Recombinant Coxiella burnetii 23S rRNA (guanosine-2'-O-)-methyltransferase RlmB (rlmB)

What is the structural characterization of Coxiella burnetii RlmB?

While specific structural data for C. burnetii RlmB is limited, insights can be derived from studies on homologous proteins like the E. coli RlmB. The E. coli enzyme consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain, forming a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region and lacks the classic AdoMet (S-adenosylmethionine) binding site features typically seen in other methyltransferases . The N-terminal domain shares structural similarities with ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition . Based on sequence homology, the C. burnetii RlmB likely shares these core structural features, though species-specific variations may exist.

How does RlmB contribute to C. burnetii pathogenicity?

RNA modifications catalyzed by methyltransferases like RlmB may contribute to C. burnetii pathogenicity through several mechanisms. These modifications can affect ribosomal function and consequently protein synthesis efficiency, which may be particularly important during different growth phases or stress conditions within host cells . Additionally, since C. burnetii must adapt to the harsh environment of phagolysosomes, proper ribosomal function supported by RNA modifications may be critical for survival. The bacterium undergoes a developmental cycle with metabolically active large cell variants (LCVs) and resistant small cell variants (SCVs) , and RlmB activity might be differentially regulated during these distinct developmental stages to support the specific protein synthesis requirements of each form.

What expression systems are suitable for producing recombinant C. burnetii RlmB?

Based on successful expression of other C. burnetii proteins, cell-free expression systems using E. coli components have proven effective for producing recombinant C. burnetii proteins . Specifically, systems like the RTS 100 E. coli HY kit and RTS 500 ProteoMaster E. coli have been successfully employed . For RlmB expression, a vector system such as pIVEX2.4d, which introduces an N-terminal 6-histidine tag for purification, would be appropriate . The expressed protein can be purified using Ni-NTA magnetic agarose beads under native conditions and stored in 25% glycerol at -80°C for optimal stability . For larger-scale productions, the RTS 500 ProteoMaster E. coli system may be more suitable .

How does the catalytic mechanism of C. burnetii RlmB compare to other bacterial 2'-O-methyltransferases?

The catalytic mechanism of C. burnetii RlmB likely shares fundamental features with other bacterial 2'-O-methyltransferases, particularly its E. coli homolog. In E. coli RlmB, the conserved residues cluster in the knotted region of the C-terminal domain, suggesting this area contains both the catalytic site and the AdoMet binding site . The enzyme likely transfers a methyl group from S-adenosylmethionine (AdoMet) to the 2'-hydroxyl group of the target guanosine in 23S rRNA.

The unique knotted topology of RlmB distinguishes it from many other methyltransferases and may confer specificity for its target site in the peptidyltransferase domain of 23S rRNA . While the general mechanism is likely conserved, C. burnetii RlmB may have evolved specific adaptations to function optimally within the unique intracellular environment of this pathogen, particularly considering the acidic conditions of the phagolysosome where the bacterium resides during infection . Careful enzymatic characterization comparing activity at different pH levels and under various ionic conditions would help elucidate these potential adaptations.

What are the implications of RlmB activity for C. burnetii antibiotic resistance?

The methylation of 23S rRNA by RlmB could potentially influence antibiotic susceptibility in C. burnetii, particularly against drugs targeting the ribosome. Modification of nucleosides within the peptidyltransferase center may alter the binding of antibiotics that target this region, such as macrolides, lincosamides, or pleuromutilins. Current treatment protocols for Q fever typically include doxycycline (tetracycline class), sometimes in combination with hydroxychloroquine . For chronic Q fever, this combination is administered for 18 to 36 months .

How does post-translational regulation affect RlmB activity in C. burnetii during different growth phases?

