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Syphilis, a sexually transmitted infection caused by the spirocheteTreponema pallidum(Tp), is resurging globally. Opsonic antibodies (Abs) targeting surface-exposed epitopes of the spirochete’s outer membrane proteins (OMPs) are believed to promote macrophage-mediated clearance of the bacterium during infection and are presumed to be key to vaccine development.Tp’s repertoire of outer membrane proteins includes BamA (β-barrelassemblymachinery subunitA/TP0326), the central component of the molecular machine that inserts newly exported OMP precursors into the OM lipid bilayer. BamA is a bipartite protein consisting of an 18-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptidetransport-associated) domains. Antisera directed against BamA ECL4 promote internalization ofTpby rabbit peritoneal macrophages. Herein, we employed a novel two-stage, phage display strategy, termed “Epivolve” (forepitopeevolution), to generate five site-directed murine monoclonal Abs (mAbs) targeting a centrally located peptide (S2) of BamA ECL4. Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of BamA ECL4 displayed by aPyrococcus furiosusthioredoxin (PfTrx) scaffold (PfTrxBamA/ECL4). One mAb containing a unique amino acid sequence in both light and heavy chains showed activity in an opsonophagocytosis assay employing murine bone marrow-derived macrophages. Mice and rabbits hyperimmunized withPfTrxBamA/ECL4produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides including S2. In contrast, Abs generated duringTpinfection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Epivolve, which circumvents the natural immune response, can be utilized for the generation of mAbs that target subdominant opsonic epitopes in ECLs ofTpOMPs.

Sequencing of most Treponema pallidum (T. pallidum) genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modelling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β- sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Background Development of a vaccine is a necessary step towards curtailing the global syphilis epidemic. Towards this end, our group has utilized bioinformatic and biophysical methods to characterize T. pallidum’s repertoire of outer membrane proteins (OMPs): the two ‘stand-alones’, BamA and LptD, and the T. pallidum repeat (Tpr), 8-stranded β-barrel, outer membrane factor (OMF) for efflux pump, and FadL paralogous families. We have hypothesized that antibodies against extracellular loops (ECLs) of OMPs are responsible for the opsonic and protective activities of immune rabbit serum (IRS). We used Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to present ECLs for assessment of reactivity with syphilitic sera and quantitation of circulating ECL-specific B-cells. To develop this system, we focused on BamA/ECL4 which we previously have shown is immunogenic and a target for opsonic antibodies. Methods PfTrx was engineered to express BamA-ECL4 (PfTrx-BamA/ECL4) with a N-terminal His6- and C-terminal Avi-tags for Ni-NTA purification and in vivo biotinylation, respectively. Reactivity of IRS and human syphilitic sera (HSS) with PfTrx-BamA/ECL4 was assessed by immunoblotting. To determine the accessibility of the BamA/ECL4 epitope, a pull-down assay was performed by incubating PfTrx-BamA/ECL4 or PfTrx with IgG from IRS or normal rabbit serum (NRS) immobilized on protein G agarose beads. BamA/ECL4-specific IgG+ B-cells were identified by flow cytometry using PfTrx-BamA/ECL4 conjugated with streptavidin (SP)-Alexa Fluor 647 and SP-Brilliant Violet-421. PfTrx was conjugated to SP-APC-Cy7 to exclude non-specific binding to scaffold. Results IRS and HSS recognized PfTrx-BamA/ECL4 but not PfTrx. PfTrx-BamA/ECL4 was pulled down by IgG from IRS but not NRS, while PfTrx was not pulled down by either. 1.66% of circulating IgG+ B-cells were PfTrx-BamA/ECL4-specific. Conclusion When used in conjunction with OMP structural models, PfTrx-ECL is a promising platform for identification of antigenic ECLs and isolation of ECL-specific B-cells for the generation of anti-ECL monoclonal antibodies with opsonic activity.

