Chlamydia HSP70 (462-503 a.a.)

What is Chlamydia HSP70 (462-503 a.a.) and what is its significance in research?

Chlamydia HSP70 (462-503 a.a.) is an E. coli-derived recombinant protein containing specific epitopes of the Chlamydia trachomatis Heat Shock Protein 70, spanning amino acids 462-503. This protein fragment is particularly significant because it demonstrates high immunoreactivity with sera from Chlamydia trachomatis-infected individuals, making it valuable for both diagnostic applications and immunological research .

The full HSP70 protein in Chlamydia trachomatis has been associated with the outer membrane complexes of infectious elementary bodies (EB) and may play a crucial role in the attachment to or entry into endometrial epithelial cells during infection . The 462-503 amino acid region likely contains important immunogenic epitopes that are specifically recognized by the host immune system during infection.

How is Chlamydia HSP70 structurally and functionally characterized?

The Chlamydia trachomatis HSP70 is a 70-kilodalton heat shock protein with multiple functional domains. Research indicates that the protein contains distinct regions including:

• ATPase domain

• Substrate-binding domain (which includes the 462-503 a.a. region)

While the complete HSP70 protein is associated with the outer membrane complexes of elementary bodies, it is not naturally displayed on the surface under normal conditions. The substrate-binding domain becomes accessible only after structural changes occur in the EB outer membrane, particularly when disulfide bonds are reduced . This suggests that the protein undergoes important conformational changes during the infection process.

The purified recombinant fragment (462-503 a.a.) maintains >95% purity as determined by PAGE and RP-HPLC analysis, making it suitable for precise immunological studies .

What are the specific applications of Chlamydia HSP70 (462-503 a.a.) in laboratory research?

The recombinant Chlamydia HSP70 (462-503 a.a.) has several important research applications:

• Immunological assays: The protein is particularly suitable for ELISA applications to detect antibodies in samples from infected individuals .

• Infection mechanism studies: The fragment can be used to investigate the role of HSP70 in cellular attachment and entry processes during Chlamydia infection .

• Antibody development: The protein can serve as an antigen for generating specific antibodies against this immunogenic region of HSP70.

• Diagnostic tool development: Given its immunoreactivity with sera from infected individuals, it serves as a valuable component in developing diagnostic tests for Chlamydia trachomatis infections .

It's important to note that while the recombinant protein is excellent for laboratory research, it is explicitly not intended for use as drugs, agricultural or pesticidal products, food additives, or household chemicals .

What are the optimal conditions for expressing and purifying Chlamydia HSP70?

Based on research methodologies documented in the literature, the following approach has proven effective for expressing and purifying Chlamydia HSP70:

Expression System:

• The open reading frame encoding HSP70 can be amplified from C. trachomatis serovar E genome using PCR with specific primers .

• For efficient expression, the E. coli strain LMG194 containing the plasmid pPBW120 has been successfully employed .

• Expression can be induced using 0.002% (vol/vol) l-arabinose .

Purification Method:

• Histidine-tagged HSP70 can be efficiently purified using nickel affinity chromatography .

• For the specific 462-503 a.a. fragment, proprietary chromatographic techniques have been used to achieve >95% purity .

Storage Conditions:

• Optimal storage is below -18°C to maintain stability .

• While the protein shows stability at 4°C for up to 1 week, freeze-thaw cycles should be prevented .

• The recommended formulation for storage is 50mM Tris-HCl, pH 8.0, 8M Urea, 60 mM NaCl, and 50% glycerol .

How can researchers effectively design antibodies against specific domains of Chlamydia HSP70?

Designing effective antibodies against Chlamydia HSP70 domains requires careful consideration of several factors:

Selection of Target Epitopes:

• Peptide selection should be based on predictive protein modeling techniques .

• Researchers should reference established three-dimensional structures of homologous proteins, such as the 70-kDa bovine heat shock cognate (Hsc70) ATPase domain .

• Previously defined linear and immunogenic epitopes of the chlamydial HSP70 should be considered .

Verification of Antibody Specificity:

• Western blot analysis against total C. trachomatis EB proteins should be performed to verify specificity .

• Immunoprecipitation assays with recombinant proteins can confirm recognition of native (non-denatured) forms of HSP70 .

• Cross-reactivity with host HSP70 should be assessed to ensure specificity for the bacterial protein.

Experimental Validation:

• Antibodies should be tested across multiple experimental platforms including Western blotting, immunoprecipitation, and microscopy techniques to ensure versatility .

