Recombinant Chlamydophila caviae Sulfur-rich protein (srp)

What is the Sulfur-rich protein (srp) from Chlamydophila caviae?

Sulfur-rich protein (srp) is a 160 amino acid protein found in Chlamydophila caviae, a Gram-negative obligate intracellular bacterium. The full amino acid sequence is MLTGVENSESGVIDLIKPGLDDVMKNETVQVTLVNSVLGWCKAHIVDPIKTSKIVQSRAFQITMVVLGVILLIAGLALTFVLQGQLGKNAFLFLIPAVIGLVKLLTTSVFMEKPCTPEKWRLCKRLLATTEDILDDGQINQSNTIFTTESSDVTNTATQS . This protein belongs to a conserved family of proteins found across Chlamydia species, with homologs present in other chlamydial organisms such as C. trachomatis . The protein contains numerous cysteine residues, contributing to its "sulfur-rich" designation.

What are the structural characteristics of C. caviae srp protein?

The C. caviae srp protein is characterized by its high sulfur content due to numerous cysteine residues. Structurally, it is a full-length protein (160 amino acids) that can be expressed with various tags for research purposes, commonly with an N-terminal His-tag . Based on its amino acid sequence, the protein likely contains both hydrophilic and hydrophobic regions, suggesting potential membrane association. The sequence indicates transmembrane domains with the segment "VVLGVILLIAGLALTFVLQ" showing characteristics of a membrane-spanning region . This structural feature is consistent with its potential role in the developmental cycle of Chlamydia species.

How does C. caviae srp compare to srp proteins in other Chlamydia species?

When comparing C. caviae srp (160 amino acids) with C. trachomatis serovar A srp (152 amino acids), both share similar structural features and functional domains . Sequence alignment shows conservation in key regions, especially in transmembrane domains and cysteine-rich areas. C. trachomatis srp is also known as cysteine-rich protein A (crpA) , highlighting the importance of sulfur-containing residues in this protein family. Both proteins can be recombinantly expressed with His-tags in E. coli expression systems . The high conservation of these proteins across Chlamydia species suggests they may serve similar functions in the chlamydial developmental cycle, potentially related to elementary body (EB) to reticulate body (RB) transitions.

What expression systems are optimal for recombinant C. caviae srp production?

The optimal expression system for recombinant C. caviae srp production appears to be E. coli, as demonstrated in multiple studies and commercial preparations . This bacterial expression system offers several advantages for srp protein expression:

• High yield of recombinant protein

• Well-established protocols for induction and harvest

• Compatibility with N-terminal His-tagging for purification

• Cost-effectiveness and reproducibility

What are the recommended methods for purification of recombinant C. caviae srp?

For purification of recombinant C. caviae srp, affinity chromatography using the His-tag is the recommended primary method . A step-by-step purification protocol should include:

• Expression in E. coli and cell lysis under conditions that maintain protein solubility

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resin to capture the His-tagged protein

• Washing with increasing imidazole concentrations to remove non-specifically bound proteins

• Elution with high imidazole buffer

• Buffer exchange to remove imidazole and optimize storage conditions

• Quality control via SDS-PAGE to confirm purity greater than 90%

For researchers requiring higher purity, additional purification steps such as size exclusion chromatography or ion exchange chromatography may be implemented. The final purified protein is typically stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . To maintain protein stability, addition of 5-50% glycerol and storage at -20°C/-80°C with minimal freeze-thaw cycles is recommended .

What are the critical factors for maintaining stability of purified srp protein?

Maintaining stability of purified srp protein requires careful attention to several critical factors:

• Storage buffer composition: Optimal stability is achieved in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

• Cryoprotectants: Addition of glycerol (5-50%, with 50% being commonly used) is crucial for long-term storage stability .

• Temperature conditions: Store at -20°C/-80°C for long-term storage, with working aliquots maintained at 4°C for up to one week only .

• Aliquoting strategy: Dividing the purified protein into single-use aliquots is essential as repeated freeze-thaw cycles significantly reduce protein stability and activity .

• Reconstitution protocol: When using lyophilized protein, brief centrifugation before opening is recommended, followed by reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

The presence of multiple cysteine residues in srp means that reducing conditions may also be important for maintaining proper protein folding and preventing disulfide-mediated aggregation during storage and handling.

What is the role of srp in the developmental cycle of Chlamydia species?

