Recombinant Chlamydophila abortus SsrA-binding protein (smpB)

What is the function of SmpB in Chlamydophila abortus?

SmpB is a unique RNA-binding protein essential for the SsrA (also known as tmRNA) quality-control system in bacteria. In Chlamydophila abortus, as in other bacteria, SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo . The SmpB-SsrA complex recognizes ribosomes stalled on defective mRNAs and facilitates the addition of a peptide tag to the C-terminus of partially synthesized polypeptide chains. This tagging marks the incomplete proteins for degradation by C-terminal-specific proteases, preventing the accumulation of potentially harmful truncated proteins .

How conserved is smpB across bacterial species compared to Chlamydophila abortus?

SmpB is highly conserved throughout the bacterial kingdom, including in Chlamydophila species. Comparative sequence analysis reveals that SmpB proteins share significant sequence homology across diverse bacterial phyla, indicating its fundamental importance in bacterial physiology . In Chlamydophila abortus, the smpB gene is similarly conserved, although it may have specific adaptations related to the obligate intracellular lifestyle of this pathogen. Deletion studies in model organisms like Escherichia coli have demonstrated that smpB-deficient strains exhibit phenotypic defects identical to those of ssrA-defective strains, confirming its essential role in the trans-translation system .

What are the implications of smpB function in Chlamydophila abortus pathogenesis?

The SmpB-SsrA system likely plays a crucial role in the pathogenesis of Chlamydophila abortus, which causes enzootic abortion in sheep and can infect humans . During the developmental cycle of Chlamydophila species, which involves conversion between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) within host cells , the SmpB-SsrA system may be particularly important for managing translational stress. It may contribute to the bacterium's ability to survive under stressful conditions, including the immune response, by ensuring proper protein quality control. In Salmonella typhimurium, disruption of the smpB gene was reported to decrease virulence and reduce bacterial survival within macrophages , suggesting a similar role might exist in C. abortus virulence.

What are the optimal expression systems for producing recombinant Chlamydophila abortus smpB?

The production of recombinant C. abortus smpB presents unique challenges due to the obligate intracellular nature of the organism. Based on successful approaches with other chlamydial proteins, the following expression systems are recommended:

• E. coli-based expression systems: Using pET vectors with T7 promoters can yield high levels of recombinant smpB. Codon optimization for E. coli is crucial due to potential codon usage bias differences between Chlamydophila and E. coli .

• Cell-free expression systems: These can be particularly valuable when the protein might be toxic to host cells.

• Chlamydia-specific vectors: Recent advances in Chlamydia genetics now permit the development of Chlamydia-specific expression systems using transformation protocols that utilize calcium chloride . These systems are particularly valuable when studying protein function in the native organism.
  Adding purification tags (His6 or GST) at either the N- or C-terminus facilitates subsequent purification, though careful consideration should be given to potential interference with SmpB function if functional studies are planned.

What purification strategies yield the highest quality recombinant C. abortus smpB protein?

Based on the characteristics of SmpB as an RNA-binding protein, a multi-step purification protocol is recommended:

• Initial capture: Affinity chromatography using nickel columns for His-tagged smpB or glutathione sepharose for GST-tagged constructs.

• Intermediate purification: Ion exchange chromatography (typically cation exchange as SmpB proteins often have a basic isoelectric point).

• Polishing step: Size exclusion chromatography to remove aggregates and ensure monomeric protein preparation.
  Critical considerations include:

• Maintaining low temperatures (4°C) throughout purification

• Including RNase inhibitors to prevent contaminating RNA from binding to smpB

• Using buffers containing 5-10% glycerol and 1-5 mM DTT or 2-ME to maintain protein stability

• For functional studies, ensuring removal of potential bacterial endotoxins using polymyxin B columns
  Typical yields from optimization studies range from 5-15 mg of purified protein per liter of E. coli culture.

How can researchers verify the structural integrity and activity of recombinant C. abortus smpB?

Multiple complementary approaches should be employed:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal shift assays to determine protein stability

  • Limited proteolysis to verify proper folding

  • Dynamic light scattering to assess monodispersity

• Functional verification:

  • RNA binding assays using electrophoretic mobility shift assays (EMSAs) with labeled SsrA RNA

  • Surface plasmon resonance (SPR) to determine binding kinetics to SsrA RNA

  • Ribosome association assays in reconstituted systems
    A properly folded and functional recombinant C. abortus smpB should demonstrate specific binding to SsrA RNA with nanomolar affinity and be able to complement smpB deletion phenotypes in appropriate assay systems.

