David L. Cox

The general transition metal (Tro) and Zn2+ (Znu) transporters in Treponema pallidum: analysis of metal specificities and expression profiles

Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster‐9 (C9) ABC transporter (troABCD) whose solute‐binding protein component (TroA) ligands Zn2+ and Mn2+ with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034–36) orthologous to Zn2+‐uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His‐rich tract characteristic of C9 Zn2+‐binding proteins. Metal analysis and metal‐reconstitution studies demonstrated that ZnuA is a Zn2+‐binding protein; parallel studies confirmed that TroA binds Zn2+, Mn2+ and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn2+. Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn2+ and Mn2+ with affinities approximately 100‐fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn2+‐binding sites, two of which are high‐affinity and presumed to be located within the binding pocket and His‐rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn2+ (Znu) transporters. Sequestration of periplasmic Zn2+ by ZnuA may free up TroA binding capacity for the importation of Fe and Mn2+.

Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum

ABSTRACT Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochetes “stealth pathogenicity,” we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely β-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of β-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and β-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochetes remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.

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 spirochaetes 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.

Diagnosis of Gastric Syphilis by Direct Immunofluorescence Staining and Real-Time PCR Testing

ABSTRACT We report on a case of gastric syphilis in a patient with chronic dyspepsia. The diagnosis was established by serology and the demonstration of spirochetes in diffusely inflammed gastric mucosa by staining with a fluorescent monoclonal antibody specific for pathogenic treponemes and by the detection of specific treponemal DNA sequences by a real-time PCR.

Secondary syphilis in cali, Colombia: new concepts in disease pathogenesis.

Venereal syphilis is a multi-stage, sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum (Tp). Herein we describe a cohort of 57 patients (age 18–68 years) with secondary syphilis (SS) identified through a network of public sector primary health care providers in Cali, Colombia. To be eligible for participation, study subjects were required to have cutaneous lesions consistent with SS, a reactive Rapid Plasma Reagin test (RPR-titer ≥1∶4), and a confirmatory treponemal test (Fluorescent Treponemal Antibody Absorption test- FTA-ABS). Most subjects enrolled were women (64.9%), predominantly Afro-Colombian (38.6%) or mestizo (56.1%), and all were of low socio-economic status. Three (5.3%) subjects were newly diagnosed with HIV infection at study entry. The duration of signs and symptoms in most patients (53.6%) was less than 30 days; however, some patients reported being symptomatic for several months (range 5–240 days). The typical palmar and plantar exanthem of SS was the most common dermal manifestation (63%), followed by diffuse hypo- or hyperpigmented macules and papules on the trunk, abdomen and extremities. Three patients had patchy alopecia. Whole blood (WB) samples and punch biopsy material from a subset of SS patients were assayed for the presence of Tp DNA polymerase I gene (polA) target by real-time qualitative and quantitative PCR methods. Twelve (46%) of the 26 WB samples studied had quantifiable Tp DNA (ranging between 194.9 and 1954.2 Tp polA copies/ml blood) and seven (64%) were positive when WB DNA was extracted within 24 hours of collection. Tp DNA was also present in 8/12 (66%) skin biopsies available for testing. Strain typing analysis was attempted in all skin and WB samples with detectable Tp DNA. Using arp repeat size analysis and tpr RFLP patterns four different strain types were identified (14d, 16d, 13d and 22a). None of the WB samples had sufficient DNA for typing. The clinical and microbiologic observations presented herein, together with recent Cali syphilis seroprevalence data, provide additional evidence that venereal syphilis is highly endemic in this region of Colombia, thus underscoring the need for health care providers in the region to be acutely aware of the clinical manifestations of SS. This study also provides, for the first time, quantitative evidence that a significant proportion of untreated SS patients have substantial numbers of circulating spirochetes. How Tp is able to persist in the blood and skin of SS patients, despite the known presence of circulating treponemal opsonizing antibodies and the robust pro-inflammatory cellular immune responses characteristic of this stage of the disease, is not fully understood and requires further study.

Tp0453, A Concealed Outer Membrane Protein Of Treponema Pallidum, Enhances Membrane Permeability

The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

Broad specificity AhpC-like peroxiredoxin and its thioredoxin reductant in the sparse antioxidant defense system of Treponema pallidum

Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium’s antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 μM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 ± 1 and -192 ± 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection.