The biphasic life cycle of C. burnetii, alternating between SCVs and LCVs, likely requires differential regulation of various cellular processes, including RNA modification . RlmB activity may be regulated post-translationally to match the distinct metabolic states and protein synthesis requirements of these different forms. Potential regulatory mechanisms could include:

• Phosphorylation or other post-translational modifications that alter enzyme activity

• Protein-protein interactions that modulate RlmB function

• Compartmentalization or localization changes within the bacterial cell

• Altered stability or turnover rates of the enzyme during different growth phases

Proteomic studies comparing RlmB modification states between SCVs and LCVs, coupled with in vitro activity assays under conditions mimicking each growth phase, would provide insights into these regulatory mechanisms. Additionally, examining RlmB activity in response to environmental stressors that trigger phase transitions could reveal how this enzyme contributes to C. burnetii adaptability.

What is the impact of RlmB deletion or mutation on C. burnetii virulence in experimental models?

While no direct studies on RlmB knockout in C. burnetii have been reported in the provided search results, targeted gene disruption or mutation studies would be valuable for understanding this enzyme's contribution to virulence. Based on studies with other bacterial pathogens, several experimental approaches could be considered:

• Creation of RlmB knockout strains using targeted mutagenesis

• Introduction of point mutations affecting catalytic activity

• Conditional expression systems to regulate RlmB levels

These modified strains could be evaluated in established infection models for C. burnetii. Guinea pigs represent a model for acute Q fever, while mice develop chronic infections that could be useful for studying long-term effects of RlmB deficiency . Macaque models provide the closest parallel to human clinical manifestations and could offer insights into how RlmB affects pathogenesis in a physiologically relevant system . Virulence assessment should examine bacterial replication rates, host immune responses, and pathological changes in infected tissues.

What are the optimal purification methods for obtaining active recombinant C. burnetii RlmB?

Based on successful approaches with other C. burnetii proteins, a multi-step purification protocol is recommended:

• Initial Expression System Selection: The RTS 500 ProteoMaster E. coli HY kit has been effectively used for large-scale expression of C. burnetii proteins .

• Construct Design: Include an N-terminal 6-histidine tag in the expression construct using vectors like pIVEX2.4d for affinity purification .

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin under native conditions

  • Size exclusion chromatography to separate dimeric active enzyme from aggregates and monomers

  • Ion exchange chromatography for final polishing and removal of nucleic acid contaminants

• Storage Conditions: Store purified enzyme in buffer containing 25% glycerol at -80°C to maintain activity .

• Activity Validation: Assess 2'-O-methyltransferase activity using synthetic RNA oligonucleotides corresponding to the target region of 23S rRNA.

For highest purity preparations intended for structural studies, additional steps like sucrose gradient ultracentrifugation may be necessary to ensure sample homogeneity.

What are the challenges in designing enzymatic assays for C. burnetii RlmB activity?

Several challenges exist in developing reliable assays for RlmB methyltransferase activity:

• Substrate Complexity: The natural substrate is a specific site within the folded structure of 23S rRNA, which is difficult to replicate with synthetic substrates. Successful approaches might include:

  • Using in vitro transcribed 23S rRNA fragments containing the target site

  • Designing minimal substrate RNA oligonucleotides that maintain the necessary structural features

  • Isolating native ribosomes from C. burnetii or heterologous hosts

• Detecting Methylation: The 2'-O-methylation does not significantly alter RNA mobility or UV absorbance, making detection challenging. Methods to consider include:

  • Radiolabeled AdoMet (S-adenosyl-L-[methyl-³H]methionine) incorporation assays

  • Mass spectrometry-based detection of methylated RNA products

  • Reverse transcription-based methods that exploit the tendency of reverse transcriptase to pause at 2'-O-methylated sites

• Reaction Conditions: Determining optimal conditions (pH, ion concentrations, temperature) that reflect the unique intracellular environment of C. burnetii is critical for physiologically relevant activity measurements.

• Control Experiments: Include appropriate controls such as catalytically inactive RlmB mutants and pre-methylated substrates to validate assay specificity.

How can researchers overcome the biosafety challenges in studying C. burnetii RlmB?