• Both preimmune and immune sera should be compared to validate specificity .

How does the surface accessibility of Chlamydia HSP70 change during the infection process?

Research has revealed important insights into the dynamic surface accessibility of Chlamydia HSP70 during infection:

Initial State:
Immunofluorescence microscopy and transmission electron microscopy have demonstrated that chlamydial HSP70 is not a surface-displayed ligand on purified elementary bodies (EB) under normal conditions .

Conformational Changes:
Brief exposure of EB to thiol reducing agents such as dithiothreitol (DTT) leads to surface accessibility of the HSP70 substrate-binding domain . This suggests that reduction of disulfide bonds in EB outer membrane proteins causes conformational changes that expose previously hidden epitopes.

Infection Process Dynamics:
The research supports a model where:

• The structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during initial attachment stages .

• Reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells .

• Subsequent conformational changes in EB ultrastructure allow productive infection to proceed .

This suggests that HSP70 may not function as a primary surface-displayed adhesin, but rather plays a role in events following the initial stage of attachment .

How can researchers reconcile contradictory findings about Chlamydia HSP70's role in infection?

The literature contains seemingly contradictory observations about HSP70's role in Chlamydia infection that can be reconciled through careful experimental design and interpretation:

Apparent Contradictions:

Reconciliation Framework:

• Temporal considerations: HSP70 accessibility changes throughout the infection process, becoming accessible only at specific stages .

• Methodological differences: Different experimental approaches may detect HSP70 at different stages or under different conditions.

• Multifunctional nature: HSP70 likely plays multiple roles during infection, some requiring surface exposure and others occurring internally.

• Redox environment: The localized reducing environment at the host-pathogen interface may differ from experimental conditions used in vitro .

These considerations suggest that HSP70 has a dynamic role that changes as infection progresses from initial attachment through productive infection.

What experimental design considerations are critical when studying the reduction-dependent conformational changes in Chlamydia HSP70?

When investigating how reduction affects HSP70 conformation and function, researchers should consider several critical experimental design factors:

Choice of Reducing Agents:

• Dithiothreitol (DTT) has been successfully used to induce conformational changes that expose the HSP70 substrate-binding domain .

• Researchers should compare different reducing agents to determine if the effects are specific to certain chemical properties.

Control Experiments:

• Compare membrane-permeable versus membrane-impermeable reducing agents to distinguish between surface and internal effects .

• Include thiol-alkylating reagents such as 5,5′-dithiobis(2-nitrobenzoic acid) as controls to determine the specific role of thiol groups .

Detection Methods:

• Employ domain-specific antibodies targeting different regions of HSP70 to map which domains become accessible under different conditions .

• Use multiple visualization techniques including immunofluorescence microscopy and transmission electron microscopy to confirm findings .

Physiological Relevance:

• Design experiments that mimic the microenvironment at the host-pathogen interface, including pH, temperature, and redox conditions.

• Consider the temporal dynamics of the infection process, testing HSP70 accessibility at different time points during infection.

Functional Validation:

• Correlate changes in HSP70 accessibility with functional outcomes such as attachment efficiency and infectivity .

• Employ site-directed mutagenesis of specific cysteine residues to determine which disulfide bonds are critical for the conformational changes.

What bioinformatic approaches can be used to predict functional domains within Chlamydia HSP70?

Several bioinformatic approaches have proven valuable for analyzing the structure and function of Chlamydia HSP70:

Sequence Analysis Tools:

• BLAST analysis can identify sequence homology with HSP70 proteins from other organisms, highlighting conserved domains .

• Multiple sequence alignment tools can identify regions of high conservation across bacterial species, suggesting functional importance.

Structural Prediction Methods:

• Predictive protein modeling has been successfully employed to identify potential epitopes and functional domains .

• Reference to established three-dimensional structures, such as the 70-kDa bovine heat shock cognate (Hsc70) ATPase domain, provides structural templates .

Epitope Prediction:

• Computational tools can identify linear epitopes within the HSP70 sequence, which is particularly relevant for the immunogenic 462-503 amino acid region .

• Surface accessibility prediction algorithms can determine which regions are likely exposed in the native protein conformation.

These bioinformatic approaches should be validated experimentally to confirm predicted functional properties and domain structures.

How do specific mutations in Chlamydia HSP70 affect its function during the infection process?

While the provided search results don't directly address specific mutations in Chlamydia HSP70, research into protein domains and surface accessibility allows us to make informed predictions about the effects of mutations:

Critical Regions for Investigation:

• Mutations in the substrate-binding domain (which includes the 462-503 a.a. region) would likely impact interactions with host cell components during infection .

