Recombinant Coxiella burnetii Phosphoribosylformylglycinamidine cyclo-ligase (purM)

What is Phosphoribosylformylglycinamidine cyclo-ligase (purM) and what is its significance in Coxiella burnetii?

Phosphoribosylformylglycinamidine cyclo-ligase (purM) is an enzyme belonging to the AIR synthase family found in Coxiella burnetii, a gram-negative obligate intracellular bacterial pathogen that causes Q fever in humans . The protein is encoded by the purM gene (CBU_0963) in C. burnetii strain RSA 493 / Nine Mile phase I and has a length of 352 amino acids with a molecular mass of approximately 38.2 kDa . As a component of the purine biosynthesis pathway, purM plays a critical role in bacterial metabolism and potentially contributes to pathogen survival within the harsh, acidic environment of the host cell's parasitophorous vacuole where C. burnetii resides during infection . Understanding this protein's structure, function, and expression patterns is essential for gaining insights into C. burnetii pathogenesis and potential therapeutic targets.

How is recombinant purM typically expressed and purified for research purposes?

Recombinant purM from Coxiella burnetii is commonly expressed in Escherichia coli expression systems using appropriate expression vectors. Based on established protocols for similar C. burnetii proteins, the gene encoding purM is typically cloned into vectors containing an N-terminal His6-tag, such as pQE30, to create an in-frame translational fusion that facilitates purification . The protein is then expressed in E. coli under optimized induction conditions, followed by cell lysis and purification via nickel affinity chromatography to isolate the His-tagged recombinant protein . The complete amino acid sequence of purM (MQSMPDSTRHPPLSYCKAGVDIEKAADLVEAIKPIAKRTRRPGVLSGIGGFGGLFELPRGYKQPVLVSGTDGVGTKLKLAVELNRHDTIGIDLVAMCVNDVITTGAEPLFFLDYYATGHLNNEQAKQILTGIGAGCELAEVALIGGETAEMPGLYRQKDYDLAGFCVGVVEKEKIIDGSRVRVGDALIGIASSGPHSNGYSLIRKILARAKIPLSQSFENKSLADGLLAPTRIYVKTIKRLFSEINVHALAHITGGGLIENVPRVLPSYTQAVIDSNGWEWPAIFHWLQKQGKVPIEEMWRTFNMGVGMVLCLDKKEVRKTLELLAALGETAWILGEIQSSSEEQPRVTITP) provides important information for designing expression constructs and optimizing purification strategies .

What analytical techniques are most effective for characterizing purified recombinant purM?

Multiple analytical techniques are essential for comprehensive characterization of recombinant Coxiella burnetii purM. SDS-PAGE analysis is fundamental for confirming protein purity, integrity, and approximate molecular weight (expected around 38.2 kDa plus any fusion tags) . Western blotting using anti-His antibodies can verify the presence of His-tagged recombinant protein. More sophisticated characterization includes mass spectrometry techniques such as MALDI-TOF/TOF MS, which can confirm protein identity through peptide mass fingerprinting and sequence coverage . Functional characterization often involves enzymatic activity assays specific to phosphoribosylformylglycinamidine cyclo-ligase function. Additionally, circular dichroism spectroscopy can provide insights into secondary structure, while thermal shift assays may evaluate protein stability. For structural studies, X-ray crystallography or cryo-electron microscopy might be employed to determine the three-dimensional structure, though no such structure has yet been reported for C. burnetii purM in the available search results.

How does purM potentially contribute to Coxiella burnetii pathogenesis and survival within the parasitophorous vacuole?

The contribution of purM to Coxiella burnetii pathogenesis likely centers on its role in purine biosynthesis, which is critical for bacterial survival and replication within the challenging environment of the parasitophorous vacuole (PV). The PV is characterized by its acidic pH (approximately 4.5) and potential oxidative stress conditions that C. burnetii must overcome to establish successful infection . While purM itself has not been directly implicated in virulence in the provided search results, other metabolic enzymes in C. burnetii have demonstrated multifunctional roles beyond their canonical metabolic activities. For example, the peroxiredoxin BCP (also encoded by C. burnetii) exhibits both enzymatic activity and DNA-binding capabilities that protect against oxidative stress . Similarly, purM may have evolved additional functions beyond purine biosynthesis that contribute to bacterial persistence within host cells. Research investigating potential moonlighting functions of purM, particularly in the context of C. burnetii's adaptation to intracellular life, would provide valuable insights into the protein's role in pathogenesis.

What are the key considerations when designing experiments to evaluate potential moonlighting functions of purM?