C. burnetii is classified as a BSL-3 pathogen due to its high infectivity and potential for aerosol transmission . Several approaches can mitigate biosafety concerns when studying RlmB:

• Recombinant Expression: Express recombinant RlmB in non-pathogenic hosts like E. coli using the gene sequence rather than working with the native protein from C. burnetii cultures .

• Phase II Strains: For studies requiring the whole organism context, consider using avirulent Phase II strains which have an incomplete lipopolysaccharide (LPS) and can sometimes be handled at lower biosafety levels with appropriate permissions .

• Cell-Free Systems: Utilize cell-free protein expression systems like the RTS E. coli HY kits, which eliminate the need for viable C. burnetii cultures .

• Surrogate Models: For certain research questions, studying RlmB homologs in related but less pathogenic organisms may provide valuable insights.

• Collaboration with Specialized Facilities: Partner with laboratories equipped with BSL-3 facilities for experiments requiring virulent C. burnetii.

Regardless of approach, researchers must adhere to institutional biosafety protocols and obtain necessary approvals before initiating work with C. burnetii-derived materials.

How can RlmB be utilized as a potential diagnostic target for Q fever?

RlmB could potentially serve as a novel diagnostic target for Q fever, complementing existing approaches. Current Q fever diagnostics primarily rely on serological methods detecting antibodies against whole cell antigens, which are hazardous and laborious to produce . The potential of RlmB as a diagnostic target can be evaluated through the following approaches:

• Serological Detection: Test if natural C. burnetii infections elicit antibodies against RlmB that could be detected by ELISA or other immunoassays.

• Recombinant Protein Production: Express and purify recombinant RlmB using systems like those described for other C. burnetii proteins, including cell-free expression systems and histidine-tagged constructs for simplified purification .

• Assay Development: Develop ELISA or multiplexed bead-based assays incorporating RlmB alongside other immunoreactive C. burnetii proteins.

• Performance Evaluation: Assess sensitivity and specificity using well-characterized serum panels from confirmed Q fever cases and appropriate controls.

Based on studies with other C. burnetii recombinant proteins, diagnostic assays typically achieve sensitivities of 21-71% and specificities of 90-100% . If RlmB proves useful, it could be incorporated into multiplex assays alongside established antigens like CBU_1718, CBU_0307, and CBU_1398, which have shown promise in diagnostic applications .

What are the considerations for developing inhibitors targeting C. burnetii RlmB?

Developing selective inhibitors against C. burnetii RlmB could provide new therapeutic options for Q fever. Current treatment relies heavily on doxycycline, sometimes combined with hydroxychloroquine for 18-36 months in chronic cases . RlmB inhibitors might offer complementary approaches, particularly for resistant infections. Key considerations include:

• Structural Insights: Leveraging the unique features of RlmB's structure, particularly the knotted region containing the catalytic and AdoMet binding sites, to design selective inhibitors .

• Selectivity: Ensuring inhibitors target C. burnetii RlmB with minimal effects on human RNA methyltransferases to reduce toxicity.

• Compound Classes:

  • AdoMet analogs that compete for the cofactor binding site

  • RNA mimetics that occupy the substrate binding pocket

  • Allosteric inhibitors that disrupt protein dimerization or conformational changes

• Delivery Challenges: Developing compounds that can penetrate both host cell membranes and bacterial cell walls to reach the intracellular pathogen.

• Resistance Potential: Assessing the likelihood of resistance development through target modification or efflux mechanisms.

Initial screening could employ in vitro enzymatic assays with purified recombinant RlmB, followed by cell culture models and eventually animal models of C. burnetii infection to validate promising candidates.

How might CRISPR-Cas9 technologies be applied to study RlmB function in C. burnetii?

While CRISPR-Cas9 applications in C. burnetii research are still emerging, this technology offers promising approaches for investigating RlmB function:

• Gene Knockout Studies: Generate precise rlmB deletion mutants to assess its essentiality and phenotypic consequences, including growth rates in different media, cell variant transitions, and virulence in infection models.