• Cysteine residues involved in disulfide bonding would be particularly interesting targets, as their mutation would affect the redox-dependent conformational changes observed in the protein .

• Residues identified through homology with known functional sites in HSP70 proteins from other organisms should be prioritized for mutagenesis studies.

Experimental Approaches for Mutation Analysis:

• Site-directed mutagenesis targeting specific amino acids within the 462-503 region

• Recombinant expression of mutant proteins for functional assays

• Assessment of mutant protein effects on attachment, entry, and productive infection

A systematic mutagenesis approach would provide valuable insights into structure-function relationships in Chlamydia HSP70.

What proteomics approaches are most effective for studying Chlamydia HSP70 interactions with host proteins?

Advanced proteomics approaches can reveal critical insights into HSP70's interactions during infection:

Immunoprecipitation-Based Methods:

• Co-immunoprecipitation using antibodies against specific domains of HSP70 can identify host protein interaction partners .

• The successful immunoprecipitation of recombinant chlamydial HSP70 using peptide antibodies demonstrates the feasibility of this approach .

Cross-Linking Proteomics:

• Chemical cross-linking followed by mass spectrometry can capture transient interactions between HSP70 and host proteins during the infection process.

• This approach is particularly valuable given the dynamic nature of HSP70 accessibility during infection.

Protein-Protein Interaction Screening:

• Yeast two-hybrid or bacterial two-hybrid systems using the substrate-binding domain (462-503 a.a.) as bait could identify specific host cell interaction partners.

• Validation of identified interactions could be performed using purified recombinant proteins in vitro.

Accessibility-Dependent Proteomics:

• Comparing protein interactions under reducing versus non-reducing conditions would identify redox-dependent interaction partners, particularly relevant given the observed DTT-dependent accessibility of the substrate-binding domain .

These proteomics approaches, combined with structural and functional analyses, would provide a comprehensive understanding of Chlamydia HSP70's role in the infection process.

How can Chlamydia HSP70 (462-503 a.a.) be utilized in developing improved diagnostic tools for Chlamydia trachomatis infections?

The recombinant Chlamydia HSP70 (462-503 a.a.) presents significant potential for diagnostic applications:

ELISA-Based Diagnostics:

• The protein is specifically suitable for ELISA applications and demonstrates immunoreactivity with sera from Chlamydia Trachomatis infected individuals .

• Optimizing working titers for specific diagnostic applications could improve sensitivity and specificity .

Multiplexed Detection Systems:

• Combining detection of antibodies against HSP70 (462-503 a.a.) with other Chlamydia antigens could improve diagnostic accuracy.

• This approach would be particularly valuable given that approximately 1.5 million cases of chlamydia are reported each year, with many more likely undiagnosed due to asymptomatic nature .

Point-of-Care Testing:

• The high immunogenicity of this specific region makes it a promising candidate for rapid point-of-care diagnostic tools that could address the high prevalence of undiagnosed infections .

The development of such diagnostic tools would be particularly valuable given that 50-70% of chlamydia infections are asymptomatic, leading to missed diagnoses and continued transmission .

What is the potential for targeting Chlamydia HSP70 in therapeutic interventions?

Based on the structural and functional insights from the research, Chlamydia HSP70 presents several potential therapeutic targets:

Attachment and Entry Inhibition:

• The role of HSP70 in EB attachment or entry processes suggests that compounds blocking the substrate-binding domain could potentially inhibit infection .

• Understanding the redox-dependent accessibility of HSP70 domains provides a potential mechanism for targeted intervention during specific infection stages .

Therapeutic Antibodies:

• Antibodies specifically targeting the accessible domains of HSP70 during infection could potentially neutralize the protein's function in pathogenesis.

• The peptide antibodies described in the research provide a foundation for developing such therapeutic approaches .

Small Molecule Modulators:

• Compounds that stabilize the outer membrane structure and prevent the redox-dependent conformational changes might inhibit infection progression.

• Conversely, compounds that trigger premature exposure of HSP70 domains could potentially render the bacteria vulnerable to immune clearance before productive infection.

These potential therapeutic approaches require further investigation but represent promising avenues for intervention in Chlamydia trachomatis infections.

Reference Table: Chlamydia HSP70 Peptide Antibodies and Their Properties

The following table summarizes key information about antibodies targeting different domains of Chlamydia HSP70: