Recombinant Treponema pallidum Chemotaxis protein CheA (cheA), partial
Product Specs
Target Background
KEGG: tpa:TP_0363
STRING: 243276.TP0363
Q&A
What is the functional role of CheA protein in Treponema pallidum?
CheA serves as the central histidine kinase in the bacterial chemotaxis pathway of Treponema pallidum, the causative agent of syphilis. This protein plays a critical role in sensing and responding to environmental chemical gradients, which is essential for bacterial survival and pathogenesis. In the chemotaxis signaling cascade, CheA undergoes ATP-dependent autophosphorylation in response to stimuli detected by methyl-accepting chemotaxis proteins (MCPs). The phosphoryl group is subsequently transferred to the response regulator CheY, which controls flagellar motor rotation and thereby bacterial movement. As demonstrated in studies of related spirochetes, this phosphorelay system is crucial for coordinated cellular movement and directional motility .
How is the cheA gene organized in the Treponema pallidum genome?
In T. pallidum, the cheA gene is organized within an operon containing four open reading frames that encode proteins involved in bacterial chemotaxis. This operon includes cheA, cheW, cheX, and cheY genes. Reverse transcriptase-PCR data indicate that these genes are co-transcribed, confirming they comprise a functional operon . This genomic organization allows for coordinated expression of the chemotaxis machinery, ensuring appropriate stoichiometry of the signaling components. The presence of this putative che operon strongly suggests that T. pallidum possesses the molecular machinery for a complete chemotactic response, which is likely important for its survival and dissemination in the host .
Why is studying CheA protein important for understanding T. pallidum pathogenesis?
Studying CheA is critical for understanding T. pallidum pathogenesis because chemotaxis likely plays a significant role in bacterial dissemination within the host during syphilis infection. Several factors make CheA research particularly valuable:
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T. pallidum cannot be continuously cultivated in vitro, making the study of conserved pathways like chemotaxis especially important for understanding its biology .
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The organism has no known mechanisms of genetic exchange, limiting traditional approaches to studying virulence .
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Evidence from related spirochetes indicates that chemotaxis significantly impacts tissue invasion and disease progression.
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As demonstrated in T. denticola, disruption of cheA results in decreased swarming ability and failure to respond chemotactically to nutrient mixtures, suggesting CheA's importance in bacterial behavior .
These insights suggest that CheA-mediated chemotaxis may be essential for T. pallidum's ability to navigate host tissues, potentially offering new therapeutic targets for syphilis treatment.
What expression systems are optimal for producing recombinant T. pallidum CheA protein?
Based on successful approaches with other treponemal proteins, several expression systems can be considered for recombinant T. pallidum CheA production:
| Expression System | Advantages | Disadvantages | Recommended Conditions |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, simple protocols | Potential inclusion body formation | Induction: 0.5mM IPTG, 16°C, 18h |
| E. coli Rosetta(DE3) | Accommodates rare codons | Higher cost | Same as BL21, beneficial for T. pallidum's different codon usage |
| Cell-free systems | Avoids toxicity issues | Lower yield, expensive | Consider for domains that are toxic to E. coli |
For optimal expression, codon optimization for E. coli is recommended given T. pallidum's different codon bias. Expression vectors with cleavable affinity tags (His6, MBP, or GST) facilitate purification while allowing tag removal for functional studies. Lower induction temperatures (16-25°C) typically result in better protein folding and increased solubility of treponemal proteins .
What are the key challenges in purifying recombinant CheA protein and their solutions?
Purification of recombinant T. pallidum CheA presents several challenges that can be addressed through specific methodological approaches:
| Challenge | Solution | Rationale |
|---|---|---|
| Poor solubility | Use fusion partners (MBP, SUMO); Add 10-20% glycerol to buffers | Enhances folding; Stabilizes protein structure |
| Protein instability | Include ATP or analogs during purification; Maintain 4°C throughout | Stabilizes native conformation; Reduces degradation |
| Low purity | Multi-step purification: affinity→ion exchange→size exclusion | Removes contaminants with different properties |
| Loss of activity | Buffer optimization (pH 7.5-8.0, 150-300mM NaCl); Include reducing agents | Maintains native conformation and prevents oxidation |
| Aggregation | Add low concentrations of non-ionic detergents (0.01-0.05% Triton X-100) | Prevents non-specific hydrophobic interactions |
When purifying CheA, it's essential to remove imidazole quickly after affinity purification through dialysis or desalting, as high imidazole concentrations can interfere with protein activity and stability. Additionally, including protease inhibitors throughout the purification process helps preserve protein integrity .
What analytical methods should be used to verify recombinant CheA identity and activity?
A comprehensive validation of recombinant T. pallidum CheA requires multiple analytical approaches:
Identity and Purity Verification:
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SDS-PAGE: Should show a predominant band at approximately 76-80 kDa
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Western blotting: Using anti-His tag or CheA-specific antibodies
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Mass spectrometry: For accurate mass determination and sequence coverage
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N-terminal sequencing: To confirm protein identity and proper processing
Functional Validation:
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Autophosphorylation assay: Incubate purified CheA with [γ-32P]ATP and monitor incorporation by autoradiography
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Phosphotransfer assay: Measure transfer of phosphate from CheA to purified CheY
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ATPase activity: Quantify ATP hydrolysis using coupled enzyme assays
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Binding studies: Assess interaction with CheW and chemoreceptor fragments
Structural Integrity Assessment:
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Circular dichroism: Evaluate secondary structure elements
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Size-exclusion chromatography: Confirm monomeric/dimeric state
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Thermal shift assays: Determine protein stability and optimal buffer conditions
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Dynamic light scattering: Assess homogeneity and aggregation state
These analytical approaches ensure that the purified recombinant CheA is correctly folded, pure, and functionally active before proceeding with more complex experimental applications .
How can CheA kinase activity be measured in vitro?
Several complementary approaches can be used to measure T. pallidum CheA kinase activity in vitro:
Table: Protocols for Measuring CheA Kinase Activity
| Assay Type | Method | Readout | Advantages | Limitations |
|---|---|---|---|---|
| Radiometric Autophosphorylation | Incubate CheA with [γ-32P]ATP; SDS-PAGE separation; autoradiography | 32P incorporation | Gold standard; quantitative | Requires radioactive materials; discontinuous |
| Phosphotransfer | Pre-phosphorylate CheA; add CheY; monitor phosphate transfer | Decrease in CheA-P; increase in CheY-P | Measures complete pathway | Complex analysis; requires both proteins |
| FRET-based | Fluorescently labeled CheA and CheY; monitor energy transfer | Fluorescence change | Real-time; continuous | Requires protein labeling; potential interference |
| ATPase Activity | Coupled enzyme (PK/LDH) or malachite green assay | NADH oxidation or color change | Non-radioactive; continuous | Indirect measure; potential interference |
| Phos-tag™ SDS-PAGE | Mobility shift of phosphorylated CheA | Band position change | No radioactivity; simple | Semi-quantitative; lower sensitivity |
Optimal reaction conditions typically include: 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 2 μM CheA, and 1 mM ATP at 25°C. For accurate kinetic measurements, time-course sampling at 0, 0.5, 1, 2, 5, and 10 minutes is recommended to capture the initial rate of autophosphorylation .
How does the T. pallidum chemotaxis system compare to other bacterial species?
The T. pallidum chemotaxis system shares core components with model organisms but exhibits several distinct features:
Table: Comparative Analysis of Bacterial Chemotaxis Systems
| Feature | T. pallidum | E. coli | B. burgdorferi | T. denticola |
|---|---|---|---|---|
| CheA Copies | Single copy | Single copy | Two paralogs (CheA1, CheA2) | Single copy |
| Chemotaxis Operons | Single operon (cheA-cheW-cheX-cheY) | Multiple operons | Multiple operons | Similar to T. pallidum |
| Unique Components | CheX (putative phosphatase) | CheZ (phosphatase) | CheX; multiple CheYs | CheX |
| Motility Structure | Periplasmic flagella | External flagella | Periplasmic flagella | Periplasmic flagella |
| Cellular Response | Likely reversal of rotation | Run and tumble | Run and flex | Reversal of direction |
| Receptor Types | Limited repertoire | Multiple MCPs | Multiple MCPs | Multiple MCPs |
Research in T. denticola has demonstrated that CheA controls cellular reversal frequency, with cheA mutants exhibiting significantly reduced reversal rates while maintaining coordinated cell movement . This contrasts with E. coli, where CheA inactivation leads to constant smooth swimming. These findings suggest that the spirochetal chemotaxis system, while sharing core components with model organisms, has evolved unique mechanisms adapted to their distinctive motility patterns and environmental niches .
What methods can be used to create and validate cheA mutants in spirochetes?
