T.pallidum p17 (Partial)
Description
Diagnostic Applications in Syphilis
p17 is evaluated as a biomarker in enzyme-linked immunosorbent assays (ELISA) for syphilis detection. Below are key performance metrics from clinical studies:
Stage-Specific Performance:
-
Secondary Syphilis: Highest sensitivity (97%) for TpN17 compared to TmpA (84–97%) .
-
Latent Syphilis: TpN17 sensitivity drops to 93.2% in recent latent stages .
Antibody Response Patterns
Studies demonstrate stage-dependent IgG titers to p17:
-
Primary Syphilis: Low total antibody levels but elevated IgG to p41 (not p17) .
-
Early Latent Syphilis: Predominant IgG to p17 with high total antibody titers .
-
Secondary Syphilis: High total antibodies with diverse IgG ratios to p17 and other antigens (e.g., p41) .
T Cell Responses
p17 elicits CD4+ T cell responses detectable in blood and skin lesions during secondary syphilis, persisting post-treatment . These T cells may contribute to immune memory and partial protection against reinfection .
Cross-Reactivity and Clinical Implications
While p17 exhibits high specificity, rare cross-reactivity occurs with non-treponemal pathogens:
| Pathogen | Cross-Reactivity Rate | Assay | Source |
|---|---|---|---|
| Chagas disease | 1.5% | ELISA | |
| Human Papillomavirus (HPV) | Not reported | – | – |
| HIV | 9.5% (via TmpA, not p17) | ELISA |
This low cross-reactivity supports its use in syphilis diagnostics, particularly in regions with overlapping endemic diseases .
Product Specs
Q&A
How do E. coli expression systems influence the immunogenicity of recombinant T. pallidum p17?
Recombinant T. pallidum p17 is typically expressed in E. coli as a 22 kDa protein fused to a 6xHis tag at the C-terminus or beta-galactosidase at the N-terminus . While this approach enhances solubility and purification efficiency, it may alter tertiary structure and expose non-native epitopes. For instance, the His tag can introduce metal-binding sites, potentially interfering with antibody binding . Studies suggest optimizing folding conditions (e.g., refolding buffers) to preserve conformational epitopes critical for serological assays .
Key considerations:
What challenges arise in producing recombinant T. pallidum p17, and how are they addressed?
Key challenges include low solubility and instability during purification. T. pallidum p17 is purified via proprietary chromatography, often in PBS with 50 mM arginine to stabilize the protein . Stability studies recommend storage at -18°C to prevent aggregation, with freeze-thaw cycles strictly avoided . For functional assays, renaturation protocols (e.g., gradual dialysis from urea) are critical to restore activity .
What methods are used to genetically engineer T. pallidum for studying p17 function, and what limitations exist?
Genetic engineering in T. pallidum involves transforming in vitro-cultivated strains with suicide vectors. For example, a kanamycin resistance (kan<sup>R</sup>) cassette was inserted into the tprA pseudogene (TP0009) using homology-directed repair . Challenges include the bacterium’s reduced genome (1.14 Mb) and limited metabolic pathways, necessitating selection markers like kan<sup>R</sup> under a weak promoter (e.g., tp0574) .
Key steps:
-
Vector design: Suicide plasmid with homology arms flanking the target gene.
-
Transformation: CaCl<sub>2</sub> treatment to enhance membrane permeability.
-
Selection: Kanamycin (25 µg/mL) to eliminate non-transformed cells .
Limitations:
-
Pseudogene targets may lack phenotypic readouts.
-
Low transformation efficiency due to T. pallidum’s slow growth (~44-hour generation time) .
How can researchers overcome pseudogene limitations in T. pallidum knockout studies?
To address the lack of phenotypic markers in pseudogene knockouts (e.g., tprA), researchers employ complementary techniques:
-
PCR validation: Confirm cassette integration via locus-specific primers .
-
Whole-genome sequencing: Verify off-target mutations.
-
Mass spectrometry: Detect kan<sup>R</sup> expression in transformed strains .
What parameters are critical for optimizing ELISA using T. pallidum p17 (TpN17)?
For indirect ELISA, key parameters include antigen coating concentration (typically 1–2 µg/mL), blocking agents (e.g., 5% BSA), and detection antibodies (anti-human IgG/IgM). TpN17 has demonstrated 97.2% sensitivity and 100% specificity in syphilis diagnosis, outperforming TmpA in latent stages .
Optimization Protocol:
How does TpN17 compare to other antigens (e.g., TmpA) in cross-reactivity and diagnostic accuracy?
TpN17 exhibits minimal cross-reactivity (1.5% in Chagas disease sera), while TmpA shows higher sensitivity (90.6%) but lower specificity in latent syphilis .
