Recombinant Coxiella burnetii Oligoribonuclease (orn)

What is Coxiella burnetii Oligoribonuclease (ORN) and what is its function in bacterial cells?

Oligoribonucleases (ORNs) are highly conserved DnaQ-fold 3′-5′ exoribonucleases that catalyze the final step of RNA degradation by hydrolyzing small RNA molecules (≤5 nucleotides) in the 3′-5′ direction. In bacteria like C. burnetii, ORN plays a critical role in RNA metabolism, particularly in the complete degradation of small nucleic acids .

Additionally, bacterial ORNs have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, converting pGpG to GMP. This function is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can inhibit phosphodiesterases and perturb cellular signaling pathways regulated by c-di-GMP .

Perturbations in c-di-GMP levels can affect bacterial survival under various stress conditions (hypoxic, reductive, or nutrient-limiting conditions) and influence pathogenicity and antibiotic responses in bacterial species .

What is the structural basis of oligoribonuclease activity in bacteria?

The structural basis of oligoribonuclease activity has been well-characterized in several bacterial species. From studies on Colwellia psychrerythraea oligoribonuclease (CpsORN), we know that:

• Bacterial ORNs form stable dimers with two separated active sites

• Each active site contains one divalent metal cation ion essential for catalysis

• The active site is formed by swapping residues from the other subunit in the dimer

• Ligand binding induces conformational changes important for enzymatic function

Crystal structures of CpsORN have been determined in multiple forms, including:

• Unliganded structure

• Thymidine 5′-monophosphate p-nitrophenyl ester (pNP-TMP)-bound form

• Two separated uridine-bound form

• Two linked uridine (U-U)-bound form

These structures provide snapshots of the enzymatic reaction process and support a one-metal-dependent reaction mechanism for bacterial ORNs, which likely applies to C. burnetii ORN as well .

How is oligoribonuclease activity characterized biochemically?

Biochemical characterization of oligoribonuclease activity typically involves the following methodological approaches:

• Substrate specificity analysis: Using 5′-fluorescein-labeled RNA substrates (e.g., 5′-F-UUUUU-3′) to detect intermediate products of degradation. Researchers can observe time-course degradation to confirm 3′-5′ directionality. Additionally, specificity for RNA versus DNA can be tested using equivalent DNA substrates (e.g., 5′-F-TTTTT-3′) .

• Length specificity assessment: Testing activity on longer substrates (e.g., 24-mer RNA) to confirm the enzyme's preference for small RNA molecules. For example, CpsORN showed degradation of 5-mer RNA but not 24-mer RNA, demonstrating clear length specificity .

• Metal dependence evaluation: Assaying enzyme activity in the presence of different divalent cations (typically manganese or magnesium ions) to determine metal cofactor requirements .

• Site-directed mutagenesis: Mutating active site residues to assess their importance in RNA binding and hydrolysis .

Table 1. Example of oligoribonuclease activity assay results:

What expression systems are typically used for recombinant production of bacterial oligoribonucleases?

For recombinant production of bacterial oligoribonucleases, researchers typically employ the following methodological approaches:

• Expression vectors: pET expression systems (e.g., pET21c) are commonly used, as demonstrated in the successful expression of a 27-kDa outer membrane protein from C. burnetii . For oligoribonucleases specifically, pET vectors with N-terminal affinity tags have been used successfully .

• Affinity tags: His6-MBP (hexahistidine-maltose binding protein) tags followed by TEV protease cleavage sites allow for efficient purification and tag removal. After hydrolytic removal of the N-terminal tag, a few additional amino acid residues typically remain at the N-terminus due to the protease recognition site .

• Host cells: E. coli strains such as BL21(DE3) are commonly used for protein expression, with growth in Luria-Bertani (LB) broth .

• Purification strategy: One-step affinity purification using the introduced tag can yield purified recombinant protein suitable for enzymatic and structural studies .

How do researchers confirm the identity and purity of recombinant oligoribonucleases?

Researchers employ multiple techniques to confirm the identity and purity of recombinant oligoribonucleases:

• SDS-PAGE and immunoblotting: To verify the molecular weight and presence of the target protein. For example, fusion proteins with molecular masses of 30 and 32 kDa were evident in recombinant C. burnetii outer membrane protein preparations by SDS-PAGE and immunoblotting .

