Recombinant Chlamydophila abortus 30S ribosomal protein S15 (rpsO)

What is Chlamydophila abortus and what is the significance of studying its ribosomal proteins?

Chlamydophila abortus (formerly known as Chlamydia psittaci abortion subtype) is an obligate intracellular bacterial pathogen with significant veterinary and zoonotic importance. It is the primary cause of ovine enzootic abortion (OEA) in sheep and goats in the United Kingdom and Northern Europe, and can also infect pregnant women, resulting in acute illness and miscarriage .
The study of ribosomal proteins like S15 (rpsO) is significant because ribosomes are essential for protein synthesis, and differences between bacterial and eukaryotic ribosomes can be exploited for therapeutic purposes. The Chlamydophila abortus genome contains 961 predicted coding sequences within its 1,144,377-bp circular chromosome, and possesses single copies of the 23S, 16S, and 5S rRNA genes, in contrast to Chlamydia species which have two copies . Understanding the structure and function of ribosomal proteins can provide insights into the pathogen's biology, evolution, and potential drug targets.

What expression systems are most effective for producing recombinant Chlamydial ribosomal proteins?

For the expression of recombinant Chlamydial proteins, multiple expression systems have been utilized successfully. Based on information about related Chlamydial proteins, the following expression systems can be considered for rpsO production:

How can recombinant Chlamydophila abortus rpsO be utilized in vaccine development research?

Recombinant Chlamydophila abortus rpsO could potentially be utilized in vaccine development through several strategic approaches:

• As a vaccine antigen: While there is no direct evidence in the search results that rpsO has been used as a vaccine antigen for Chlamydophila abortus, other recombinant proteins from this pathogen have shown promise. For example, research has demonstrated that a Vibrio cholerae ghost (VCG)-based subunit vaccine harboring the N-terminal portion of the Chlamydia abortus Pmp18D protein (rVCG-Pmp18.3) effectively prevented Chlamydia abortus-induced neonatal mortality in a pregnant mouse model .

• As a carrier protein: Ribosomal proteins can potentially serve as carrier proteins for antigenic peptides or as part of multivalent vaccine constructs.

• For serological assays: Recombinant proteins can be used to develop serological assays to monitor vaccine efficacy, similar to how recombinant protein fragments of polymorphic outer membrane proteins have been used in enzyme-linked immunosorbent assays for the diagnosis of ovine enzootic abortion.
  When developing vaccines against Chlamydophila abortus, it's important to note that the pathogen lacks toxin genes and certain metabolic pathways (such as tryptophan metabolism and nucleotide salvaging), which suggests that its niche adaptation is distinct from other chlamydial species . These unique characteristics should inform vaccine design strategies.

What is the potential role of rpsO in Chlamydophila abortus pathogenesis and host adaptation?

While the search results don't provide specific information about the role of rpsO in Chlamydophila abortus pathogenesis, we can make some informed inferences based on general bacterial pathogenesis principles and the information provided about Chlamydophila abortus.
Ribosomal proteins primarily function in protein synthesis, but in some bacteria, they can have secondary roles in pathogenesis. The potential roles of rpsO in Chlamydophila abortus pathogenesis could include:

• Essential role in bacterial survival and replication: As part of the ribosome, rpsO is essential for protein synthesis, which is necessary for bacterial growth and virulence factor production.

• Potential moonlighting functions: Some ribosomal proteins have been shown to have secondary "moonlighting" functions outside of the ribosome, potentially interacting with host components.

• Adaptation to host environment: Chlamydophila abortus has shown specific adaptations to its niche, including the absence of genes involved in tryptophan metabolism and nucleotide salvaging (guaB is present as a pseudogene) . The precise configuration of ribosomal proteins might contribute to these adaptations by optimizing protein synthesis under the specific conditions of its ecological niche.

• Contribution to host-pathogen interactions: Chlamydophila abortus possesses several gene families encoding surface proteins, such as polymorphic membrane proteins (Pmps) and TMH/Inc proteins, which are important for host-pathogen interactions . Efficient synthesis of these proteins, mediated by ribosomal proteins including rpsO, would be crucial for successful infection.

How can structural studies of recombinant Chlamydophila abortus rpsO inform drug development?