The srp protein appears to play a significant role in the unique biphasic developmental cycle of Chlamydia species, which transitions between the elementary body (EB) and reticulate body (RB) forms . Based on phosphoproteomic analysis, the function of srp may be regulated through post-translational modifications during this developmental cycle. Research has shown that Chlamydia species possess a complex phosphoprotein network with 41 of 42 identified C. caviae phosphoproteins being conserved across Chlamydia species .

The developmental regulation appears to involve different phosphorylation patterns between EBs and RBs. EBs contained threefold more phosphorylated proteins than RBs (34 versus 11), which correlates with physiological differences between these developmental forms . This differential phosphorylation pattern may serve as a mechanism for rapid activation of proteins upon infection without requiring immediate protein synthesis, as proteins could be pre-packaged in the EB form and activated through dephosphorylation upon infection .

While the specific function of srp has not been fully characterized, its conservation across Chlamydia species and its potential regulation through phosphorylation suggest it may be involved in the structural or functional transitions during the developmental cycle.

How does phosphorylation affect the function of srp in different developmental stages?

Phosphoproteomic analysis of C. caviae has revealed distinct phosphorylation patterns between the elementary body (EB) and reticulate body (RB) forms . While the specific phosphorylation status of srp was not individually detailed in the search results, the study identified 42 non-redundant phosphorylated proteins across both developmental stages, with only three phosphoproteins found in both EB and RB phosphoproteomes .

The developmental stage-specific phosphorylation patterns suggest two possible mechanisms for how phosphorylation might affect srp function:

• Inhibitory phosphorylation in EBs: Phosphorylation may inhibit protein activity in the EB stage, allowing for rapid activation upon infection through dephosphorylation, without requiring new protein synthesis .

• Functional modulation: Phosphorylation might modify protein function to meet specific needs in different environments—the extracellular environment for EBs or the intracellular environment for RBs .

The table below summarizes the phosphoproteome differences between developmental stages:

This stage-specific phosphorylation suggests that protein phosphorylation may be a crucial mechanism of developmental regulation in Chlamydia species .

What are the potential interactions of srp with host cell proteins during infection?

While the search results don't specifically address srp interactions with host proteins, we can infer potential interaction mechanisms based on the protein's characteristics and chlamydial infection biology:

• Membrane localization: The amino acid sequence of srp contains hydrophobic regions consistent with transmembrane domains , suggesting it may localize to the bacterial membrane where it could interact with host cell components.

• Developmental regulation: The phosphoproteomic analysis indicates that protein phosphorylation patterns differ significantly between the infectious EB form and the intracellular RB form . This suggests that proteins like srp may undergo functional changes during the infection process that could affect host-pathogen interactions.

• Virulence-related function: Several proteins involved in virulence, including type III secretion system (T3SS) components, were found to be phosphorylated in the EB stage . If srp is involved in virulence pathways, it may participate in modulating host cell responses.

Research on related chlamydial proteins suggests that interactions with host proteins often involve manipulating host signaling pathways, cytoskeletal arrangements, or cellular trafficking to establish the intracellular niche required for chlamydial replication. Further studies using techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity labeling would be needed to identify specific host protein interactions with srp.

How can recombinant srp be utilized in vaccine development against chlamydial infections?

Recombinant srp protein could serve as a potential vaccine candidate against chlamydial infections based on several characteristics:

• Conservation across species: The high conservation of srp across Chlamydia species suggests that immune responses against this protein might provide cross-species protection.

• Developmental importance: If srp plays a role in the developmental cycle transitions that are essential for chlamydial infectivity, antibodies or cellular immune responses targeting this protein might disrupt the infection process .

• Potential surface exposure: Based on its amino acid sequence containing transmembrane domains , portions of srp might be exposed on the bacterial surface, making them accessible to antibodies.

A methodological approach for exploring srp as a vaccine candidate would include:

• Immunogenicity testing using recombinant srp in animal models

• Evaluation of both humoral and cell-mediated immune responses

• Challenge studies to assess protection against live chlamydial infection

• Comparative studies with other chlamydial antigens to determine relative efficacy

• Investigation of adjuvant formulations to enhance immunogenicity

Additionally, epitope mapping of srp could identify specific regions that elicit protective immune responses, potentially leading to peptide-based vaccine approaches rather than using the full-length protein.