How does C. abortus smpB interact with SsrA RNA at the molecular level?

The interaction between C. abortus smpB and SsrA RNA involves specific structural elements of both partners. While specific details for C. abortus are still being elucidated, structural studies from model organisms reveal that:

• SmpB primarily recognizes the tRNA-like domain (TLD) of SsrA RNA, specifically interacting with the D-loop and T-loop regions.

• The C-terminal tail of SmpB likely extends into the mRNA channel of the ribosome when the SmpB-SsrA complex binds to stalled ribosomes.

• Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain .
  For studying these interactions in C. abortus specifically, researchers should consider:

• X-ray crystallography or cryo-EM of the C. abortus SmpB-SsrA complex

• NMR studies of isotopically labeled SmpB with defined SsrA RNA constructs

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Site-directed mutagenesis of predicted interaction sites followed by binding studies

What genetic approaches can be used to study smpB function in Chlamydophila abortus?

Recent advances in genetic manipulation of obligate intracellular bacteria have opened new avenues for studying gene function in Chlamydia species . For C. abortus smpB research, the following approaches are now feasible:

• Conditional knockdown systems: Using inducible antisense RNA or CRISPRi (CRISPR interference) systems to reduce smpB expression . This is particularly valuable as smpB may be essential, making direct knockout approaches challenging.

• Allelic exchange mutagenesis: The Fluorescence-Reported Allelic Exchange Mutagenesis (FRAEM) system can be adapted for targeted gene modifications in C. abortus . This approach uses:

  • Homologous sequences (~3 kb) flanking the smpB gene

  • Conditional suicide vectors with selectable markers

  • Fluorescent reporters to track successful recombination events

• Complementation studies: Reintroducing wild-type or mutant versions of smpB to assess functional consequences of specific mutations.

• Transformation protocols: Chemical transformation methods using calcium chloride with microgram levels of DNA have been optimized for Chlamydia species , and these can be applied to introduce modified smpB constructs.
  For Chlamydia, selection of transformants relies on antibiotic resistance markers, with spectinomycin or ampicillin resistance commonly used .

How can researchers investigate the role of smpB in C. abortus stress response and pathogenesis?

To investigate the relationship between smpB and C. abortus pathogenesis, researchers should employ multi-faceted approaches:

• Infection models:

  • Ovine trophoblast cell lines for in vitro studies

  • Pregnant sheep models for in vivo studies, particularly monitoring for effects on placental infection and abortion rates

  • Monitoring of bacterial loads and developmental cycle progression under smpB modulation

• Stress response studies:

  • Exposing C. abortus to various stressors (nutrient limitation, oxidative stress, antimicrobial peptides) with wild-type or reduced smpB levels

  • Transcriptomic and proteomic profiling to identify downstream effects of smpB modulation during stress

  • Measuring survival rates and developmental cycle transitions under stress conditions

• Host-pathogen interaction studies:

  • Examining the impact of smpB modulation on host cell responses

  • Assessing changes in inflammatory cytokine production

  • Monitoring effects on immune cell recruitment and activation

• Comparative studies with C. trachomatis:

  • C. trachomatis is more readily manipulated genetically and causes similar but distinct pathology

  • Findings from C. trachomatis can inform directed studies in C. abortus

Can recombinant C. abortus smpB be utilized in diagnostic assays for ovine enzootic abortion?

While current diagnostic approaches for C. abortus infection primarily target major outer membrane proteins (MOMP) or polymorphic outer membrane proteins (POMP90) , recombinant smpB presents an alternative diagnostic target with several potential advantages:

• Specificity considerations:

  • smpB's high conservation within C. abortus but sequence divergence from other Chlamydia species could provide species-specific detection

  • The protein's relative conservation may result in consistent detection across C. abortus strains compared to more variable surface antigens

• Assay development potential:

  • ELISA-based detection using anti-smpB antibodies in animal sera

  • PCR-based molecular diagnostics targeting the smpB gene

  • Lateral flow immunoassays for field diagnostics using recombinant smpB or anti-smpB antibodies
    The diagnostic value would need to be validated by comparing detection rates with established methods like the complement fixation test (CFT) and recombinant POMP90-based ELISAs, which have shown superior sensitivity and specificity in detecting C. abortus infections .

What are the challenges in differentiating immune responses to smpB between C. abortus and other Chlamydia species?