Structural studies of recombinant Chlamydophila abortus rpsO can provide valuable insights for drug development through the following methodological approaches:

• Identification of unique structural features: Determining the three-dimensional structure of Chlamydophila abortus rpsO using X-ray crystallography or cryo-electron microscopy can reveal structural elements that differ from the host (human or animal) counterparts. These differences can be exploited for selective targeting by antimicrobial compounds.

• Structure-based drug design: Once the structure is determined, computational methods can be used to identify potential binding pockets and design small molecules that specifically interact with these sites to disrupt ribosome assembly or function.

• Comparative structural analysis: Comparing the structure of Chlamydophila abortus rpsO with those from other bacteria can help identify conserved features across bacterial species, potentially leading to broad-spectrum antibiotics, or unique features that would allow species-specific targeting.

• Functional mapping through site-directed mutagenesis: By creating variants of recombinant rpsO with specific amino acid substitutions and analyzing their effects on structure and function, researchers can identify critical residues that might serve as optimal drug targets.

• Interaction studies with known antibiotics: Structural studies can reveal how existing ribosome-targeting antibiotics interact with Chlamydophila abortus ribosomes, providing insights for the development of more effective derivatives.

What are the optimal conditions for expression and purification of functional recombinant Chlamydophila abortus rpsO?

• Temperature: Lower temperatures (15-25°C) often favor proper folding of recombinant proteins.

• Induction conditions: For IPTG-inducible systems, optimize IPTG concentration (typically 0.1-1.0 mM) and induction time (4-16 hours).

• Media composition: Enriched media (like LB or 2xYT) typically provide better yields.
  Purification Strategy:

• Affinity tags: A His-tag is commonly used for initial purification, allowing for immobilized metal affinity chromatography (IMAC).

• Solubility considerations: Include appropriate buffers and additives to maintain protein solubility (typically 20-50 mM Tris-HCl, pH 7.5-8.0, 100-300 mM NaCl).

• Sequential purification: Follow IMAC with size exclusion chromatography to achieve higher purity.

• Tag removal: If the affinity tag might interfere with functional studies, include a protease cleavage site for tag removal after initial purification.
  Quality Control:

• SDS-PAGE and Western blotting: To confirm protein identity and purity.

• Mass spectrometry: For accurate molecular weight determination and confirmation of primary structure.

• Circular dichroism: To assess secondary structure and proper folding.

• Functional assays: Such as RNA binding assays, to confirm that the recombinant protein retains its biological activity.

What methods can be used to study the interaction between Chlamydophila abortus rpsO and ribosomal RNA?

To study the interaction between Chlamydophila abortus rpsO and ribosomal RNA, researchers can employ several methodological approaches:

• Electrophoretic Mobility Shift Assay (EMSA):

  • This technique allows visualization of protein-RNA complexes based on their reduced mobility in non-denaturing gels.

  • Purified recombinant rpsO is incubated with labeled ribosomal RNA fragments at varying protein concentrations.

  • The formation of protein-RNA complexes is detected as a mobility shift of the RNA band.

• Surface Plasmon Resonance (SPR):

  • Provides real-time measurement of binding kinetics without labels.

  • Either the protein or RNA is immobilized on a sensor chip, and the binding partner is flowed over the surface.

  • Allows determination of association and dissociation rate constants and equilibrium binding constants.

• Isothermal Titration Calorimetry (ITC):

  • Measures the heat released or absorbed during binding.

  • Provides thermodynamic parameters (ΔH, ΔS, ΔG) in addition to binding constants.

  • Requires no labeling or immobilization.

• RNA Footprinting:

  • Identifies specific RNA regions protected from chemical or enzymatic cleavage when bound to the protein.

  • Methods include chemical modification (DMS, CMCT), hydroxyl radical footprinting, or nuclease protection assays.

• Cross-linking studies:

  • UV or chemical cross-linking followed by mass spectrometry can identify specific contact points between the protein and RNA.

• Structural studies:

  • X-ray crystallography or cryo-electron microscopy of the rpsO-RNA complex provides atomic-level details of the interaction interface.

  • NMR spectroscopy can be used for smaller complexes to study the interaction dynamics.
    These methods can provide complementary information about the specificity, affinity, and structural basis of the interaction between Chlamydophila abortus rpsO and ribosomal RNA.

