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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 01 March 2011, Vol.108(9), pp.3683-8
    Description: 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.
    Keywords: Signal Transduction ; Borrelia Burgdorferi -- Physiology ; Interferon-Beta -- Genetics ; Monocytes -- Microbiology ; Phagosomes -- Metabolism ; Toll-Like Receptor 2 -- Metabolism ; Toll-Like Receptor 8 -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2012, Vol.109(8), pp.3059-3064
    Description: The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process. ; p. 3059-3064.
    Keywords: Dermis ; Ticks ; Borrelia Burgdorferi ; Rheological Properties ; Ixodes Scapularis ; Pathogens ; Swimming ; Mice ; Gelatin ; Bacteria ; Hosts ; Microscopy ; Adhesion ; Basement Membrane ; Midgut
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Biophysical Journal, 19 November 2013, Vol.105(10), pp.2273-2280
    Description: The spirochetes that cause Lyme disease ( ) and syphilis ( ) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20–40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of and decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens.
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
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  • 4
    Language: English
    In: The Journal of infectious diseases, November 2011, Vol.204(9), pp.1295-6
    Keywords: Antigens, Bacterial ; Recombinant Proteins ; Mass Screening -- Methods ; Syphilis -- Diagnosis ; Syphilis Serodiagnosis -- Methods ; Treponema Pallidum -- Immunology
    ISSN: 00221899
    E-ISSN: 1537-6613
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2012, Vol.109(4), pp.1228-1232
    Description: Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. B. burgdorferi has been shown to bind to different receptors on the surface of phagocytic cells, including the β2 integrin, complement receptor 3 (CR3). However, whether these receptors mediate the phagocytosis of the spirochete remains unknown. We now demonstrate that CR3 mediates the phagocytosis of the spirochete by murine macrophages and human monocytes. Interaction of B. burgdorferi with the integrin is not sufficient, however, to internalize the spirochete; phagocytosis requires the interaction of CR3 with the GPI-anchored protein, CD14, independently of TLR/MyD88-induced or inside-out signals. Interestingly, the absence of CR3 leads to marked increases in the production of TNF in vitro and in vivo, despite reduced spirochetal uptake. Furthermore, the absence of CR3 during infection with B. burgdorferi results in the inefficient control of bacterial burdens in the heart and increased Lyme carditis. Overall, our data identify CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. These data also establish a unique mechanism of CR3-mediated phagocytosis that requires the direct cooperation of GPI-anchored proteins. ; p. 1228-1232.
    Keywords: Heart ; Borrelia Burgdorferi ; Myocarditis ; Humans ; Receptors ; Phagocytosis ; Mice ; Macrophage-1 Antigen ; Immune Response ; Tumor Necrosis Factors ; Macrophages ; Monocytes ; Lyme Disease
    ISSN: 0027-8424
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  • 6
    Language: English
    In: Journal of bacteriology, May 2013, Vol.195(9), pp.2060-71
    Description: The major outer sheath protein (Msp) is a primary virulence determinant in Treponema denticola, as well as the parental ortholog for the Treponema pallidum repeat (Tpr) family in the syphilis spirochete. The Conserved Domain Database (CDD) server revealed that Msp contains two conserved domains, major outer sheath protein(N) (MOSP(N)) and MOSP(C), spanning residues 77 to 286 and 332 to 543, respectively, within the N- and C-terminal regions of the protein. Circular dichroism (CD) spectroscopy, Triton X-114 (TX-114) phase partitioning, and liposome incorporation demonstrated that full-length, recombinant Msp (Msp(Fl)) and a recombinant protein containing MOSP(C), but not MOSP(N), form amphiphilic, β-sheet-rich structures with channel-forming activity. Immunofluorescence analysis of intact T. denticola revealed that only MOSP(C) contains surface-exposed epitopes. Data obtained using proteinase K accessibility, TX-114 phase partitioning, and cell fractionation revealed that Msp exists as distinct OM-integrated and periplasmic trimers. Msp(Fl) folded in Tris buffer contained slightly less β-sheet structure than detergent-folded Msp(Fl); both forms, however, partitioned into the TX-114 detergent-enriched phase. CDD analysis of the nine Tpr paralogs predicted to be outer membrane proteins (OMPs) revealed that seven have an Msp-like bipartite structure; phylogenetic analysis revealed that the MOSP(N) and MOSP(C) domains of Msp are most closely related to those of TprK. Based upon our collective results, we propose a model whereby a newly exported, partially folded intermediate can be either processed for OM insertion by the β-barrel assembly machinery (BAM) or remain periplasmic, ultimately forming a stable, water-soluble trimer. Extrapolated to T. pallidum, our model enables us to explain how individual Tprs can localize to either the periplasmic (e.g., TprK) or OM (e.g., TprC) compartments.