What structural biology techniques are most suitable for determining the three-dimensional structure of srp?

Determining the three-dimensional structure of srp presents specific challenges due to its membrane-associated domains . A multi-technique approach would be most effective:

• X-ray crystallography: For soluble domains of srp, after removing transmembrane regions. This would require:

  • Expression of truncated constructs focusing on soluble domains

  • High-purity protein preparation (>95%)

  • Crystallization screening with various precipitants and conditions

  • X-ray diffraction data collection and structure solution

• Nuclear Magnetic Resonance (NMR) spectroscopy: Particularly useful for smaller domains or peptides derived from srp:

  • Isotopic labeling with 15N and 13C

  • Solution NMR for structure determination of soluble domains

  • Solid-state NMR for membrane-associated regions

• Cryo-Electron Microscopy: For full-length protein, especially if it forms oligomeric complexes:

  • Sample preparation in detergent micelles or nanodiscs

  • Single-particle analysis for structure determination

  • Potential for visualizing the protein in different conformational states

• Computational modeling: To integrate experimental data and predict full structure:

  • Homology modeling based on related proteins

  • Molecular dynamics simulations to understand dynamic properties

  • Integration of limited experimental constraints from other methods

Each method provides complementary information, and combining multiple approaches would yield the most comprehensive structural characterization of srp.

How can CRISPR-Cas9 gene editing be applied to study srp function in Chlamydia species?

Applying CRISPR-Cas9 gene editing to study srp function in Chlamydia species presents unique challenges due to their obligate intracellular lifestyle but offers powerful opportunities for functional characterization. A methodological approach would include:

• Design of transformation system:

  • Development of a shuttle vector containing CRISPR-Cas9 components that can function in Chlamydia

  • Selection of appropriate promoters for expression in the chlamydial background

  • Incorporation of selection markers compatible with intracellular growth

• Guide RNA design:

  • Targeting specific regions of the srp gene

  • Consideration of PAM site availability in the AT-rich chlamydial genome

  • Testing multiple guide RNAs to identify efficient targeting

• Delivery methods:

  • Calcium chloride transformation during the RB stage when Chlamydia are more amenable to DNA uptake

  • Electroporation protocols optimized for chlamydial transformation

  • Potential use of cell-penetrating peptides to facilitate nucleic acid delivery

• Phenotypic analysis of mutants:

  • Evaluation of developmental cycle progression in srp mutants

  • Investigation of infectivity and intracellular growth

  • Transcriptomic and proteomic profiling to identify affected pathways

  • Protein interaction studies to identify partners of srp

• Complementation studies:

  • Re-introduction of wild-type srp to confirm phenotypes

  • Introduction of srp variants with modified phosphorylation sites to study the role of post-translational modifications

This approach would build upon recent advances in the genetic manipulation of previously genetically intractable Chlamydia species and could provide definitive insights into the function of srp in chlamydial biology.

How can researchers overcome solubility issues when working with recombinant srp protein?

Solubility challenges are common when working with proteins containing transmembrane domains like srp . A systematic approach to overcome these issues includes:

• Expression optimization:

  • Testing multiple expression temperatures (16°C, 25°C, 30°C, 37°C)

  • Varying induction conditions (IPTG concentration, induction time)

  • Using specialized E. coli strains designed for membrane protein expression (C41/C43)

  • Evaluating co-expression with chaperones to assist folding

• Fusion tags and constructs:

  • While His-tags are commonly used , alternative solubility-enhancing tags could be tested:

    • MBP (maltose-binding protein) fusion

    • SUMO tag

    • Thioredoxin fusion

  • Creating truncated constructs that exclude transmembrane domains for studies requiring only soluble domains

• Solubilization strategies:

  • Screening detergent panels (non-ionic, zwitterionic, and mild ionic detergents)

  • Testing detergent concentrations above critical micelle concentration

  • Evaluating mixed micelle systems with lipids

  • Using newer solubilization agents like nanodiscs, amphipols, or styrene maleic acid lipid particles (SMALPs)

• Buffer optimization:

  • Testing pH ranges (typically 7.0-8.5)

  • Evaluating ionic strength variations

  • Adding stabilizing agents (glycerol, arginine, trehalose)

  • Including reducing agents to prevent disulfide-mediated aggregation

• Refolding protocols:

  • If inclusion body formation occurs, developing refolding protocols from denatured protein

  • Utilizing dialysis with decreasing denaturant concentration

  • On-column refolding during purification

Each protein preparation should be validated for proper folding using techniques such as circular dichroism or limited proteolysis to ensure that solubilized srp maintains its native structure.

