Recombinant Coxiella burnetii UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA)

What is the biological function of UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) in Coxiella burnetii?

UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) in Coxiella burnetii plays a crucial role in cell wall formation. It catalyzes the addition of enolpyruvyl to UDP-N-acetylglucosamine, which represents an essential step in peptidoglycan biosynthesis pathway . This enzyme is localized in the cytoplasm and belongs to the EPSP synthase family, specifically in the MurA subfamily . As an obligate intracellular Gram-negative bacterium, C. burnetii requires a functional cell wall for structural integrity and survival, making murA an important component of its cellular machinery.

How does murA contribute to Coxiella burnetii pathogenesis?

While the search results don't directly address murA's specific contribution to pathogenesis, we can infer its importance based on established bacterial pathogenesis mechanisms. As C. burnetii can be transmitted by aerosol with a very low infectious dose (1-10 viable organisms are sufficient to induce infection via the aerogenic route), the integrity of its cell wall structures is crucial for environmental persistence and host infection . The murA enzyme's role in peptidoglycan synthesis makes it fundamentally important for bacterial survival within macrophages, where C. burnetii typically replicates in parasitophorous vacuoles. The bacterium's ability to establish chronic infections suggests that cell wall components, dependent on murA activity, may contribute to evasion of host immune responses and persistence within host cells.

What expression systems are typically used to produce recombinant C. burnetii murA?

Based on the product information provided, recombinant C. burnetii UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) can be expressed in various heterologous systems including yeast and E. coli . The search results mention specific product codes for different expression systems:

• Yeast-expressed recombinant murA (Product code: CSB-YP774415DXP)

• E. coli-expressed recombinant murA (Product code: CSB-EP774415DXP)

• E. coli-expressed recombinant murA with Avi-tag Biotinylation (Product code: CSB-EP774415DXP-B)

E. coli systems offer advantages for high yield protein production, while yeast expression may provide certain post-translational modifications. The availability of biotinylated versions suggests applications requiring specific protein labeling and detection methodologies.

What are the optimal purification strategies for recombinant C. burnetii murA?

While the search results don't detail specific purification protocols, the available recombinant products suggest standard approaches for purification. For E. coli-expressed recombinant murA proteins, a typical purification strategy would involve:

• Affinity chromatography: Using His-tag or other fusion tags that appear to be incorporated into the recombinant proteins

• Size exclusion chromatography: To ensure high purity and remove aggregates

• Ion exchange chromatography: For further purification if needed

For biotinylated versions (CSB-EP774415DXP-B), the AviTag-BirA technology mentioned indicates that the protein was biotinylated in vivo, where "BirA catalyzes amide linkage between the biotin and the specific lysine of the AviTag" . This suggests that streptavidin-based affinity purification could be an effective additional or alternative purification step.

How can researchers effectively assess the enzymatic activity of purified recombinant murA?

An effective methodology for assessing recombinant murA enzymatic activity would include:

• Substrate conversion assay: Measuring the rate of conversion of UDP-N-acetylglucosamine to enolpyruvyl-UDP-N-acetylglucosamine in the presence of phosphoenolpyruvate (PEP)

• Spectrophotometric monitoring: Following the reaction at 232 nm to detect the formation of the enolpyruvyl product

• Coupled enzyme assays: Using pyruvate kinase and lactate dehydrogenase to monitor NADH oxidation as an indirect measure of murA activity

• Inhibition studies: Evaluating activity in the presence of known inhibitors like fosfomycin to confirm specificity

A typical reaction mixture would contain:

• Purified recombinant murA (1-10 μg/ml)

• UDP-N-acetylglucosamine (0.5-2 mM)

• Phosphoenolpyruvate (1-5 mM)

• Buffer system (typically phosphate buffer, pH 7.0-7.5)

• Divalent cations (Mg²⁺ or Mn²⁺, 5-10 mM)

Temperature optimization studies (typically 25-37°C) would be necessary to determine optimal conditions for the C. burnetii enzyme.

How effective is recombinant murA as an immunogen for vaccine development against C. burnetii?

Based on these findings, researchers working with recombinant murA as a potential vaccine candidate should consider the following methodological approach:

• Evaluate antigenicity in appropriate animal models (e.g., BALB/c mice)

• Test different adjuvant formulations to enhance immune response

• Perform challenge studies with virulent C. burnetii strains (e.g., Nine Mile RSA493)

• Assess protection through clinical examinations and measurements of spleen and liver weights

• Compare results against established vaccines like Q-VaxTM as a positive control

It's important to note that previous attempts to develop Q fever vaccines have had limitations, either causing unacceptable side effects or failing to be protective , suggesting careful consideration of safety and efficacy parameters when evaluating murA as a vaccine component.