How can recombinant Chlamydophila abortus rpsO be used to develop diagnostic assays for ovine enzootic abortion?

Recombinant Chlamydophila abortus rpsO could potentially be used to develop diagnostic assays for ovine enzootic abortion through the following methodological approaches:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Recombinant rpsO can be used as an antigen to detect anti-rpsO antibodies in sheep sera.

  • This approach is similar to existing diagnostic ELISAs that use recombinant protein fragments of polymorphic outer membrane proteins (POMPs) for the diagnosis of ovine enzootic abortion.

  • The methodology would involve coating ELISA plates with purified recombinant rpsO, blocking non-specific binding sites, incubating with sheep sera, and detecting bound antibodies using labeled secondary antibodies.

• Lateral Flow Assays:

  • Rapid point-of-care tests can be developed using recombinant rpsO as the capture antigen.

  • These tests would be particularly valuable for field diagnostics.

• Multiplex Serological Assays:

  • Recombinant rpsO could be included in multiplex assays alongside other Chlamydophila abortus antigens to improve diagnostic sensitivity and specificity.

  • Techniques like Luminex bead-based assays allow simultaneous detection of antibodies against multiple antigens.

• PCR-Based Detection:

  • While not directly using the recombinant protein, knowledge gained from studying rpsO could inform the design of nucleic acid-based diagnostic tests targeting the rpsO gene or its transcripts.
    The development of such assays would require validation using samples from infected and uninfected animals to establish sensitivity, specificity, and appropriate cut-off values for distinguishing positive from negative results. It would also be important to evaluate potential cross-reactivity with related Chlamydial species.

How can I analyze contradictory results in studies of recombinant Chlamydophila abortus rpsO function?

When faced with contradictory results in studies of recombinant Chlamydophila abortus rpsO function, researchers should follow a systematic approach to identify potential sources of discrepancy and resolve the contradictions:

What bioinformatic approaches are useful for predicting potential functional domains in Chlamydophila abortus rpsO?

Several bioinformatic approaches can be employed to predict potential functional domains in Chlamydophila abortus rpsO:

What quality control methods should be used to ensure the functionality of purified recombinant Chlamydophila abortus rpsO?

To ensure the functionality of purified recombinant Chlamydophila abortus rpsO, researchers should implement a comprehensive quality control workflow including:

• Physical characterization:

  • SDS-PAGE: To assess purity and confirm the expected molecular weight

  • Western blotting: To verify protein identity using specific antibodies

  • Mass spectrometry: For accurate molecular weight determination and identification of potential post-translational modifications or degradation products

  • Dynamic light scattering (DLS): To check for aggregation and determine the hydrodynamic radius

  • Analytical size exclusion chromatography: To assess oligomeric state and homogeneity

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy: To confirm proper secondary structure formation

  • Fluorescence spectroscopy: To assess tertiary structure integrity by monitoring intrinsic tryptophan fluorescence

  • Thermal shift assays: To evaluate protein stability and proper folding

  • Limited proteolysis: To verify the compact folding of the protein

• Functional assays:

  • RNA binding assays: Since rpsO is a ribosomal protein, its ability to bind specific RNA sequences should be verified

    • Electrophoretic mobility shift assays (EMSA)

    • Filter binding assays

    • Surface plasmon resonance (SPR)

  • Ribosome assembly assays: To confirm the ability of rpsO to incorporate into ribosomal subunits

  • In vitro translation assays: To assess the functionality of ribosomes containing the recombinant rpsO

• Storage stability tests:

  • Accelerated stability studies: To determine optimal storage conditions

  • Freeze-thaw stability: To assess whether the protein maintains functionality after multiple freeze-thaw cycles

  • Long-term storage tests: To establish shelf-life under different conditions

• Batch consistency analysis:

  • Lot-to-lot comparison: To ensure reproducibility in protein production

  • Certificate of analysis: Documentation of key quality parameters for each batch
    By implementing these quality control methods, researchers can ensure that their purified recombinant Chlamydophila abortus rpsO maintains its native functionality and is suitable for downstream applications in research or diagnostic development.

How might comparative analysis of rpsO across Chlamydial species contribute to understanding evolutionary relationships within Chlamydiaceae?