• Domain Function Analysis: Create partial deletions or targeted mutations affecting specific functional domains to dissect the roles of N-terminal versus C-terminal regions and the significance of the knotted structure.

• Regulated Expression: Develop CRISPR interference (CRISPRi) systems to conditionally repress rlmB expression, allowing temporal control to study its role during different growth phases.

• Reporter Fusions: Insert fluorescent protein tags or epitope tags at the endogenous locus to monitor RlmB expression, localization, and dynamics during infection.

• High-Throughput Screening: Combine CRISPR techniques with transposon mutagenesis to identify genetic interactions and compensatory pathways related to RlmB function.

Implementation would require optimization of transformation protocols for C. burnetii and careful design of guide RNAs specific to the rlmB locus, while avoiding off-target effects in the relatively small C. burnetii genome.

What comparative genomics approaches can reveal about RlmB conservation and evolution among Coxiella species?

Comparative genomics offers valuable insights into the evolutionary significance of RlmB:

• Sequence Conservation Analysis: Compare rlmB sequences across:

  • Different C. burnetii isolates (human, animal, and environmental)

  • Related Coxiella-like endosymbionts

  • Other members of the Legionellales order

  • More distant bacterial lineages with confirmed 2'-O-methyltransferase activity

• Structural Comparisons: Examine conservation of key structural features like the knotted region and domains involved in substrate recognition.

• Synteny Analysis: Investigate the genomic context of rlmB across species to identify conserved gene neighborhoods that might suggest functional relationships.

• Selection Pressure: Calculate dN/dS ratios to determine whether rlmB is under purifying selection (suggesting essential function) or positive selection (indicating adaptive evolution).

• Horizontal Gene Transfer Assessment: Evaluate whether rlmB shows evidence of horizontal acquisition based on GC content, codon usage bias, or phylogenetic incongruence.

This research could clarify whether RlmB represents a core function maintained throughout Coxiella evolution or an adaptation specific to certain ecological niches or host environments.

What are the key knowledge gaps in our understanding of C. burnetii RlmB?

Despite advances in understanding bacterial methyltransferases, several critical knowledge gaps remain regarding C. burnetii RlmB:

• Structural Characterization: The three-dimensional structure of C. burnetii RlmB has not been determined, limiting structure-based drug design efforts and mechanistic understanding.

• Substrate Specificity: The precise target site(s) within C. burnetii 23S rRNA have not been experimentally confirmed, nor has the specificity of the enzyme been thoroughly characterized.

• Physiological Role: The importance of RlmB-mediated methylation for C. burnetii growth, phase transitions, and virulence remains largely speculative without targeted gene disruption studies.

• Regulation: The mechanisms controlling RlmB expression and activity during different phases of the C. burnetii life cycle are unknown.

• Host Interactions: Whether RlmB or its activity affects host cell responses during infection has not been investigated.

Addressing these gaps would significantly advance our understanding of this enzyme's role in C. burnetii biology and potentially open new avenues for Q fever diagnostics or therapeutics.

How do methodological advances in Q fever research impact future studies of RlmB?

Recent methodological advances in C. burnetii research will facilitate more detailed studies of RlmB:

• Axenic Culture: The development of cell-free culture media like ACCM-2 enables growth of C. burnetii outside host cells, simplifying experiments and reducing biosafety concerns .

• Improved Genetic Tools: Enhanced transformation protocols and genetic manipulation techniques allow for more efficient creation of mutants and tagged constructs.

• Advanced Imaging: Super-resolution microscopy and electron cryotomography provide unprecedented visualization of C. burnetii structures and protein localization.

• Systems Biology Approaches: Integration of transcriptomics, proteomics, and metabolomics data offers comprehensive views of how RlmB fits into broader cellular networks.

• Improved Animal Models: Refinement of animal models, including macaque models that closely mimic human disease, provides better systems for testing hypotheses about RlmB's role in pathogenesis .