What role might murA play in host-pathogen interactions during C. burnetii infection?

While the search results don't specifically address murA's role in host-pathogen interactions, understanding can be inferred from general C. burnetii infection biology. As a cell wall biosynthesis enzyme, murA contributes to the structural components recognized by pattern recognition receptors (PRRs) of the innate immune system.

C. burnetii's interaction with host cells, particularly macrophages, is complex. The bacterium can:

• Replicate in large, typical Coxiella-containing vacuoles (CCVs) in human alveolar macrophages

• Require a functional type IV secretion system for CCV formation and bacterial growth

• Modulate host cell functions through effector proteins

• Induce different cytokine responses depending on bacterial strain virulence

Avirulent C. burnetii strains promote pro-survival kinase activation and robust pro-inflammatory responses (TNF-α and IL-6), while virulent isolates elicit reduced cytokine secretion . The persistence of bacterial components, including cell wall elements dependent on murA activity, may contribute to the "Immunomodulatory complex" (IMC) that has been implicated in chronic Q fever pathogenesis .

How can structural analysis of murA inform rational drug design targeting C. burnetii?

Although the search results don't provide structural information about C. burnetii murA specifically, a methodological approach to structure-based drug discovery would include:

• Homology modeling: Generating a structural model based on crystallized murA proteins from related bacteria

• Molecular docking studies: Identifying potential binding sites and interactions with substrate analogs or known inhibitors

• Structure-activity relationship analysis: Systematically modifying inhibitor structures to optimize binding affinity and specificity

• In vitro validation: Testing predicted inhibitors against purified recombinant murA enzyme

• Cellular studies: Evaluating compounds in C. burnetii infection models

The relationship between murA structure and function is particularly important since MurA is the target of fosfomycin, a clinically used antibiotic. Understanding structural differences between human and bacterial enzymes is crucial for developing selective inhibitors with minimal off-target effects.

What are the challenges in expressing and purifying enzymatically active recombinant murA from C. burnetii?

While specific challenges aren't detailed in the search results, common difficulties in expressing bacterial enzymes like murA include:

• Protein solubility: Optimizing expression conditions to prevent inclusion body formation

  • Solution: Testing different solubility tags, expression temperatures, and induction parameters

• Proper folding: Ensuring native-like structure in heterologous expression systems

  • Solution: Co-expression with molecular chaperones or slow refolding protocols

• Enzymatic activity: Maintaining catalytic function through purification steps

  • Solution: Including stabilizing agents and avoiding harsh conditions during purification

• Post-translational modifications: Identifying if any modifications are required for function

  • Solution: Comparing expression in prokaryotic vs. eukaryotic systems (as suggested by the availability of both E. coli and yeast expression systems)

• Protein yield: Obtaining sufficient quantities for structural and functional studies

  • Solution: Optimizing codon usage for the expression host and scaling up production

How does C. burnetii murA compare with orthologs from other bacterial pathogens?

The search results don't provide direct comparative information, but a methodological approach to ortholog comparison would include:

• Sequence alignment analysis: Identifying conserved catalytic residues and variable regions

• Phylogenetic analysis: Determining evolutionary relationships between murA proteins from different bacteria

• Substrate specificity comparison: Evaluating kinetic parameters (Km, Vmax, kcat) across orthologs

• Inhibition profile analysis: Comparing sensitivity to known inhibitors

• Structural comparison: Analyzing differences in protein folding and active site architecture

Expected findings would likely reveal high conservation of catalytic domains given the essential nature of the enzymatic function, with potential variations in regulatory regions that might influence expression patterns or activity modulation in response to environmental conditions.

What is the potential of C. burnetii murA as a therapeutic target for Q fever treatment?

While the search results don't directly address murA as a therapeutic target, its essential role in cell wall biosynthesis makes it a promising candidate. A methodological approach to evaluating its therapeutic potential would include:

• Target validation: Confirming essentiality through genetic approaches (if possible in C. burnetii)

• Druggability assessment: Evaluating the active site for potential binding pockets suitable for small molecule inhibitors

• Selective inhibition: Developing compounds that specifically target C. burnetii murA without affecting human enzymes or beneficial microbiota

• Efficacy testing: Evaluating potential inhibitors in:

  • Enzymatic assays with purified recombinant protein

  • Cellular infection models (e.g., human alveolar macrophages)

  • Animal models of acute and chronic Q fever

• Combination therapy evaluation: Testing murA inhibitors alongside current treatments (doxycycline plus hydroxychloroquine)

The available data on C. burnetii infection biology suggests that targeting cell wall biosynthesis could be particularly effective given the bacterium's reliance on structural integrity for survival within host cells and environmental persistence .