What are the best approaches for detecting post-translational modifications of srp?

Detecting post-translational modifications (PTMs) of srp, particularly phosphorylation which appears important in chlamydial biology , requires specialized techniques:

• Mass Spectrometry-Based Approaches:

  • Phosphopeptide enrichment using:

    • Titanium dioxide (TiO2) chromatography

    • Immobilized metal affinity chromatography (IMAC)

    • Phosphospecific antibody immunoprecipitation

  • LC-MS/MS analysis with collision-induced dissociation (CID) or electron transfer dissociation (ETD)

  • Quantitative phosphoproteomics using stable isotope labeling (SILAC, TMT, or iTRAQ)

• Gel-Based Detection:

  • Pro-Q Diamond phosphoprotein staining as used in the C. caviae phosphoproteome study

  • Phos-tag SDS-PAGE for mobility shift detection of phosphorylated proteins

  • 2D gel electrophoresis to separate protein variants with different PTMs

• Site-Specific Analysis:

  • Site-directed mutagenesis of potential modification sites followed by functional studies

  • Phosphospecific antibodies if common phosphorylation sites are identified

  • In vitro kinase assays to identify responsible kinases

• Phosphorylation Dynamics:

  • Time-course studies during development cycle transitions

  • Comparison between EB and RB forms as demonstrated in previous research

  • Inhibitor studies using kinase or phosphatase inhibitors

The 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis used in the C. caviae phosphoproteome study provides a validated methodological framework that could be adapted specifically for srp analysis.

How can researchers design effective experiments to study the kinetics of srp phosphorylation during the chlamydial developmental cycle?

Designing experiments to study srp phosphorylation kinetics during the chlamydial developmental cycle requires careful planning and integration of multiple techniques:

• Synchronized infection system:

  • Establish a protocol for synchronizing chlamydial infection in host cells

  • Define clear timepoints spanning the developmental cycle (EB to RB transition and back)

  • Include controls for uninfected cells and heat-inactivated Chlamydia

• Sample collection strategy:

  • Harvest samples at defined intervals (e.g., 0, 2, 4, 8, 12, 24, 36, 48 hours post-infection)

  • Separate EB and RB forms using density gradient centrifugation at each timepoint

  • Prepare samples with phosphatase inhibitors to preserve in vivo phosphorylation state

• Analytical techniques:

  • Quantitative phosphoproteomics using:

    • SILAC or TMT labeling for comparing timepoints

    • Parallel reaction monitoring (PRM) for targeted analysis of srp phosphopeptides

  • Western blotting with phospho-specific antibodies (if available)

  • Phos-tag gel electrophoresis to visualize phosphorylation-dependent mobility shifts

• Data integration:

  • Correlate srp phosphorylation status with developmental stage markers

  • Analyze phosphorylation site occupancy changes over time

  • Map kinetics data to structural models to infer functional implications

• Validation experiments:

  • In vitro dephosphorylation assays to confirm phosphorylation sites

  • Site-directed mutagenesis of identified phosphorylation sites to assess functional impact

  • Treatment with kinase inhibitors at specific timepoints to disrupt normal phosphorylation patterns

This comprehensive approach would build upon the methodology used in previous phosphoproteomic studies of C. caviae , extending it to capture the dynamic nature of phosphorylation throughout the developmental cycle rather than providing static snapshots of the EB and RB forms.

How might single-molecule techniques advance our understanding of srp function?