Creating cheA mutants in T. pallidum is challenging due to cultivation difficulties, but related spirochetes provide valuable models. The following approaches have been validated:
Table: Methods for cheA Mutation in Spirochetes
In T. denticola, successful cheA mutation involved:
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Insertion of ermF-ermAM gene cassette into a naturally occurring NheI site at position 885 of the approximately 2.4-kb cheA gene
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Electroporation of linearized plasmid into T. denticola
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Selection on plates containing erythromycin (25 μg/ml)
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Confirmation by colony PCR and RT-PCR to verify the downstream genes (cheW, cheX, cheY) were still expressed
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Functional validation through swarming assays and chemotactic response tests
This approach yielded approximately 1,000 Erm resistant colonies per μg of DNA, though results vary between experiments. The mutants exhibited decreased swarming on soft-agar plates and failed to respond chemotactically to nutrient mixtures, confirming CheA's role in spirochetal chemotaxis .
How can structural biology approaches be applied to study T. pallidum CheA?
Multiple structural biology approaches can be employed to elucidate the structure-function relationships of T. pallidum CheA:
Table: Structural Biology Approaches for T. pallidum CheA Analysis
Given the modular nature of CheA, a domain-by-domain approach is often most successful. The P1-P5 domains can be expressed individually for structural determination, followed by integrative modeling to assemble the complete structure. Crystal structures of CheA domains from other organisms (such as Thermotoga maritima) provide valuable templates for homology modeling if experimental determination proves challenging .
What is the potential role of CheA in T. pallidum pathogenesis and host interaction?
CheA likely contributes significantly to T. pallidum pathogenesis through several mechanisms:
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Tissue Dissemination: By controlling directional motility, CheA enables T. pallidum to navigate through host tissues. Studies in related spirochetes demonstrate that cheA mutants exhibit impaired tissue penetration abilities .
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Nutrient Acquisition: Given T. pallidum's limited metabolic capabilities, CheA-mediated chemotaxis likely directs the bacterium toward essential nutrients in the host environment.
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Immune Evasion: Directed movement may allow T. pallidum to evade host immune responses, contributing to its remarkable ability to establish persistent infection.
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Tissue Tropism: CheA may facilitate preferential migration to specific tissues, explaining the diverse clinical manifestations of syphilis in different organ systems.
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Transmission: Efficient movement within host tissues may enhance the organism's ability to reach sites important for transmission to new hosts.
Evidence from T. denticola indicates that CheA controls cellular reversal frequency, which is critical for efficient bacterial movement through complex environments . The lack of cheA results in decreased swarming and failure to respond chemotactically to nutrients, suggesting similar impairments would significantly reduce T. pallidum virulence .
How can high-throughput approaches be used to identify CheA inhibitors as potential therapeutics?
A systematic high-throughput screening (HTS) approach for T. pallidum CheA inhibitors would involve:
Table: High-Throughput Screening Cascade for CheA Inhibitors
| Stage | Assay Type | Format | Hit Criteria | Purpose |
|---|---|---|---|---|
| Primary Screen | ATPase activity (luminescence-based) | 384-well plates; 10μM compound | >50% inhibition | Identify initial hits |
| Dose-Response | ATPase activity | 8-point curves (0.1-100μM) | IC50 <10μM | Confirm activity; potency ranking |
| Counter-Screen | Human kinase panel | 10μM single point | <30% inhibition of human kinases | Selectivity assessment |
| Secondary Validation | Autophosphorylation (32P or Phos-tag) | 96-well format | IC50 confirmation | Orthogonal confirmation |
| Tertiary Validation | CheA-CheY phosphotransfer | FRET-based assay | Inhibition of phosphotransfer | Pathway relevance |
| Biophysical Validation | Thermal shift; SPR | 96-well format | Direct binding confirmation | Binding mechanism |
Table: Compound Libraries for CheA Inhibitor Discovery
| Library Type | Examples | Advantages | Success Probability |
|---|---|---|---|
| Kinase-focused | Published kinase inhibitor sets | Higher hit rate; known scaffolds | Medium (bacterial/human differences) |
| Natural products | Microbial extracts; plant derivatives | Novel chemical space; historical anti-spirochete activity | Medium-high |
| Fragment-based | Low MW (150-300 Da) diverse fragments | Efficient chemical space coverage; optimization potential | Low initial potency, high long-term |
| Diversity-oriented | Synthetic libraries with diverse scaffolds | Broad coverage; novel chemotypes | Low-medium |
| Virtual screening hits | Computationally selected compounds | Cost-effective; structure-based | Medium (depends on model quality) |
Lead compounds identified through this cascade would require further optimization for:
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Potency against CheA (target IC50 <100nM)
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Selectivity over human kinases (>100-fold)
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Antimicrobial activity against related cultivable spirochetes
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Appropriate physicochemical properties (solubility, permeability)
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In vivo efficacy in animal models of spirochetal infection
This approach could identify novel anti-virulence therapeutics targeting bacterial motility and chemotaxis, potentially complementing traditional antibiotics for treating syphilis infections .