• Mass spectrometry: Whole-cell mass spectrometry can be used to confirm protein identity and detect post-translational modifications .

• Enzyme activity assays: Functional testing using oligonucleotide substrates as described in question 3 confirms that the recombinant enzyme is active .

• Structural analysis: X-ray crystallography or other structural techniques can confirm proper folding and structural integrity of the recombinant protein .

What role might oligoribonuclease play in Coxiella burnetii pathogenesis and lifecycle?

While the specific role of oligoribonuclease in C. burnetii pathogenesis is not directly addressed in the search results, we can make informed hypotheses based on related research:

• RNA metabolism during developmental cycle: C. burnetii undergoes a unique biphasic developmental cycle where bacteria transition from a replicating large cell variant (LCV) form to a nonreplicating small cell variant (SCV) form . RNA degradation enzymes like ORN may play crucial roles during this transition, potentially helping to recycle nucleotides during developmental changes.

• Regulation of c-di-GMP signaling: In other bacteria, ORN is involved in c-di-GMP degradation, which affects survival under stress conditions and pathogenicity . The RpoS sigma factor regulates genes involved in C. burnetii SCV development and intracellular growth , and ORN might be part of this regulatory network.

• Potential link to toxin-antitoxin systems: C. burnetii has been reported to encode 11 toxin-antitoxin systems, which is unusual for intracellular bacteria . Since toxin-antitoxin systems often target RNA, ORN might interact with these systems during infection cycles.

How can site-directed mutagenesis be used to study oligoribonuclease function?

Site-directed mutagenesis is a powerful tool for studying oligoribonuclease function, as demonstrated in studies of related bacterial ORNs:

• Active site mutation strategy: Creating inactive mutants (e.g., D163A in CpsORN) that can bind but not hydrolyze RNA substrates. This allows researchers to capture enzyme-substrate complexes for structural studies, as demonstrated with the thymidine 5′-monophosphate p-nitrophenyl ester (pNP-TMP)-bound form and U-U-bound form of CpsORN .

• Mutation design protocol:

  • Identify conserved residues through sequence alignment

  • Design primers containing the desired mutations

  • Perform PCR amplification using high-fidelity polymerases like AccuPrime Pfx

  • Clone the mutated sequences using systems like the In-Fusion cloning system

  • Transform into E. coli competent cells and select transformants on appropriate antibiotic plates

  • Verify mutations by sequencing

• Functional assessment of mutations: Comparing the RNA-binding and hydrolysis activities of wild-type and mutant enzymes to identify residues crucial for substrate recognition and catalysis .

What methods can be used to study the interaction between oligoribonuclease and RNA substrates?

Several methodological approaches can be used to study ORN-RNA interactions:

• Crystallography: X-ray crystallography with various ligands (as done with CpsORN) provides atomic-level details of protein-RNA interactions. This has revealed how conformational changes occur upon ligand binding .

• Fluorescence-based assays: Using 5′-fluorescein-labeled RNA substrates allows real-time monitoring of degradation activity .

• Binding assays: Techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can determine binding affinities and thermodynamic parameters of ORN-RNA interactions.

• Yeast two-hybrid approaches: While primarily used for protein-protein interactions, this method has been successfully applied to study interactions involving C. burnetii proteins, such as the identification of vimentin as an interactor for the AnkF effector protein .

How might the cold-active properties of some oligoribonucleases be leveraged in molecular biology applications?

Cold-active enzymes like CpsORN from the psychrophilic bacterium Colwellia psychrerythraea have high catalytic efficiency at low and moderate temperatures, making them valuable for biotechnological applications . For molecular biology applications:

• Complete RNA digestion: Cold-active ORN could be used to completely digest small RNA molecules in molecular biology and cell biology experiments, particularly at lower temperatures that may preserve other biomolecules .

• RNA sample preparation: ORN could potentially be used to remove small RNA contaminants from RNA samples destined for sequencing or other analyses.

• Methodological advantages:

  • Reaction can proceed at lower temperatures (reducing non-specific reactions)

  • Lower energy requirements for reaction conditions

  • Potential for higher specific activity than mesophilic counterparts at moderate temperatures

  • Easier inactivation by moderate heat, allowing better control of reaction termination

What approaches can be used to study the potential role of oligoribonuclease in Coxiella burnetii antimicrobial resistance?

