It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-β and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-α, IL-6, IL-10 and IL-1β in monocytes than did lysates. Secreted IL-18, which, like IL-1β, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-β and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.Spirochetes are an ancient and extremely successful eubacterial phylum characterized by distinctive helical or planar wave-form morphology and flagellar filaments confined to the periplasmic space (55, 87). Spirochetes from the genera Leptospira, Treponema, and Borrelia are highly invasive pathogens that pose public health problems of global dimensions (1,6,57,109). Treponema denticola and numerous other treponemal species, most of which remain uncultivated, are major components of the polymicrobial biofilms that cause periodontal disease (34, 56) and also have been implicated as risk factors for atherosclerosis (4,125). The treponemal symbionts that dwell in the hindguts of termites, where they provide their insect host with essential nutrients (10), are one of the most striking examples of the extraordinary biodiversity achieved by spirochetes. It is readily apparent, therefore, that in the course of their complex evolution, spiroc...

Background The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum ( Tp ) to escape immune recognition while simultaneously inducing inflammation. Methods To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo . Results Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp -DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized. Conclusions Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

A new strain of Babesia microti (KR-1) was isolated from a Connecticut resident with babesiosis by hamster inoculation and adapted to C3H/HeJ and BALB/c mice. To examine the relative importance of humoral and cellular immunity for the control of B. microti infection, we compared the course of disease in wild-type BALB/c mice with that in BALB/c SCID mice, JHD-null (B-cell-deficient) mice, and T-cell receptor ␣␤ (TCR␤ ؊/؊ ) or gamma interferon (IFN-␥) (IFN-␥ ؊/؊ ) knockout mice following inoculation with the KR-1-strain. SCID mice and TCR␣␤ knockouts sustained a severe but nonlethal parasitemia averaging 35 to 45% infected erythrocytes. IFN-␥-deficient mice developed a less severe parasitemia but were able to clear the infection. In contrast, in six of eight JHD-null mice, the levels of parasitemia were indistinguishable from those in the wild-type animals. These data indicate that cellular immunity is critical for the clearance of B. microti in BALB/c mice but that disease resolution can occur even in the absence of IFN-␥.

1 strains provides insights into the evolution of Treponema pallidum subsp. 2 pallidum, the syphilis spirochete 3 4 ABSTRACT 28In recent years, considerable progress has been made in topologically and 29 functionally characterizing integral outer membrane proteins (OMPs) of Treponema 30 pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-31 exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are 32 known to be highly variable. We examined the sequence diversity of β-barrel-encoding 33 regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, 34 California; and the Czech Republic and compared them to allelic variants in the 41 35 reference genomes in the NCBI database. To establish a phylogenetic framework, we 36 used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 37 clades. We found that (i) β-barrels in clinical strains could be grouped according to 38 allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains 39 within the Nichols or SS14 clades often harbored β-barrel variants that differed from the 40 Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in 41 predicted extracellular loops containing B-cell epitopes. Based upon structural models, 42 non-conservative amino acid substitutions in predicted transmembrane -strands of 43 TprC and TprD2 could give rise to functional differences in their porin channels. OMP 44 profiles of some clinical strains were mosaics of different reference strains and did not 45 correlate with results from enhanced molecular typing. Our observations suggest that 46 human host selection pressures drive TPA OMP diversity and that genetic exchange 47 IMPORTANCE 51Despite recent progress characterizing outer membrane proteins (OMPs) of 52 Treponema pallidum (TPA), little is known about how their surface-exposed, β-barrel-53 forming domains vary among strains circulating within high-risk populations. In this 54 study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and 55 bamA, in TPA from a large number of patient specimens from geographically disparate 56 sites were examined. Structural models predict that sequence variation within β-barrel 57 domains occurred predominantly within predicted extracellular loops. Amino acid 58 substitutions in predicted transmembrane strands that could potentially affect porin 59 channel function also were noted. Our findings suggest that selection pressures exerted 60 by human populations drive TPA OMP diversity and that recombination at OMP loci 61 contributes to the evolutionary biology of syphilis spirochetes. These results also set the 62 stage for topology-based analysis of antibody responses that promote clearance of TPA 63 and frame strategies for vaccine development based upon conserved OMP extracellular 64 loops. 65 66 67 After years of steady decline during the 1990s, syphilis, a sexually transmitted 68 infection caused by...