Background The upsurge of syphilis cases is a public health threat, particularly in people living with HIV (PLWH). The aim of this case series was to describe the histological and immunohistochemical changes in skin lesions of patients with secondary syphilis (SS) by HIV status. Methods Clinical data and skin biopsies from SS patients who were consented and enrolled in Cali, Colombia between 2003 and 2018 were included in the analysis. Clinical data was extracted using a standardized form. Four-micrometer sections of paraffin embedded skin biopsies were stained with haematoxylin and eosin (H&E) or labelled with antibodies to detect CD4, CD8, CD56, CD68, CD138 or Treponema pallidum (Tp). Data from PLWH and people without HIV (PWH) were compared using the Fisher’s exact test for qualitative variables, the Student’s t test or the Wilcoxon rank-sum test for quantitative variables according to their distribution. The Kruskal-Wallis test was used to compare cellular populations according to categories of Tp burden (absent, sparse or moderate to severe). Results Of 114 subjects with SS, 28 PWH and 10 PLWH subjects had available skin biopsies. Most participants were male (65.8%), the mean age was 27 years (range 18–60). PLWH were more likely to identify themselves as mestizo (100% vs 52%, p=0.01), there were no differences in age, sex, symptoms or signs of syphilis. PLWH were more likely to have acanthosis (70% vs 32%, p=0.06) and epidermal hyperplasia (70% vs 18%, p=0.005). There were no differences in other H&E findings. PLWH had lower CD4 cells in the skin (p=0.0001), without differences in other cellular populations or Tp positivity. There were no differences in the cell counts by categories of Tp burden. Conclusions HIV infection was associated with non-specific epidermal hyperplasia and lower CD4 cell counts in the skin, but not with Tp positivity.

Background The outer membrane (OM) of Treponema pallidum (Tp) serves as the interface between the syphilis spirochete and its obligate human host and also is key to developing a vaccine with worldwide efficacy. We used structural modeling and bioinformatics to delineate the repertoire of OM proteins (OMPs) in the Tp Nichols strain. The Tp ‘OMPeome’ consists of two ‘stand-alone’ proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in influx/efflux of small molecules: 8-stranded β-barrels, long-chain fatty acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and Tp repeat protein (Tprs). Design/Methods Three-dimensional structural OMP models were generated with I-TASSER, Phyre2, and trRosetta. SAXS was used to solve solution structures for TprK and T. denticola MOSP, the Tpr family parental ortholog. Results Tp’s BamA (TP0326) is the central component of a hybrid BAM system in which POTRA1–5 interacts with the DUF domain of TamB (TP0325). Although Tp lacks lipopolysaccharide, its genome encodes a nearly complete LPS transporter apparatus. The LptD ortholog (TP0515) contains a large unstructured C-terminal domain, which models LptE-like inside the β-barrel. Tp encodes four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Surprisingly, Tp encodes five FadL orthologs, all of which display features (i.e., hatch domain and NPA motifs) that are characteristic of Gram-negative FadLs. Three (TP0548, TP0859 and TP0865) have α-helical extensions that could interact with periplasmic components. The Tp genome encodes Mac and RND efflux pumps that presumably achieve combinatorial diversity via co-expression of four paralogous OMFs. Lastly, we confirmed the bipartite membrane topology of Tprs using SAXS to solve solution structures for MOSPN domains of TprK and MOSP. Conclusions The Tp Nichols OMPeome provides a structural framework to (i) elucidate Tp’s enigmatic parasitic strategies, (ii) identify targets of natural immunity, and (iii) interrogate candidate vaccinogens.

Older adults suffer from weakened and delayed bone healing due to age-related alterations in bone cells and in the immune system. Given the interaction between the immune system and skeletal cells, therapies that address deficiencies in both the skeletal and the immune system are required to effectively treat bone injuries of older patients. The sequence of macrophage activation observed in healthy tissue repair involves a transition from a pro-inflammatory state followed by a pro-reparative state. In older patients, inflammation is slower to resolve and impedes healing. The goal of this study was to design a novel drug delivery system for temporal guidance of the polarization of macrophages using bone grafting materials. A biomimetic calcium phosphate coating (bCaP) physically and temporally separated the pro-inflammatory stimulus interferon-gamma (IFNγ) from the pro-reparative stimulus simvastatin (SIMV). Effective doses were identified using a human monocyte line (THP-1) and testing culminated with bone marrow macrophages obtained from old mice. Sequential M1-to-M2 activation was achieved with both cell types. These results suggest that this novel immunomodulatory drug delivery system holds potential for controlling macrophage activation in bones of older patients.