Given that deletion of oligoribonuclease in some bacteria (e.g., P. aeruginosa) has been associated with reduced susceptibility to antibacterial drugs , the following approaches could be used to investigate its role in C. burnetii:

• Gene knockout studies: Generate targeted mutations in C. burnetii ORN using techniques established for other C. burnetii genes:

  • Plasmid-based mutant generation using vectors like pUC19

  • CRISPR interference (CRISPRi) systems, which have been successfully developed for C. burnetii to examine the role of individual genes

  • Transposon mutagenesis, which has been used to create C. burnetii mutants

• Antibiotic susceptibility testing: Compare the minimum inhibitory concentrations (MICs) of various antibiotics between wild-type and ORN-deficient C. burnetii. As a reference point, the MIC for doxycycline (a recommended treatment for C. burnetii infection) is 0.01 μg per mL (22.5 nM) .

• RNA accumulation analysis: Determine if oligonucleotides accumulate in ORN-deficient strains, as has been observed in E. coli , and whether this affects antibiotic susceptibility.

• Alternative compound screening: Test compounds that have shown activity against C. burnetii, such as the tetrahydropyrimidine pyrantel pamoate, which demonstrated an inhibitory effect at concentrations as low as 1 μg per mL (1.68 μM) .

How can recombinant oligoribonuclease be used for developing molecular diagnostic tools for C. burnetii?

Several molecular diagnostic approaches for C. burnetii have been developed, and recombinant ORN could potentially enhance these methods:

• PCR-based detection systems: A nested PCR method targeting the com1 gene encoding a 27-kDa outer membrane protein has been developed for detecting C. burnetii in human serum samples, with 87% sensitivity compared to immunofluorescence tests . Recombinant ORN could potentially be used in sample preparation to remove small RNA contaminants.

• Recombinase polymerase amplification (RPA) with lateral flow (LF) detection: This method targeting the 23S rRNA gene of C. burnetii can be completed in 30 minutes at 37°C with visually judged results. It demonstrated high specificity and sensitivity (detecting as few as 7 copies/reaction) . Integrating recombinant ORN in sample processing could potentially improve detection limits.

• ELISA-based approaches: Using recombinant proteins as antigens. A 27-kDa recombinant outer membrane protein from C. burnetii has been successfully used in ELISA for detecting anti-C. burnetii antibodies in human sera with high sensitivity and specificity . Similar approaches could be developed using recombinant ORN if it proves to be immunogenic.

• Genomotyping methods: Novel rapid genomotyping methods based on spacer sequences could be enhanced by enzymatic sample preparation involving recombinant ORN .

Table 2: Comparison of molecular detection methods for C. burnetii

What challenges exist in studying C. burnetii proteins like ORN and how can they be addressed?

Several challenges exist in studying C. burnetii proteins, including ORN:

• Biosafety requirements: C. burnetii is classified as a biological warfare agent and requires appropriate containment facilities . Alternative approaches include:

  • Using axenic media culture methods rather than cell culture systems

  • Developing cell-free protein expression systems for recombinant production

  • Focusing on recombinant protein production in standard E. coli systems

• Phase variation: C. burnetii undergoes lipopolysaccharide (LPS) phase transition similar to Enterobacteriaceae upon in vitro passage, which can affect protein expression and bacterial properties . Methods to address this include:

  • Using Vero cell culture to selectively enrich solutions with phase I and intermediate phase LPS-expressing bacteria

  • Carefully monitoring and documenting the phase status of the bacteria used in experiments

• Obligate intracellular lifestyle: C. burnetii's natural growth environment is within host cells, specifically within an acidic phagolysosomal vacuole (the C. burnetii-containing vacuole or CCV) . Strategies include:

  • Using cell lines like Vero epithelial cells or THP-1 macrophage-like cells for propagation

  • Developing improved axenic culture systems that better mimic intracellular conditions

• Protein purification challenges: Ensuring proper folding and activity of recombinant C. burnetii proteins. Approaches include:

  • Optimization of expression conditions (temperature, induction timing, etc.)

  • Testing various affinity tags and purification strategies

  • Inclusion of appropriate cofactors (e.g., divalent metal ions) during purification