Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N⁶-adenine) methyltransferases. T. pallidum Dam can be assigned to group α DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam⁺ phenotype).     

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.     

Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease,Pallilysin

Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin''s active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹⁹⁸A], HAXXH [E¹⁹⁹A], and HEXXA [H²⁰²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.     

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lacto-ferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.     

Treponema pallidum Infection in the Wild Baboons of East Africa: Distribution and Genetic Characterization of the Strains Responsible

It has been known for decades that wild baboons are naturally infected with Treponema pallidum, the bacterium that causes the diseases syphilis (subsp. pallidum), yaws (subsp. pertenue), and bejel (subsp. endemicum) in humans. Recently, a form of T. pallidum infection associated with severe genital lesions has been described in wild baboons at Lake Manyara National Park in Tanzania. In this study, we investigated ten additional sites in Tanzania and Kenya using a combination of macroscopic observation and serology, in order to determine whether the infection was present in each area. In addition, we obtained genetic sequence data from six polymorphic regions using T. pallidum strains collected from baboons at two different Tanzanian sites. We report that lesions consistent with T. pallidum infection were present at four of the five Tanzanian sites examined, and serology was used to confirm treponemal infection at three of these. By contrast, no signs of treponemal infection were observed at the six Kenyan sites, and serology indicated T. pallidum was present at only one of them. A survey of sexually mature baboons at Lake Manyara National Park in 2006 carried out as part of this study indicated that roughly ten percent displayed T. pallidum-associated lesions severe enough to cause major structural damage to the genitalia. Finally, we found that T. pallidum strains from Lake Manyara National Park and Serengeti National Park were genetically distinct, and a phylogeny suggested that baboon strains may have diverged prior to the clade containing human strains. We conclude that T. pallidum infection associated with genital lesions appears to be common in the wild baboons of the regions studied in Tanzania. Further study is needed to elucidate the infection''s transmission mode, its associated morbidity and mortality, and the relationship between baboon and human strains.     

Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.     

Molecular typing of Treponema pallidum: a systematic review and meta-analysis

Background

Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum) with three objectives: (1) to determine specimen types most suitable for molecular typing; (2) to determine T. pallidum subtype distribution across geographic areas; and (3) to summarize available information on subtypes associated with neurosyphilis and macrolide resistance.

Methodology/Principal Findings

Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included “Treponema pallidum,” or “syphilis,” combined with the subject headings “molecular,” “subtyping,” “typing,” “genotype,” and “epidemiology.” Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ² = 247.6, p<0.001) and an average higher efficiency of full molecular typing (80.9% vs. 43.1%, χ² = 102.3, p<0.001) compared to plasma, whole blood, and cerebrospinal fluid. A pooled analysis of subtype distribution based on country location showed that 14d was the most common subtype, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited.

Conclusions/Significance

Primary lesion was a better specimen for obtaining T. pallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further validation of ear lobe blood for obtaining T. pallidum DNA would be useful for future molecular studies of syphilis.     

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B₁), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (K_d) values (TDE_0143, K_d of 36.50 nM; TP_0144, K_d of 32.62 nM). Finally, heterologous expression of TP_0144 in a ΔTDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well.     

Rapid Treponema pallidum Clearance from Blood and Ulcer Samples following Single Dose Benzathine Penicillin Treatment of Early Syphilis

Currently, the efficacy of syphilis treatment is measured with anti-lipid antibody tests. These can take months to indicate cure and, as a result, syphilis treatment trials require long periods of follow-up. The causative organism, Treponema pallidum (T. pallidum), is detectable in the infectious lesions of early syphilis using DNA amplification. Bacteraemia can likewise be identified, typically in more active disease. We hypothesise that bacterial clearance from blood and ulcers will predict early the standard serology-measured treatment response and have developed a qPCR assay that could monitor this clearance directly in patients with infectious syphilis. Patients with early syphilis were given an intramuscular dose of benzathine penicillin. To investigate the appropriate sampling timeframe samples of blood and ulcer exudate were collected intensively for T. pallidum DNA (tpp047 gene) and RNA (16S rRNA) quantification. Sampling ended when two consecutive PCRs were negative. Four males were recruited. The mean peak level of T. pallidum DNA was 1626 copies/ml whole blood and the mean clearance half-life was 5.7 hours (std. dev. 0.53). The mean peak of 16S rRNA was 8879 copies/ml whole blood with a clearance half-life of 3.9 hours (std. dev. 0.84). From an ulcer, pre-treatment, 67,400 T. pallidum DNA copies and 7.08x107 16S rRNA copies were detected per absorbance strip and the clearance half-lives were 3.2 and 4.1 hours, respectively. Overall, T. pallidum nucleic acids were not detected in any sample collected more than 56 hours (range 20–56) after treatment. All patients achieved serologic cure. In patients with active early syphilis, measuring T. pallidum levels in blood and ulcer exudate may be a useful measure of treatment success in therapeutic trials. These laboratory findings need confirmation on a larger scale and in patients receiving different therapies.     