Comparative analysis of rpsO across Chlamydial species can significantly contribute to understanding evolutionary relationships within Chlamydiaceae through several methodological approaches:

What are the potential applications of recombinant Chlamydophila abortus rpsO in developing novel antimicrobial strategies?

Recombinant Chlamydophila abortus rpsO offers several promising avenues for developing novel antimicrobial strategies:

• Target-based drug screening:

  • Purified recombinant rpsO can be used in high-throughput screening assays to identify small molecules that specifically bind to or interfere with its function.

  • Such compounds could potentially disrupt ribosome assembly or function, inhibiting bacterial protein synthesis.

  • The advantage of targeting ribosomal proteins is the essential nature of protein synthesis for bacterial survival.

• Structure-based drug design:

  • The three-dimensional structure of rpsO (determined using the recombinant protein) can guide rational design of inhibitors that specifically target this protein.

  • Focusing on structural differences between bacterial and host ribosomal proteins can lead to selective antibacterial agents with minimal host toxicity.

• Peptide inhibitors:

  • Identifying peptides that mimic interaction interfaces of rpsO with other ribosomal components could lead to the development of peptide-based inhibitors.

  • These peptides could potentially disrupt ribosome assembly or stability.

• Vaccine development:

  • While ribosomal proteins are typically not primary vaccine candidates due to their intracellular location, recombinant rpsO could potentially be included in multi-component subunit vaccines.

  • This approach would be similar to the use of other Chlamydial proteins in vaccine development, such as the N-terminal portion of the Pmp18D protein used in a VCG-based vaccine against Chlamydia abortus .

• Antisense strategies:

  • Knowledge gained from studying rpsO could inform the design of antisense oligonucleotides targeting the rpsO mRNA.

  • Such oligonucleotides could potentially inhibit rpsO expression and thereby disrupt ribosome assembly.

• CRISPR-Cas antimicrobials:

  • The rpsO gene could serve as a target for CRISPR-Cas-based antimicrobials, which are emerging as a novel class of highly specific antibacterial agents.
    These approaches take advantage of the essential nature of ribosomal proteins for bacterial survival and could potentially lead to new treatments for Chlamydophila abortus infections, addressing the limitations of current antibiotic therapies. The development of such targeted antimicrobials is particularly important given the intracellular lifestyle of Chlamydophila abortus and its significant impact on animal and human health .

How might recombinant Chlamydophila abortus rpsO contribute to understanding host-pathogen interactions during placental infection?

Recombinant Chlamydophila abortus rpsO could contribute to understanding host-pathogen interactions during placental infection through several methodological approaches:

• Immune response profiling:

  • Recombinant rpsO can be used to stimulate immune cells isolated from placental tissues to study cytokine and chemokine responses.

  • This approach can reveal how the host immune system recognizes and responds to Chlamydial components during infection.

  • Comparing responses in different host species (e.g., sheep, humans) could provide insights into species-specific aspects of immunity.

• Translocation studies:

  • Investigating whether rpsO, despite being primarily a ribosomal protein, might be exposed to the host during infection.

  • Some bacterial ribosomal proteins have been found to have "moonlighting" functions outside the ribosome, potentially interacting with host components.

  • Fluorescently labeled recombinant rpsO could be used to track potential translocation in cell culture models.

• Placental barrier models:

  • Using in vitro placental barrier models to study how Chlamydophila abortus components, including potentially rpsO, might affect placental integrity.

  • This could provide insights into the mechanisms by which the pathogen crosses the placental barrier to cause abortion.

• Comparative proteomics:

  • Using recombinant rpsO in pull-down assays to identify potential host interaction partners in placental tissues.

  • Mass spectrometry analysis of the pulled-down complexes could reveal unexpected interactions.

• Antibody response analysis:

  • Investigating whether natural infection induces antibodies against rpsO.

  • If antibodies are detected, studying their potential protective effects could inform vaccine development strategies.

• Transcriptomic studies:

  • Analyzing how exposure to recombinant rpsO affects gene expression in placental cells.

  • This could reveal host response pathways activated during infection.
    These approaches could contribute to our understanding of how Chlamydophila abortus causes placental infection and subsequent abortion, which is particularly important given that this pathogen can cause zoonotic infections resulting in miscarriage in pregnant women . Furthermore, understanding these mechanisms could inform the development of more effective preventive and therapeutic strategies for both veterinary and human medicine.