    Keywords: Bacterial Outer Membrane Proteins -- Genetics ; Periplasm -- Metabolism ; Treponema Denticola -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 7
    Language: English
    In: Journal of Bacteriology, Feb, 2014, Vol.196(3-4), p.859(14)
    Description: The Borrelia burgdorferi outer membrane (OM) contains numerous surface-exposed lipoproteins but a relatively low density of integral OM proteins (OMPs). Few membrane-spanning OMPs of B. burgdorferi have been definitively identified, and none are well characterized structurally. Here, we provide evidence that the borrelial OMP P66, a known adhesin with pore-forming activity, forms a beta-barrel in the B. burgdorferi OM. Multiple computer-based algorithms predict that P66 forms a beta-barrel with either 22 or 24 transmembrane domains. According to our predicted P66 topology, a lysine residue (K487) known to be sensitive to trypsin cleavage is located within a surface-exposed loop. When we aligned the mature P66 amino acid sequences from B. burgdorferi and B. garinii, we found that K487 was present only in the B. burgdorferi P66 protein sequence. When intact cells from each strain were treated with trypsin, only B. burgdorferi P66 was trypsin sensitive, indicating that K487 is surface exposed, as predicted. Consistent with this observation, when we inserted a c-Myc tag adjacent to K487 and utilized surface localization immunofluorescence, we detected the loop containing K487 on the surface of B. burgdorferi. P66 was examined by both Triton X-114 phase partitioning and circular dichroism, confirming that the protein is amphiphilic and contains extensive (48%) beta-sheets, respectively. Moreover, P66 also was able to incorporate into liposomes and form channels in large unilamellar vesicles. Finally, blue native PAGE (BN-PAGE) revealed that under nondenaturing conditions, P66 is found in large complexes of ~400 kDa and ~600 kDa. Outer surface lipoprotein A (OspA) and OspB both coimmunoprecipitate with P66, demonstrating that P66 associates with OspA and OspB in B. burgdorferi. The combined computer-based structural analyses and supporting physicochemical properties of P66 provide a working model to further examine the porin and integrin-binding activities of this OMP as they relate to B. burgdorferi physiology and Lyme disease pathogenesis.
    Keywords: Biophysics -- Usage ; Borrelia Burgdorferi -- Research ; Borrelia Burgdorferi -- Physiological Aspects ; Membrane Proteins -- Analysis
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 8
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 21 February 2012, Vol.109(8), pp.3059-64
    Description: The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.
    Keywords: Borrelia Burgdorferi -- Drug Effects ; Dermis -- Drug Effects ; Gelatin -- Pharmacology ; Lyme Disease -- Microbiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 9
    Language: English
    In: Journal of bacteriology, June 2015, Vol.197(11), pp.1906-20
    Description: 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 β-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 β-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 β-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. Previously, 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.
    Keywords: Bacterial Outer Membrane Proteins -- Chemistry ; Immunodominant Epitopes -- Chemistry ; Opsonin Proteins -- Immunology ; Syphilis -- Immunology ; Treponema Pallidum -- Chemistry
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 10
    Language: English
    In: The Journal of biological chemistry, 08 May 2015, Vol.290(19), pp.12313-31
    Description: We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.
    Keywords: Electron Microscopy (Em) ; Microbial Pathogenesis ; Outer Membrane ; Outer Membrane Proteins ; Porins ; Small Angle X-Ray Scattering (Saxs) ; Syphilis ; Treponema Pallidum ; X-Ray Scattering ; Beta-Barrel ; Bacterial Outer Membrane Proteins -- Chemistry ; Porins -- Chemistry ; Treponema Pallidum -- Chemistry
    E-ISSN: 1083-351X
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