Biophysical and Bioinformatic Analyses Implicate the Treponema pallidum Tp34 Lipoprotein (Tp0971) in Transition Metal Homeostasis

Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to Zn²⁺, which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions (Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺) readily induce the dimerization of Tp34; Cu²⁺ (50% effective concentration [EC₅₀] = 1.7 μM) and Zn²⁺ (EC₅₀ = 6.2 μM) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34''s likely role in metal ion homeostasis in T. pallidum.     

Treponema pallidum subsp. pallidum TP0136 Protein Is Heterogeneous among Isolates and Binds Cellular and Plasma Fibronectin via its NH2-Terminal End

Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum (T. pallidum), the agent of syphilis. Among T. pallidum adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of T. pallidum binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein’s central and COOH-terminal regions. Additionally, message quantification studies show that tp0136 is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in T. pallidum persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.     

Genetic engineering of Treponema pallidum subsp. pallidum,the Syphilis Spirochete

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan^R gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan^R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan^R cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl₂-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan^R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan^R message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA^ko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.     

Whole Genome Sequence of Treponema pallidum ssp. pallidum,Strain Mexico A,Suggests Recombination between Yaws and Syphilis Strains

Background

Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains.

Methodology/Principal Findings

The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains.

Conclusions/Significance

The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.     

Identification of Functional Candidates amongst Hypothetical Proteins of Treponema pallidum ssp. pallidum

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.     

Treponema pallidum fibronectin-binding proteins

Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.     

The Tryptophanase Gene Cluster of Haemophilus influenzae Type b: Evidence for Horizontal Gene Transfer

Among strains of Haemophilus influenzae, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic H. influenzae which are homologous to the tryptophanase (tna) operon of Escherichia coli. The tna genes are located on a 3.1-kb fragment between nlpD and mutS in the H. influenzae type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from nlpD, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The tna cluster is found at the same map location in all indole-positive strains of H. influenzae surveyed and is absent from reference type d and e genomes. In contrast to H. influenzae, most other Haemophilus species lack tna genes. Phylogenetic comparisons suggest that the tna cluster was acquired by intergeneric lateral transfer, either by H. influenzae or a recent ancestor, and that E. coli may have acquired its tnaA gene from a related source. Genomes of virulent H. influenzae resemble those of pathogenic enterics in having an island of laterally transferred DNA next to mutS.     

Recombinant Treponema pallidum Protein Tp0965 Activates Endothelial Cells and Increases the Permeability of Endothelial Cell Monolayer

The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.     

The Structure of Treponema pallidum Tp0751 (Pallilysin) Reveals a Non-canonical Lipocalin Fold That Mediates Adhesion to Extracellular Matrix Components and Interactions with Host Cells

Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete Borrelia burgdorferi to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein’s lipocalin fold.     

High Prevalence of Antibodies against the Bacterium Treponema pallidum in Senegalese Guinea Baboons (Papio papio)

The bacterium Treponema pallidum is known to cause syphilis (ssp. pallidum), yaws (ssp. pertenue), and endemic syphilis (ssp. endemicum) in humans. Nonhuman primates have also been reported to be infected with the bacterium with equally versatile clinical manifestations, from severe skin ulcerations to asymptomatic. At present all simian strains are closely related to human yaws-causing strains, an important consideration for yaws eradication. We tested clinically healthy Guinea baboons (Papio papio) at Parc National Niokolo Koba in south eastern Senegal for the presence of anti-T. pallidum antibodies. Since T. pallidum infection in this species was identified 50 years ago, and there has been no attempt to treat non-human primates for infection, it was hypothesized that a large number of West African baboons are still infected with simian strains of the yaws-bacterium. All animals were without clinical signs of treponematoses, but 18 of 20 (90%) baboons tested positive for antibodies against T. pallidum based on treponemal tests. Yet, Guinea baboons seem to develop no clinical symptoms, though it must be assumed that infection is chronic or comparable to the latent stage in human yaws infection. The non-active character is supported by the low anti-T. pallidum serum titers in Guinea baboons (median = 1:2,560) versus serum titers that are found in genital-ulcerated olive baboons with active infection in Tanzania (range of medians among the groups of initial, moderate, and severe infected animals = 1:15,360 to 1:2.097e+7). Our findings provide evidence for simian infection with T. pallidum in wild Senegalese baboons. Potentially, Guinea baboons in West Africa serve as a natural reservoir for human infection, as the West African simian strain has been shown to cause sustainable yaws infection when inoculated into humans. The present study pinpoints an area where further research is needed to support the currently on-going second WHO led yaws eradication campaign with its goal to eradicate yaws by 2020.     

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X-100. Intact., encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a candidate T. pailidum outer membrane protein (TpN50) with C-terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co-incubated with detergent. In contrast, antibodies generated against isolated T, pal-lidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze-fracture electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.