Background Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88−/−Bb-stimulated bone marrow-derived macrophages (BMDMs). Results MyD88−/− BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88−/− BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. Conclusion Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded β-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a β-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the β-barrel. T. pallidum has four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete’s many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded β-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location., Author summary Development of a syphilis vaccine requires knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a paucity of integral OM proteins, while the spirochete’s lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed adhesin/protease and protective antigen. The recent increase in calycin/lipocalin structures prompted a comprehensive re-examination of TP0751. TP0751 contains two domains—an N-terminal intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin. Query of the structural databases identified related bacterial lipocalins, none known surface molecules. In silico docking studies predicted that TP0751 binds diverse ligands along the rim of its eight-stranded β-barrel and this was confirmed in heme binding assays. TP0751 is poorly expressed compared to other T. pallidum lipoproteins and barely elicits antibodies in syphilis immune rabbits or patients with secondary syphilis. TP0751 antibodies did not promote uptake of spirochetes by rabbit peritoneal macrophages, and immunization of rabbits with intact, full-length TP0751 did not prevent local or disseminated infection. Our data indicate that TP0751 resides within the periplasmic space of T. pallidum where it binds small molecules; we propose that the IDR facilitates ligand binding by and offloading from the lipocalin domain.

Background Whole-genome sequencing (WGS) of Treponema pallidum subspecies pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples. Methods We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. Results By combining parallel, pooled whole-genome amplification with hybrid selection, we generated high-quality genomes from 4 of 8 chancre-swab samples and 2 of 2 rabbit-passaged isolates, all subjected to challenging storage conditions. Conclusions This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.

Background The failure of current prevention strategies to curtail the spread of syphilis, including mother-to-child transmission, underscores the need for a vaccine with worldwide efficacy. Characterization of variation within T. pallidum (TPA) outer membrane proteins (OMPs) is critical for this goal. Methods ClustalW identified polymorphisms within 21 β-barrel forming OMPs (TPA‘s OMPeome) of 15 unique TPA strains. Structural models and conformational B-cell epitopes (BCEs) were predicted. Clade assignments were made using tp0548 and tp0558 sequences. Results TPA OMPeomes contain four paralogous families (Tpr, FadL, OmpW, and efflux pump OMPs) and orthologs for BamA and LptD. The Tprs group into Subfamilies I (C/D/I), II (E/G/J) and III (B/H/L); Tpr β-barrel domains contain 10 β-strands and five extracellular loops (ECLs) harboring BCEs. TprI is invariant, while TprC and TprD have variability located predominantly in ECLs. The closely related TprE and TprJ β-barrels exhibit extremely low variability. TprG β-barrels variants contain a 23 aa insertion derived from TprE or TprJ. The TprB and TprH β-barrels are fully conserved; there are two TprL β-barrels variants. The FadL orthologs (TP0548, TP0856, TP0858, TP0859, TP0865) consist of 14-stranded β-barrels with seven ECLs harboring predicted BCEs. Variability among FadLs range between fully conserved (TP0856) to highly variable (TP0548). Both OmpWs are fully conserved and consist of 8-stranded β-barrels with four ECLs containing BCEs. The highly conserved efflux pump OMPs (OprJ/TP0966, OprN/TP0967, TolC/TP0969) likely form homotrimeric 12-stranded β-barrels. The 18-stranded BamA/TP0326 β-barrel contains a single aa substitution in ECL4. The LptD/TP0515 β-barrel contains 24 β-strands, 12 ECLs and numerous BCEs, which cluster primarily into two variants. Most OMPs do not align with clade designations. Conclusion 1.) TPA OMPs display variable degrees of sequence and surface antigenic variation. 2.) TPA OMPeomes are mosaics likely resulting from recombination among circulating strains. 3.) Our analysis enables selection of variable and non-variable OMPs for assessment of protective capacity. Disclosure No significant relationships.