Single-molecule techniques offer powerful approaches to investigate srp function at unprecedented resolution, potentially revealing mechanistic insights not accessible through bulk measurements:

• Single-Molecule FRET (smFRET):

  • Engineering fluorescently labeled srp proteins with donor-acceptor pairs

  • Monitoring conformational changes in response to developmental cues

  • Detecting interactions with binding partners at the single-molecule level

  • Revealing potential structural heterogeneity and intermediate states

• Single-Molecule Force Spectroscopy:

  • Using atomic force microscopy (AFM) to probe mechanical properties of srp

  • Investigating how phosphorylation affects protein stability and unfolding pathways

  • Measuring interaction forces between srp and binding partners

• Single-Particle Tracking:

  • Visualization of fluorescently tagged srp in live Chlamydia during infection

  • Tracking protein localization changes during developmental transitions

  • Quantifying diffusion coefficients and confined motion patterns

• Nanopore Analysis:

  • Investigation of srp translocation through membranes

  • Detection of conformational states based on current blockade patterns

  • Analysis of interactions with other proteins or ligands

• Implementation challenges and solutions:

  • Adapting techniques for the small size of Chlamydia (0.3-1μm)

  • Developing labeling strategies compatible with the intracellular environment

  • Creating microfluidic platforms to facilitate single-bacterium analysis

These approaches would provide dynamic information about srp function that complements the static snapshots provided by traditional structural and biochemical techniques, potentially revealing how this protein contributes to the unique developmental cycle of Chlamydia species.

What are the prospects for developing srp-targeted antimicrobial therapies?

The development of srp-targeted antimicrobial therapies represents an emerging research direction with several promising aspects:

• Target validation considerations:

  • Determining essentiality of srp for chlamydial survival and infectivity

  • Assessing conservation across Chlamydia species to predict spectrum of activity

  • Evaluating potential for resistance development

• Potential therapeutic approaches:

  • Small molecule inhibitors targeting:

    • Protein-protein interactions essential for srp function

    • Conformational changes required for developmental transitions

    • Post-translational modification sites (e.g., phosphorylation sites)

  • Peptide inhibitors mimicking critical binding interfaces

  • Antibody-based therapeutics if portions of srp are surface-accessible

• Structure-based drug design strategy:

  • Utilizing 3D structural information to identify druggable pockets

  • Virtual screening of compound libraries against identified binding sites

  • Fragment-based approaches to develop high-affinity ligands

  • Molecular dynamics simulations to identify transient binding pockets

• Screening and validation methodology:

  • Development of high-throughput assays for srp function

  • Cell-based infection models to evaluate compound efficacy

  • Animal models of chlamydial infection for in vivo validation

• Delivery considerations:

  • Strategies for intracellular delivery of therapeutic agents

  • Formulations to enhance bioavailability at infection sites

  • Potential for combination therapy with existing antibiotics

This approach is supported by precedent in the field, as evidenced by the development of a C. pneumoniae-specific PknD inhibitor with demonstrated antibacterial activity . The targeting of proteins involved in developmental regulation, like srp, offers the potential for highly specific anti-chlamydial agents with reduced impact on commensal microbiota compared to broad-spectrum antibiotics.

How can systems biology approaches integrate srp function into the broader context of chlamydial developmental regulation?

Systems biology approaches can provide a comprehensive framework for understanding srp function within the complex regulatory networks governing the chlamydial developmental cycle:

• Multi-omics data integration:

  • Combining phosphoproteomics , transcriptomics, and metabolomics data

  • Temporal profiling throughout the developmental cycle

  • Development of computational models to predict regulatory relationships

  • Network analysis to position srp within signaling pathways

• Protein-protein interaction mapping:

  • High-throughput interactome studies using proximity labeling approaches

  • Yeast two-hybrid screening against the chlamydial proteome

  • Co-immunoprecipitation coupled with mass spectrometry

  • Correlation of interaction dynamics with developmental transitions

• Genetic interaction networks:

  • CRISPR interference or antisense RNA approaches for partial depletion

  • Synthetic genetic array analysis to identify genetic interactions

  • Suppressor screens to identify compensatory pathways

  • Construction of genetic dependency maps

• Mathematical modeling approaches:

  • Ordinary differential equation (ODE) models of developmental regulation

  • Bayesian network inference from multi-omics data

  • Agent-based modeling of single-cell developmental dynamics

  • Constraint-based modeling incorporating metabolic and regulatory networks

• Integration with host-pathogen interface data:

  • Dual RNA-seq to capture simultaneous host and pathogen responses

  • Proteomics of the inclusion membrane and associated proteins

  • Metabolic exchange modeling between host and pathogen

  • Signaling pathway reconstruction spanning host-pathogen boundaries

This systems-level understanding would position srp within the broader context of the 41 conserved phosphoproteins identified across Chlamydia species , potentially revealing how these proteins work in concert to orchestrate the complex developmental transitions that are essential for chlamydial pathogenesis.