Culture of Treponema pallidum

Publisher Summary This chapter discusses the culture of Treponerna pallidum ( T. pallidum ). T. pallidum can be cultivated in a variety of tissue culture vessels; 24-, 12-, and 6-well culture plates, and 25-, 75-, and 150-cm 2 tissue culture flasks, have been used successfully to cultivate T. pallidum . Cultivation in suspension cultures of Sf1Ep cells in Magna–Flex flasks has also been successful. The amount of cells varies for each type of vessel. The cultures are set up 2 days before infection and confluency is between 20 and 25%. Methods have been developed to cultivate T. pallidum on Sf1Ep cells attached to microcarrier beads. The rationale for this development was to provide a better vehicle for serial passage experiments. Suspension cultures would be much easier to transfer from flask to flask because harvesting using TV would not be required. Thus, the cells and the treponemes would experience minimal disturbance during the suspension culture passage process as compared with more rigorous harvesting using TV. The chapter illustrates the typical growth curve for T. pallidum cultivated in vitro . When incubated at 34 ° in a microaerophilic atmosphere of 3-4% oxygen, the typical lag period for treponemal growth is about 2 days. Small increases (2- to 3-fold) can be detected as early as 3 days. In tissue cultures, generation times between 35 and 40 hr are routinely observed. The stationary phase of growth in treponemal cultures is relatively short, typically lasting only 1 to 2 days.

Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes treponema pallidum and Borrelia burgdorferi

The authors examined the ability of octadecanoyl (C(18)), hexadecanoyl (C(16)) and dodecanoyl (C(12)) fatty acid (FA) conjugates of 5-aminofluorescein (OAF, HAF and DAF, respectively) to insert into the outer membranes (OMs) of Treponema pallidum, Borrelia burgdorferi and Escherichia coli. Biophysical studies have demonstrated that these compounds stably insert into phospholipid bilayers with the acyl chain within the hydrophobic interior of the apical leaflet and the hydrophilic fluorescein moiety near the phospholipid head groups. Consistent with the known poor intrinsic permeability of the E. coli OM to hydrophobic compounds and surfactants, E. coli was not labelled with any of the FA probes. OAF inserted more readily into OMs of B. burgdorferi than into those of T. pallidum, although both organisms were completely labelled at concentrations at or below 2 microg ml(-1). Intact spirochaetes were labelled with OAF but not with antibodies against known periplasmic antigens, thereby confirming that the probe interacted exclusively with the spirochaetal OMs. Separate experiments in which organisms were cooled to 4 degrees C (i.e. below the OM phase-transition temperatures) indicated that labelling with OAF was due to insertion of the probe into the OMs. B. burgdorferi, but not T. pallidum, was labelled by relatively high concentrations of HAF and DAF. Taken as a whole, these findings support the prediction that the lack of lipopolysaccharide renders T. pallidum and B. burgdorferi OMs markedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts. The data also raise the intriguing possibility that these two pathogenic spirochaetes obtain long-chain FAs, nutrients they are unable to synthesize, by direct permeation of their OMs.

A Monoclonal Antibody That Conveys In Vitro Killing and Partial Protection in Experimental Syphilis Binds a Phosphorylcholine Surface Epitope of Treponema pallidum

ABSTRACT Immunization with purified Treponema pallidum outer membrane vesicles (OMV) has previously resulted in high-titer complement-dependent serum bactericidal activity. In this study, OMV immunization resulted in the isolation of a monoclonal antibody, M131, with complement-dependent killing activity. Passive immunization of rabbits with M131 administered intravenously conferred significant immunity demonstrated by the failure of syphilitic lesions to appear at 29% of intradermal challenge sites (7/24) and a mean delay of approximately 8 days to lesion appearance at the remaining sites (17/24). M131 not only bound to OMV and to the surfaces of intact motile T. pallidum cells but also bound to organisms whose outer membranes were removed, indicating both surface and subsurface locations for the killing target. This target was determined to be a T. pallidum lipid. Lipid extracted from T. pallidum and made into liposomes bound M131. Reverse-phase high-pressure liquid chromatography separation and fraction collection mass spectrometry (LC-MS+) of T. pallidum lipid showed that the target of M131 was phosphorylcholine. M131 binding required both liposome formation and a critical concentration of phospholipid containing phosphorylcholine, suggesting that the epitope has both a conformational and a compositional requirement. M131 did not react with red blood cells, which have phosphorylcholine-containing lipids in their exterior membrane leaflets, or with Venereal Disease Research Laboratory antigen that also contains phosphorylcholine, further indicating the specificity of M131. This is the first physical demonstration of an antigen on the T. pallidum surface and indication that such a surface antigen can be a target of immunity.