Lyme disease is a tick-borne illness caused by the spirocheteBorrelia burgdorferi(Bb). It is believed that the robust inflammatory response induced by the host’s innate immune system is responsible for the clinical manifestations associated with Bb infection. The macrophage plays a central role in the immune response to many bacterial infections and is thought to play a central role in activation of the innate immune response to Bb. Previous studies have shown that following phagocytosis of spirochetes by macrophages, phagosome maturation results in degradation ofBband liberation of bacterial lipoproteins and nucleic acids, which are recognized by TLR2 and TLR8, respectively, and elicit MyD88-mediated phagosome signaling cascades. Bone marrow-derived macrophages (BMDMs) fromMyD88−/−mice show significantly reduced spirochete uptake and inflammatory cytokine production when incubated withBb ex vivo. Paradoxically, additional studies revealed that Bb-infectedMyD88−/−mice exhibit inflammation in joint and heart tissues. To determine the contribution of MyD88 to macrophage-mediated spirochete clearance, we compared wildtype (WT) andMyD88−/−mice using a murine model of Lyme disease.MyD88−/−mice showed increased Bb burdens in hearts 28 days post infection, while H&E staining and immunohistochemistry showed significantly increased inflammation and greater macrophage infiltrate in the hearts ofMyD88−/−mice. This suggests that Bb triggers MyD88-independent inflammatory pathways in macrophages to facilitate cell recruitment to tissues. Upon stimulation with Bbex vivo, WT andMyD88−/−BMDMs exhibit significant differences in bacteria uptake, suggesting that MyD88 signaling mediates cytoskeleton remodeling and the formation of membrane protrusions to enhance bacteria phagocytosis. A comprehensive transcriptome comparison in Bb-infected WT andMyD88−/−BMDMs identified a large cohort of MyD88-dependent genes that are differentially expressed in response to Bb, including genes involved in actin and cytoskeleton organization (Daam1, Fmnl1). We also identified a cohort of differentially-expressed MyD88-independent chemokines (Cxcl2, Ccl9) known to recruit macrophages. We identified master regulators and generated networks which model potential signaling pathways that mediate both phagocytosis and the inflammatory response. These data provide strong evidence that MyD88-dependent and -independent phagosomal signaling cascades in macrophages play significant roles in the ability of these cells to phagocytose Bb and mediate infection.AUTHOR SUMMARYMacrophages play prominent roles in bacteria recognition and clearance, includingBorrelia burgdorferi(Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance ofBbby macrophages, we studied Bb-infected wildtype (WT) and MyD88−/− mice and Bb-stimulated bone marrow-derived macrophages (BMDMs). Bb-infected MyD88−/− mice show increased bacterial burdens, macrophage infiltration and altered gene expression in inflamed heart tissue. MyD88−/− BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88−/− BMDMs identified a large cohort of differentially expressed MyD88-dependent genes involved in re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-independent master regulators (Cxcl2andVcam1) and MyD88-dependent intermediate proteins (RhoqandCyfip1) that are known to mediate inflammation and phagocytosis respectively. These results provide mechanistic insights into MyD88-mediated phagosomal signaling enhancing Bb uptake and clearance.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum , the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC , tprD , and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 ( tp0548 ) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum . They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum , little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC , tprD , and bamA , in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

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. IMPORTANCE 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.

Introduction Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Clinical manifestations result from the treponeme’s ability to elicit a robust immune response while at the same time evading host defenses. Syphilitic lesions are comprised of a rich cellular infiltrate, which includes IFN-gamma (IFNg) producing T cells, NK cells and activated macrophages. We previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. The purpose of this study is to establish a potential role for macrophages and opsonic Ab in clearance of Tp and generation of tissue-based inflammation during human syphilis. Methods We used monocyte-derived macrophages to develop an ex vivo model for studying spirochete-macrophage interactions. We used macrophage-colony stimulating factor and IFNg for macrophage maturation and evaluated the immunophenotypic modulations by flow cytometry. We assessed Tp uptake, in the presence or absence of HSS by confocal microscopy. We also determined the cellular responses initiated by opsonophagocytosis of Tp using targeted transcriptional array analysis and cytokine bead array. Results IFNg polarisation of macrophages led to an increase in Fcg receptors (FcgRs) expression, phagocytosis of HSS opsonized Tp and cytokine production. Blockade of CD64 significantly diminished spirochetal uptake and pro-inflammatory cytokine secretion by the macrophages. Conclusion Our ex vivo studies provide a potential role for macrophages in clearance of Tp during human syphilis. These data are the first to demonstrate that CD64 in the primary FcR involved in opsonophagocytosis of Tp and IFNg plays a critical role in the macrophages responsiveness following uptake of the spirochete. Moreover, our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that p38 MAP kinase–mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell–attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