Diagnostic Performance:
| Antigen | Sensitivity (%) | Specificity (%) | Cross-reactivity (%) |
|---|---|---|---|
| TpN17 | 97.2 | 100 | 1.5 (Chagas) |
| TmpA | 90.6 | 100 | Not reported |
How should researchers interpret discrepancies in serological data using T. pallidum p17?
Discrepancies may arise from:
-
Clinical phase variability: TpN17 detects primary (100%), secondary (100%), and latent syphilis (93.2%) with high accuracy .
-
Antigen processing: False negatives may occur due to antigen degradation or blocking by host antibodies.
-
Population diversity: Ethnic/geographic differences in T. pallidum strains could influence epitope recognition.
Mitigation: Use chimeric antigens (e.g., p15/p17/p47) to enhance epitope coverage .
What strategies improve the detection of low-abundance T. pallidum p17 in complex samples?
For low-abundance detection (e.g., early syphilis):
-
Signal amplification: Use tyramide signal amplification (TSA) in immunohistochemistry .
-
Multiplex platforms: Combine TpN17 with TmpA in bead-based assays to reduce false negatives .
-
Sample pre-treatment: Use protein precipitation to concentrate antigens in serum .
How can researchers validate T. pallidum p17 as a diagnostic biomarker across distinct syphilis stages?
Validation requires:
-
Sera panels: Include primary (chancre), secondary (rash), latent, and tertiary stages.
-
ROC curve analysis: Determine optimal cutoff values for each stage .
-
Comparison with gold standards: Treponemal tests (e.g., FTA-ABS) and non-treponemal tests (e.g., VDRL) .
What obstacles exist in studying T. pallidum p17’s role in pathogenesis?
Key challenges include:
-
Host restriction: T. pallidum cannot be cultured in artificial media, limiting in vitro studies .
-
Immune evasion mechanisms: p17 may trigger regulatory T-cell responses to suppress host immunity .
-
Antigenic variation: Limited data on T. pallidum subspecies-specific epitopes (e.g., pertenue vs. pallidum) .
How can CRISPR-based tools advance T. pallidum p17 research?
CRISPR-Cas9 systems could enable precise gene editing in T. pallidum, though challenges remain:
-
Delivery efficiency: Electroporation or conjugation systems for plasmid uptake.
-
Selection markers: Requires non-antibiotic selection (e.g., auxotrophy) due to T. pallidum’s reduced genome .
What biosafety protocols are essential when handling T. pallidum p17?
While recombinant p17 is non-infectious, BSL-2 practices are recommended:
Product Science Overview
Treponema pallidum is a spirochete bacterium responsible for syphilis, a sexually transmitted infection. The bacterium has several proteins that play crucial roles in its pathogenicity and immune evasion. One such protein is the p17 protein, also known as TpN17 or 17 kDa lipoprotein. The recombinant form of this protein, particularly the partial recombinant, has been extensively studied for its potential applications in diagnostic assays and research.
The p17 protein of Treponema pallidum is a lipoprotein with a molecular weight of approximately 17 kDa. It is encoded by the TP_0435 gene in the Nichols strain of Treponema pallidum. The recombinant form of this protein is typically expressed in Escherichia coli to ensure high yield and purity. The recombinant p17 protein is often produced with a purity greater than 90%, making it suitable for various applications such as enzyme-linked immunosorbent assays (ELISA) and Western blotting (WB) .
The p17 protein is an immunodominant antigen, meaning it elicits a strong immune response in infected individuals. This makes it a valuable target for serological tests aimed at diagnosing syphilis. The protein is involved in the bacterium’s ability to adhere to host tissues and evade the host immune system. Its lipoprotein nature allows it to integrate into the bacterial membrane, contributing to the bacterium’s structural integrity and pathogenic mechanisms.
Recombinant p17 protein has been evaluated for its performance in serological diagnosis of syphilis. Studies have shown that it can achieve high accuracy in detecting Treponema pallidum infections. For instance, the sensitivity and specificity of p17 in various diagnostic assays have been reported to be quite high, making it a reliable marker for syphilis diagnosis . The use of recombinant proteins like p17 in immunoassays provides greater reliability and consistency in test results, which is crucial for accurate diagnosis and treatment.
The recombinant p17 protein is also used in research to study the immune response to Treponema pallidum. By understanding how the immune system interacts with this protein, researchers can develop better diagnostic tools and potentially new therapeutic approaches. The protein’s role in immune evasion and pathogenicity makes it a focal point for studies aimed at understanding the mechanisms of syphilis infection.