What controls should be included in CheA functional studies?
Rigorous CheA functional studies require appropriate controls to ensure reliable and interpretable results:
Table: Essential Controls for T. pallidum CheA Experimental Studies
| Control Type | Description | Purpose | Implementation |
|---|---|---|---|
| Positive Controls | Wild-type CheA protein | Establish baseline activity | Include in all enzymatic assays |
| Negative Controls | Heat-inactivated CheA; ATP-binding site mutant (H405Q) | Confirm specificity of assay | Process identical to experimental samples |
| Substrate Controls | Non-hydrolyzable ATP analogs (AMP-PNP) | Verify ATP-dependence | Replace ATP in reaction |
| Protein Controls | Purified CheA from related species (T. denticola) | Comparative analysis | Process in parallel with T. pallidum CheA |
| System Controls | Reconstituted systems with/without receptors and CheW | Assess complete pathway | Include/exclude components systematically |
| Buffer Controls | Reaction buffer without CheA | Account for non-enzymatic effects | Process identical to experimental samples |
For CheA mutant studies (e.g., in T. denticola), additional controls should include:
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Wild-type strain processed identically to mutant strains
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Complemented mutant strains expressing the wild-type cheA gene
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RT-PCR verification that downstream genes (cheW, cheX, cheY) are still expressed in the cheA mutant to rule out polar effects
What are the key experimental design considerations when studying CheA-receptor interactions?
When investigating CheA-receptor interactions in the T. pallidum chemotaxis system, several experimental design considerations are critical:
Table: Experimental Approaches for CheA-Receptor Interaction Studies
| Approach | Methodology | Advantages | Limitations | Key Considerations |
|---|---|---|---|---|
| Reconstituted Systems | Purified components in lipid vesicles or nanodiscs | Defined composition; controllable | May not reflect native environment | Receptor:CheW:CheA ratios critical (6:2:1) |
| Pull-down Assays | His-tagged receptors with CheA and CheW | Simple; identifies stable interactions | May miss transient interactions | Requires gentle washing; consider crosslinking |
| FRET Analysis | Fluorescently labeled CheA and receptors | Real-time; detects conformational changes | Requires protein labeling | Labeling position affects sensitivity |
| Microscale Thermophoresis | Mobility changes upon complex formation | Measures in solution; low protein requirements | Requires fluorescent labeling | Temperature gradients may affect complexes |
| Surface Plasmon Resonance | Receptor immobilization; CheA as analyte | Real-time kinetics; no labeling | Surface artifacts possible | Immobilization strategy affects function |
| Hydrogen-Deuterium Exchange | Mass spectrometry after D2O exposure | Maps interaction interfaces; no labeling | Complex data analysis | Requires careful experimental design |
When designing these experiments, researchers should consider:
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The native membrane environment significantly affects receptor-CheA interactions; consider membrane mimetics
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CheW is essential for coupling CheA to receptors and should be included in most interaction studies
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Receptor clustering affects CheA activity; higher-order assemblies may be necessary for physiologically relevant results
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The methylation state of receptors modulates their interaction with CheA and should be controlled or systematically varied
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Signal molecules (attractants/repellents) dramatically impact receptor conformation and subsequent CheA activity
How can comparative analysis of CheA across different spirochetes inform T. pallidum research?
Comparative analysis of CheA across spirochete species provides valuable insights for T. pallidum research:
Table: Comparative Features of Spirochetal CheA Proteins
Cross-species approaches that can inform T. pallidum CheA research include:
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Heterologous complementation: Introducing T. pallidum cheA into T. denticola cheA mutants to assess functional conservation
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Domain swapping: Creating chimeric CheA proteins with domains from T. pallidum and T. denticola to identify species-specific functional regions
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Comparative structural analysis: Using solved structures from more tractable spirochetes to build models of T. pallidum CheA
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Inhibitor cross-reactivity: Testing CheA inhibitors against multiple spirochetal species to identify broadly effective compounds
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Receptor specificity: Comparing chemotaxis receptor repertoires across species to identify T. pallidum-specific chemotactic signals
This comparative approach leverages the experimental accessibility of related spirochetes while maintaining focus on the specific biological properties of T. pallidum, creating a more comprehensive understanding of cheA function in syphilis pathogenesis .