Complement receptor (CR) 3 is a bona fide phagocytic receptor for Borrelia burgdorferi, the causative agent of Lyme borreliosis 1, 2. CR3 is also the receptor for the opsonin iC3b, a final degradation product of the complement component C3 3, 4. The transit of B. burgdorferi through the blood during the dissemination phase exposes the spirochete to serum components, including complement 5. B. burgdorferi contains proteins with anti-complement activities that trigger the inactivation of C3b through the binding of factor H (FH) on the surface of the bacterium 6. This could lead to the accumulation of the opsonin, iC3b, and the enhanced phagocytosis of the spirochete. To test whether the presence of serum would increase the phagocytosis of B. burgdorferi, we incubated bone marrow-derived macrophages (BMMs), generated as described 1, with heat-inactivated (HI; 56 oC, 30 min) or active normal mouse sera (NMS). Phagocytosis protocols were followed according to published reports 1, 2. The presence of 10% HI serum resulted in phagocytosis levels comparable to those in the absence of serum (Fig. ​(Fig.1A,B).1A,B). However, the presence of 10 % NMS enhanced the capacity of macrophages to internalize B. burgdorferi (Fig. ​(Fig.1A).1A). The same effect was observed on RAW264.7 (RAW) cells (Fig. ​(Fig.1C).1C). Thus, the contact of B. burgdorferi with serum components results in their enhanced phagocytic uptake. Next, we assessed whether the increased phagocytic activity associated with the presence of serum was due to C3-derived opsonin generation. We used sera from C3-deficient mice (C3 KO), in which B. burgdorferi dissemination is enhanced compared to WT mice 5. The presence of C3 KO serum abrogated the increased phagocytosis observed in the presence of NMS, both in BMMs (Fig. ​(Fig.1A,B)1A,B) and RAW cells (Fig. ​(Fig.11C).

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.

Phagocytosis of the extracellular bacterium Borrelia burgdorferi (Bb) enhances toll-like receptor, NF-κB mediated cytokine responses in human and murine phagocytic cells. TLR adaptor molecule MyD88 is recruited to Bb containing phagosomes, and the spirochetal cargo does not appear to leak from the phagosome into the cytosol. Transcriptome and Network reconstruction analysis identified several master regulators of diverse cellular and molecular processes, including regulation of the TLR-MyD88 pathway, IRFs, cytoskeletal rearrangement, and resolution of inflammation. Surprisingly, genes that code for inflammasome proteins (NLRP3, NOD2 and caspase-1) were also differentially-expressed. We thereby examined the cellular localization of these molecules in Bb-infected WT BMDMs and observed that NLRP3 and NOD2 were observed to colocalize to phagosomes containing degraded spirochetes. NLRP3 also colocalized with MyD88, but only in phagosomes containing degraded spirochetal cargo. Although caspase-1 also colocalized to the phagosome, infection with Bb did not result in an increase in speck formation or secretion of IL-1β. Our results show that the phagosome is a crucial cellular location not only for degradation of the spirochete, but where components of the Myddosome and inflammasome are recruited. Our findings are in line with recent studies showing that cytosolic receptors can localize to endosomal membranes, and can potentially have an alternate role in regulating an adequate functionality of the phagosome. Our study evidences the complexity of phagosomal signaling in which MyD88 plays a critical role not only in the generation of inflammatory but inducing regulatory signals in murine macrophages upon Bb infection.

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 β 2 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.