitatively from those evoked by equivalent lipoprotein-rich lysates. Here we report that ingested B. burgdorferi induces significantly greater transcription of proinflammatory cytokine genes than do lysates and that live B. burgdorferi, but not B. burgdorferi lysate, is avidly internalized by monocytes, where the bacteria are completely degraded within phagolysosomes. In the course of these experiments, we discovered that live B. burgdorferi also induced a dose-dependent decrease in monocytes but not a decrease in dendritic cells or T cells and that the monocyte population displayed morphological and biochemical hallmarks of apoptosis. Particularly noteworthy was the finding that apoptotic changes occurred predominantly in monocytes that had internalized spirochetes. Abrogation of phagocytosis with cytochalasin D prevented the death response. Heat-killed B. burgdorferi, which was internalized as well as live organisms, induced a similar degree of apoptosis of monocytes but markedly less cytokine production. Surprisingly, opsonophagocytosis of Treponema pallidum did not elicit a discernible cell death response. Our combined results demonstrate that B. burgdorferi confined to phagolysosomes is a potent inducer of cytosolic signals that result in (i) production of NF-B-dependent cytokines, (ii) assembly of the inflammasome and activation of caspase-1, and (iii) induction of programmed cell death. We propose that inflammation and apoptosis represent mutually reinforcing components of the immunologic arsenal that the host mobilizes to defend itself against infection with Lyme disease spirochetes.

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn 2؉ , whereas DNA binding by TroR in vitro is Mn 2؉ -dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn 2؉ Treponema pallidum, the causative agent of syphilis, is a noncultivable sexually transmitted human pathogen that disseminates from a site of inoculation, usually within the genital area, to diverse organs where it can establish persistent, even lifelong, infection (1). Like all pathogens, T. pallidum presumably requires sequestered transition metals for vital structural and catalytic functions in proteins. Nevertheless, in contrast to other pathogenic bacteria in which specialized uptake systems for iron (2-4), and more recently for Zn 2ϩ and Mn 2ϩ (5-8), have been identified, there is little information about metal acquisition by the syphilis spirochete. The lack of techniques for in vitro cultivation and genetic manipulation of T. pallidum has greatly hindered investigation of treponemal physiology, including metal metabolism.Two recent developments have spawned interest in the processes by which T. pallidum acquires trace metals. One is the availability of the genomic sequence of the spirochete (9), which reveals that many genes encoding well characterized iron-containing proteins (e.g. cytochromes, superoxide dismutase, and tricarboxylic acid metalloenzymes) are lacking. The finding that T. pallidum expresses at least two experimentally confirmed iron-binding proteins, superoxide reductase (neelaredoxin) and rubredoxin, (10 -12), argues that the bacterium does require iron. The second impetus to study metal utilization by T. pallidum is provided by the discovery of the six gene tro (transport-related operon) ( Fig. 1) (13). The first gene of the operon, troA, encodes the solute binding protein (SBP) 1 component of an ATP-binding cassette transporter (13-15) belonging to a newly described cluster (cluster nine or C9) of transition metal permeases (5). In addition to encoding an ATPase (TroB), two cytoplasmic membrane permeases (TroC and TroD), and a DtxR-like metalloregulator (TroR), the operon also encodes the glycolytic enzyme phosphoglyceromutase (Gpm) (13). Because the T. pallidum Gpm has no requirement for metals (16), the physiological benefit of transcriptionally linking the spirochete's sole copy of this essential enzyme to the Tro transporter genes is unclear.The identity of the metal ligand(s) fo...

eThe total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. T oll-like receptors (TLRs) represent a diverse family of molecules that play a critical role in activating the innate immune system in response to pathogens (1, 2). Toll-like receptor 2 (TLR2) recognizes diverse molecular structures of microbial cell wall origin, including lipoteichoic acid, lipoproteins, peptidoglycan from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from yeast cell walls. TLR2 is reported to be activated by many other microbial products, including phenol-soluble modulins (3) and Porphyromonas gingivalis lipoprotein (4), lipopolysaccharide (LPS) (5-7), and fimbriae (8-10). However, two recent reports have questioned the extent to which lipoprotein, LPS, or fimbriae mediate TLR2 engagement by P. gingivalis (11,12).We previously reported that the total lipid extract of P. gingivalis promotes activation of mouse dendritic cells and inhibits osteoblast-mediated bone deposition through engagement of TLR2 (13,14). These effects were attributed to the dominant phosphorylated dihydroceramide lipids of P. gingivalis, in particular, phosphoethanolamine dihydroceramides. These studies reported engagement of TLR2 only in vitro in mouse cells. Recent reports have demonstrated TLR2-dependent periodontal bone loss in mice following oral infection with P. gingivalis (15,16). Most recently, cell adhesion mediated through the expression of fimbriae by P. gingivalis has been implicated in promoting of TLR2-dependent oral bone loss (17). In contrast, two recent reports indicated that the capacity of fimbriae to engage TLR2 is dependent on the presence of a contaminating factor that is susceptible to hydrolysis by lipoprotein lipase (11,18).In addition to effects on mouse cells, the phosphorylated dihydroceramide lipids of P. gingivalis have been shown to promote proinflammatory responses in human fibroblasts and to cause disruption of human fibroblast adherence/vitality in culture (19). However, it is not clear whether these effects require engagement of TLR2. Since the total lipid extract of P. gingivalis has been shown to activate TLR2 in mice and in mouse cells, the primary purpose of this investigation was to further identify and characterize the specific lipid classes of P. gingivalis that are responsible for engagement of TLR2 and, specifica...

We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the ␤-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the ␤-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the ␤-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu 593 ¡ Gln 593 ) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a "structure-to-pathogenesis" approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection. IMPORTANCEPreviously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis of T. pallidum's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of a T. pallidum rare OMP within the context of syphilitic infection. W ithin the outer membranes (OMs) of Gram-negative bacteria is a unique class of integral membrane proteins that fold into a ␤-barrel structure consisting of 8 to 26 antiparallel amphipathic ␤ strands; typically, extensive hydrogen bonding between the first and last strands closes and stabilizes the barrel, often creating a central channel (1, 2). ␤-Barrel outer membrane proteins (OMPs) have two principal functions: (i) insertion/transport of proteins into or across the OM and (ii) formation of aqueous pores for the passive or selective uptake of...