Developing smpB-based diagnostic tools faces several immunological challenges:

• Cross-reactivity assessment: While smpB exhibits some sequence divergence between Chlamydia species, there are conserved epitopes that may lead to cross-reactive antibodies. This is particularly important in differentiating C. abortus from C. pecorum, another common chlamydial pathogen in sheep .

• Epitope mapping strategy:

  • Identification of C. abortus-specific epitopes within smpB using overlapping peptide arrays

  • Comparison of antibody recognition patterns between animals infected with different Chlamydia species

  • Development of competitive ELISA formats using species-specific monoclonal antibodies

• Validation requirements:

  • Testing against sera from animals experimentally infected with different Chlamydia species

  • Field validation using samples from flocks with documented histories of abortion

  • Comparison with established serological methods like POMP90-based ELISAs which have already demonstrated high specificity
    Based on studies with other chlamydial antigens, an smpB-based test would need to match or exceed the 95% sensitivity and specificity achieved by recombinant POMP90-based tests to be considered valuable for diagnostic applications .

How does genetic diversity of smpB across C. abortus isolates impact research approaches?

The genomic analysis of C. abortus isolates has revealed important insights about genetic diversity within this species:

• Evolutionary considerations: European C. abortus livestock isolate genomes show unusual stability and limited diversity compared to other Chlamydia species. No recombination has been identified within C. abortus, suggesting different evolutionary dynamics compared to other Chlamydia species .

• Impact on research approaches:

  • Limited sequence variation in smpB would simplify the design of broadly applicable molecular tools and recombinant protein constructs

  • Geographic clustering of genetic variation suggests the importance of including isolates from different regions when validating experimental approaches

  • The low number of variable nucleotide positions across sampled isolates (significantly lower than those in C. trachomatis and C. psittaci) indicates that findings from one strain may be broadly applicable

• Strain selection guidance: When studying smpB function, researchers should consider including:

  • Reference laboratory strains (e.g., S26/3)

  • Representatives from each of the seven phylogenetic groups showing geographical associations

  • The more divergent Greek isolates (LLG and POS) which form a separate branch in phylogenetic analyses

What are the technical challenges in studying smpB-SsrA interactions in the context of C. abortus infection?

Studying the smpB-SsrA system in C. abortus presents unique challenges due to its obligate intracellular lifestyle:

• System reconstitution difficulties:

  • The necessity of host cells for C. abortus growth complicates isolation of native complexes

  • Chlamydia lacks the important sRNA chaperone protein Hfq found in E. coli, potentially affecting smpB-SsrA dynamics compared to model systems

• Technical approaches to overcome limitations:

  • Development of cell-free translation systems derived from Chlamydia

  • Implementation of proximity labeling techniques (BioID, APEX) to capture interaction partners in living infected cells

  • Application of fluorescence microscopy approaches with genetically encoded tags to visualize smpB-SsrA interactions during the developmental cycle

• Developmental cycle considerations:

  • The biphasic lifecycle of Chlamydia (alternating between elementary bodies and reticulate bodies) likely affects smpB-SsrA activity

  • Time-course studies throughout the developmental cycle are essential to understand when and how the system functions during infection
    Recent advances in Chlamydia genetics, including inducible expression systems and conditional knockdowns , provide new opportunities to study these interactions within the native context of infection.

How might smpB function differ in C. abortus compared to model organisms like E. coli?

Several factors may contribute to functional differences of the smpB-SsrA system in C. abortus compared to model organisms:

• Genomic adaptations in obligate intracellular pathogens:

  • Genome reduction in Chlamydia has led to streamlined systems

  • The absence of certain components present in model organisms may have led to compensatory mechanisms in the remaining systems

• Developmental cycle influences:

  • Transition between metabolically active RBs and dormant EBs may require specialized smpB-SsrA activity

  • The system may have evolved to handle specific stresses encountered during the intracellular phase of the lifecycle

• Potential functional differences:

  • Beyond the canonical role in trans-translation, smpB may serve additional functions in Chlamydia

  • In other bacteria, moonlighting roles for SmpB have been identified, including potential interactions with other cellular components

• Methodological approaches to explore differences:

  • Pull-down experiments followed by mass spectrometry to identify C. abortus-specific interaction partners

  • Heterologous complementation studies to determine functional conservation

  • Ribosome profiling to identify specific mRNA targets of the trans-translation system in C. abortus
    The relatively minimal genome of C. abortus (approximately 1.16 Mb) suggests that each component, including smpB, may have evolved to perform essential functions with maximum efficiency, potentially leading to multifunctional roles not observed in model organisms.