When investigating potential moonlighting functions of Coxiella burnetii purM beyond its canonical role in purine biosynthesis, researchers should employ multiple complementary approaches. First, protein-protein interaction studies using techniques such as pull-down assays, co-immunoprecipitation, or bacterial two-hybrid systems can identify binding partners that might suggest non-canonical functions. Drawing from the discovery of DNA-binding activity in C. burnetii BCP, researchers should evaluate potential nucleic acid binding activity of purM using Southwestern (SW) blotting and electrophoretic mobility shift assays (EMSAs) . Functional complementation studies, similar to those conducted with C. burnetii BCP in E. coli, would be valuable for assessing whether purM can substitute for homologous proteins from other species and potentially reveal additional activities . Controlled gene expression studies during different phases of C. burnetii's intracellular growth cycle, using methods like RT-qPCR or RNA-seq, could reveal temporal expression patterns that correlate with specific infection stages, as observed with the bcp gene which shows maximal expression during early exponential growth . Finally, mutagenesis studies combined with infection models would be critical for determining the impact of purM modification on bacterial fitness within host cells.

What approaches would be most effective for studying the structure-function relationship of purM in the context of C. burnetii infection?

Investigating the structure-function relationship of purM requires a multi-faceted approach combining structural biology with functional genomics and infection models. X-ray crystallography or cryo-electron microscopy would provide detailed structural information about purM, including active site architecture and potential binding interfaces for substrates or protein partners. Site-directed mutagenesis targeting conserved residues, followed by enzymatic activity assays, would help identify critical amino acids for catalytic function. To study purM in the context of infection, complementary approaches include creating defined C. burnetii purM mutants using methods like Himar1 transposon mutagenesis (as used for studying T4SS effectors) , followed by evaluation in cellular infection models such as bone marrow-derived macrophages (BMDMs) . RNA interference or CRISPR-Cas9 approaches could be used to modulate purM expression levels during infection to assess dose-dependent effects. Comparative studies between purM from different C. burnetii strains with varying virulence profiles might reveal strain-specific structural or functional adaptations. Finally, heterologous expression of purM variants in surrogate bacterial systems could enable controlled study of specific protein features outside the complexity of C. burnetii infection.

What are the optimal conditions for expressing and purifying enzymatically active recombinant purM?

Obtaining enzymatically active recombinant Coxiella burnetii purM requires careful optimization of expression and purification conditions. For expression, E. coli BL21(DE3) or similar strains are typically preferred due to their reduced protease activity . Expression vectors should include an affinity tag (such as His6) for purification, but consideration should be given to tag position (N- or C-terminal) to minimize interference with enzymatic activity. Induction conditions (temperature, IPTG concentration, and duration) must be optimized to balance protein yield with proper folding – lower temperatures (16-25°C) and longer induction times often favor proper folding of active protein. During purification, maintaining buffer conditions that preserve protein stability and activity is critical; this typically includes pH optimization (considering purM's native environment in the acidic parasitophorous vacuole), appropriate salt concentration, and the inclusion of reducing agents like DTT or β-mercaptoethanol to maintain cysteine residues in their reduced state. Size exclusion chromatography following initial affinity purification can improve purity and remove aggregates. Activity assays should be established to monitor enzymatic function throughout purification, and storage conditions (buffer composition, temperature, addition of glycerol) should be optimized to maintain long-term stability.

How can researchers effectively evaluate the potential role of purM in oxidative stress response?

To investigate whether purM contributes to oxidative stress defense in Coxiella burnetii, researchers should implement a comprehensive experimental approach similar to that used for studying the bacterioferritin comigratory protein (BCP) . In vitro assays could assess whether purified recombinant purM exhibits peroxidase activity or protects DNA from oxidative damage, as demonstrated for BCP . Complementation studies in oxidative stress-sensitive bacterial mutants (e.g., E. coli strains lacking key oxidative stress response genes) would reveal whether purM can functionally substitute for known stress response proteins . Growth inhibition assays exposing wild-type C. burnetii and purM-deficient strains to various oxidative stressors (hydrogen peroxide, tert-butyl hydroperoxide, etc.) at different concentrations could quantify the contribution of purM to stress resistance. Real-time PCR and Western blot analysis of purM expression under oxidative stress conditions would determine whether the gene is upregulated as part of a stress response. Fluorescence microscopy using oxidative stress-sensitive probes in infected cells could visualize the spatial and temporal dynamics of ROS production in relation to bacterial localization. Finally, transcriptomic and proteomic comparisons between wild-type and purM-deficient C. burnetii during oxidative stress could reveal compensatory mechanisms and associated pathways.

What techniques are most appropriate for studying potential interactions between purM and host cell components?

Investigating interactions between Coxiella burnetii purM and host cell components requires specialized techniques that can detect both direct physical interactions and functional relationships. Bacterial two-hybrid or yeast two-hybrid screens can serve as initial high-throughput approaches to identify potential host protein binding partners. For more direct evidence, co-immunoprecipitation using antibodies against purM followed by mass spectrometry analysis can identify host proteins that physically associate with purM during infection. Proximity labeling techniques such as BioID or APEX2, where purM is fused to a promiscuous biotin ligase, allow for spatially-restricted biotinylation of proximal proteins in living cells, helping identify the protein's neighborhood within the host cell environment. Fluorescence microscopy with appropriately tagged purM can visualize its localization relative to host cell structures during different stages of infection. If purM is secreted or released into the host cell, cellular fractionation followed by Western blotting could track its distribution among different host cell compartments. RNA-seq analysis comparing host cell responses to wild-type versus purM-deficient C. burnetii could reveal host pathways influenced by the protein. Finally, CRISPR screens targeting host genes could identify factors that specifically influence the function or impact of purM during infection.

What are the critical controls needed when studying purM in infection models?

When studying Coxiella burnetii purM in infection models, several critical controls must be implemented to ensure experimental validity and interpretable results. First, genetic complementation controls are essential when using purM mutants or knockdowns – restoring the wild-type gene should reverse any observed phenotypes, confirming that effects are specifically due to purM alteration rather than polar effects or secondary mutations . Positive controls using well-characterized C. burnetii strains with known infection phenotypes (such as T4SS mutants with established attenuation) should be included to validate infection model functionality . When using primary cells like bone marrow-derived macrophages (BMDMs), appropriate polarization controls (e.g., LPS/IFN-γ treatment) should be included to assess cellular activation states independent of bacterial infection . Time-course experiments with multiple sampling points are crucial, as C. burnetii exhibits distinct phase-dependent gene expression patterns, with some genes like bcp showing maximal expression during early exponential growth phase . Host cell viability controls must be monitored throughout experiments to distinguish bacterial effects from cytotoxicity. Finally, when studying potential oxidative stress responses, appropriate oxidative stress-inducing agents should be included as positive controls, and experiments should include measurements of relevant stress markers to confirm the effectiveness of treatments .

How does purM research complement studies on C. burnetii type IV secretion system (T4SS) effectors?

Research on Coxiella burnetii purM can provide valuable complementary insights to studies on the pathogen's type IV secretion system (T4SS) effectors. While T4SS effectors are dedicated virulence factors that enable stealthy colonization of immune cells by modulating host cell functions , purM represents the metabolic machinery that supports bacterial survival and replication within the intracellular niche. This complementarity creates opportunities for integrated research approaches. For instance, analyzing the temporal expression patterns of purM relative to T4SS components during different infection phases could reveal coordination between metabolic adaptation and virulence factor deployment. Investigating whether metabolic enzymes like purM are regulated by the same environmental cues that control T4SS expression might identify common regulatory networks. Additionally, determining whether purM activity is necessary for optimal T4SS function (or vice versa) could reveal functional dependencies between these systems. The different responses of host cells to purM versus T4SS effectors, as measured by transcriptional profiling, could distinguish between host responses to metabolic activities versus dedicated virulence factors . Finally, comparative studies of purM and T4SS effector mutants in infection models such as BMDMs and SCID mice would help establish their relative contributions to different aspects of C. burnetii pathogenesis .

What insights can comparative genomics provide about purM conservation and evolution across Coxiella strains?

Comparative genomics approaches offer powerful insights into the conservation, evolution, and potential functional importance of purM across different Coxiella burnetii strains. Analysis of purM sequence conservation across diverse C. burnetii isolates (such as Nine Mile RSA493, Dugway, and others) can identify core conserved regions likely essential for function versus more variable regions that might reflect strain-specific adaptations . Examination of purM in the context of genomic synteny would reveal whether the gene's chromosomal location and neighboring genes are conserved, potentially indicating functional relationships or co-regulation with adjacent genes. Assessment of selective pressure on purM through calculation of nonsynonymous to synonymous substitution ratios (dN/dS) can determine whether the gene is under purifying selection (suggesting functional conservation) or positive selection (suggesting adaptive evolution). Comparison with homologs from related intracellular pathogens might identify unique features of C. burnetii purM that relate to its specific lifestyle. Investigation of purM variants in strains with different virulence profiles or host specificities could correlate genetic differences with phenotypic variations. Finally, examination of purM in the context of C. burnetii's reductive genome evolution would provide insights into why this gene has been maintained despite the general trend toward genome reduction in obligate intracellular pathogens .

How might purM research contribute to understanding the metabolic adaptations of C. burnetii to the parasitophorous vacuole environment?

Research on Coxiella burnetii purM can significantly advance our understanding of the metabolic adaptations that enable this pathogen to thrive within the harsh environment of the parasitophorous vacuole (PV). The PV presents multiple challenges, including acidic pH (~4.5), potential nutrient limitations, and oxidative stress . As a key enzyme in purine biosynthesis, purM likely plays a critical role in nucleotide metabolism under these specialized conditions. Metabolomic profiling of wild-type versus purM-deficient C. burnetii during intracellular growth could reveal metabolic pathway reconfigurations that compensate for altered purine biosynthesis. Transcriptomic and proteomic analyses comparing purM expression levels in different environmental conditions (varying pH, nutrient availability, oxidative stress) would identify regulatory patterns that might coordinate purine metabolism with other adaptive responses. Investigation of potential moonlighting functions of purM, similar to those discovered for BCP , might reveal additional roles in stress resistance or other adaptive processes. Studies examining the impact of host cell metabolic status on purM expression and activity could uncover metabolic crosstalk between pathogen and host. Integration of purM research with studies on C. burnetii's central carbon metabolism, amino acid utilization, and energy production would provide a more comprehensive view of the pathogen's metabolic adaptation to its unique intracellular niche.

How can systems biology approaches integrate purM function into comprehensive models of C. burnetii pathogenesis?

Systems biology approaches offer powerful frameworks for integrating purM function into holistic models of Coxiella burnetii pathogenesis. Genome-scale metabolic modeling incorporating purM-catalyzed reactions could predict the systemic effects of purM perturbation on bacterial metabolism during infection, identifying potential metabolic vulnerabilities or compensatory pathways. Network analysis integrating transcriptomic, proteomic, and metabolomic data from wild-type and purM-modified bacteria could reveal regulatory connections between purine metabolism and virulence pathways. Agent-based modeling simulating individual bacteria within host cells could predict how purM-dependent processes influence population dynamics and bacterial community behaviors during infection. Multi-omics data integration across different infection time points would provide a dynamic view of how purM-related processes evolve throughout the infection cycle, particularly during the transition from small cell variant (SCV) to large cell variant (LCV) forms . Mathematical modeling of bacterial growth kinetics in response to different environmental stressors could quantify purM's contribution to stress resilience. Finally, comparative systems analysis between C. burnetii and other intracellular pathogens could identify common principles and unique features of metabolic adaptation to intracellular life, placing purM function in a broader evolutionary context.

What are the primary challenges in developing therapeutic strategies targeting purM or its associated pathways?

Developing therapeutic strategies targeting Coxiella burnetii purM faces several significant challenges that researchers must address. First, target validation remains difficult due to the limited genetic manipulation tools available for C. burnetii compared to model organisms, though recent advances in transposon mutagenesis offer improvement . The essential nature of purine biosynthesis for many organisms creates selectivity challenges – therapeutics must preferentially inhibit bacterial purM over human purine biosynthesis enzymes to avoid toxicity. C. burnetii's intracellular lifestyle presents pharmacokinetic barriers, as drugs must penetrate both host cell membranes and the parasitophorous vacuole to reach effective concentrations at the target site. The pathogen's slow growth rate (doubling time of approximately 12 hours) complicates traditional antibiotic susceptibility testing and likely influences the pharmacodynamics of potential inhibitors. C. burnetii's phase variation between small cell variant (SCV) and large cell variant (LCV) forms, with potentially different metabolic activities and permeability properties, may require drugs effective against both morphotypes . Metabolic plasticity and potential redundant pathways might allow the bacterium to bypass inhibited steps through alternative routes. Target site accessibility represents another hurdle, as the three-dimensional structure of C. burnetii purM remains unresolved, complicating structure-based drug design efforts. Finally, the relatively low global incidence of Q fever limits commercial incentives for drug development, though the pathogen's classification as a potential bioterrorism agent maintains interest in therapeutic countermeasures.

What are the key takeaways from current research on C. burnetii purM and related proteins?

Current research on Coxiella burnetii purM and related proteins reveals several important insights relevant to understanding this obligate intracellular pathogen's biology. Metabolic enzymes in C. burnetii, such as the BCP peroxiredoxin, often demonstrate multifunctional capabilities beyond their canonical roles, including DNA-binding activity and protection against oxidative stress—suggesting similar potential for purM to serve multiple functions . The bacterium's adaptation to the harsh environment of the parasitophorous vacuole involves sophisticated metabolic adjustments and stress response mechanisms that likely include purM-dependent pathways . Previous attempts at developing vaccines using recombinant C. burnetii proteins have shown limited success, highlighting the challenges in translating molecular understanding into effective interventions . C. burnetii exhibits complex gene expression patterns during its developmental cycle, with proteins like BCP showing maximal expression during early exponential growth phase—information that should guide temporal considerations in purM research . The pathogen employs multiple strategies to counter oxidative stress, including specialized enzymes that detoxify hydroperoxides, suggesting an integrated stress response network that may involve purM . The field continues to benefit from improved genetic manipulation techniques and cellular infection models, such as primary macrophages, which provide more physiologically relevant contexts for studying C. burnetii proteins including purM . These collective insights emphasize the importance of integrating molecular, cellular, and systems-level approaches to fully understand the role of purM in C. burnetii pathogenesis.