Recombinant Yersinia pseudotuberculosis serotype O:1b GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the functional role of GMP synthase (guaA) in Yersinia pseudotuberculosis?

GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2) catalyzes the amination of xanthosine 5'-monophosphate (XMP) to form guanosine monophosphate (GMP) in the presence of glutamine and ATP. This reaction is a critical step in the de novo biosynthesis of guanine nucleotides, which are essential for DNA replication, RNA synthesis, and numerous cellular signaling pathways.

The enzyme contains two functional domains that work in concert:

• The glutaminase domain: Responsible for hydrolyzing glutamine to produce the necessary amino group

• The synthetase domain: Responsible for ATP hydrolysis and the formation of GMP

The reaction can be summarized as follows:
XMP + glutamine + ATP → GMP + glutamate + AMP + PPi

Methodologically, researchers studying this enzyme frequently employ genetic knockout studies followed by complementation with recombinant guaA to determine its essentiality for bacterial growth and virulence .

What expression systems are typically used to produce recombinant Y. pseudotuberculosis guaA?

Several expression systems have proven effective for producing recombinant Yersinia proteins, including guaA:

• E. coli expression systems: Protease-deficient E. coli strains like BL21(DE3) or BL21(DE3)pLysS are commonly used for recombinant Yersinia protein expression. These strains contain a copy of the T7 polymerase gene under an IPTG-inducible promoter, allowing controlled expression . For optimal results, the guaA gene can be cloned into expression vectors such as pET21a.

• Mammalian cell expression: For applications requiring specific post-translational modifications or improved solubility, mammalian cell expression systems have been successfully used to produce recombinant Y. pseudotuberculosis proteins .

A typical expression and purification protocol involves:

• Cloning the guaA gene into an appropriate expression vector

• Transforming the construct into expression host cells

• Inducing protein expression with IPTG (for E. coli)

• Harvesting cells and lysing to release recombinant protein

• Purification using affinity chromatography (typically Ni²⁺ chelation for His-tagged proteins)

• For proteins purified under denaturing conditions, refolding through step dialysis against decreasing concentrations of urea (3M, 1M, 0.5M) and finally phosphate-buffered saline

How can I confirm the purity and identity of recombinant Y. pseudotuberculosis guaA?

Multiple complementary techniques should be employed to confirm both purity and identity:

Purity assessment:

• SDS-PAGE with Coomassie blue staining (target purity >85%)

• Size exclusion chromatography to detect aggregates or impurities

• Endotoxin testing if the protein will be used in immunological studies

Identity confirmation:

• Western blotting using anti-His antibodies (for His-tagged protein) or specific antibodies

• Mass spectrometry for peptide mass fingerprinting

• N-terminal sequencing by Edman degradation

• Enzymatic activity assays to confirm functional identity

Functional verification:

• Glutaminase activity: Measure glutamate production using glutamate dehydrogenase-coupled assays

• ATP hydrolysis: Measure inorganic phosphate release using malachite green assays

• GMP formation: Quantify using HPLC or coupled enzyme assays

For long-term storage, the purified protein should be stored at -20°C or -80°C, preferably with 5-50% glycerol as a cryoprotectant. Repeated freeze-thaw cycles should be avoided to maintain enzymatic activity .

How does the structure of Y. pseudotuberculosis guaA compare to homologous proteins?

The structure of Y. pseudotuberculosis guaA follows the conserved architecture of GMP synthases across bacterial species, with two distinct functional domains:

• The N-terminal glutaminase domain containing the glutamine-binding site and catalytic residues responsible for glutamine hydrolysis

• The C-terminal synthetase domain containing the ATP-binding and XMP-binding sites

Key structural features include:

• A conserved cysteine residue (equivalent to Cys104 in human GMP synthase) that is critical for glutamine hydrolysis and is the target of irreversible inhibitors like acivicin

• ATPase domain with conserved motifs for nucleotide binding

• XMP binding pocket in the synthetase domain

Methodologically, structural information is typically obtained through X-ray crystallography, cryo-electron microscopy, or homology modeling based on related proteins with known structures.

How can recombinant Y. pseudotuberculosis guaA be used in studying bacterial pathogenesis?

Recombinant Y. pseudotuberculosis guaA serves as a powerful tool for investigating multiple aspects of bacterial pathogenesis:

1. Drug target validation:

• High-throughput screening of potential inhibitors against purified recombinant guaA

• Structure-activity relationship studies to optimize lead compounds

• Correlation of in vitro inhibition with antimicrobial effects in cell culture

2. Host-pathogen interaction studies:

• Tracking labeled recombinant guaA to identify potential interactions with host factors

• Investigating immunomodulatory effects of guaA on host cells

• Determining whether guaA is recognized by the host immune system during infection

3. Environmental persistence studies:
Recent research has demonstrated that Y. pseudotuberculosis can colonize and persist in entomopathogenic nematodes (EPNs), with an average of 5.0 × 10³ CFUs per nematode after the first infection cycle, and similar counts maintained through multiple infection cycles . This surprising finding suggests EPNs may serve as environmental reservoirs for this pathogen.

Table 1: Persistence of Y. pseudotuberculosis in entomopathogenic nematodes across multiple infection cycles

Recombinant guaA could be used to investigate whether nucleotide metabolism plays a role in this ecological behavior by:

• Comparing wild-type and enzymatically inactive guaA variants in colonization assays

• Determining if guaA is upregulated during EPN colonization

• Testing whether guaA inhibitors can prevent colonization of these environmental reservoirs

What methodological challenges exist in purifying functional recombinant Y. pseudotuberculosis guaA?

Purifying functional recombinant Y. pseudotuberculosis guaA presents several methodological challenges that must be addressed:

1. Protein solubility issues:

• Recombinant bacterial proteins often form inclusion bodies in E. coli

• Strategies to improve solubility include:

  • Lower induction temperature (16-25°C)

  • Reduced inducer concentration (0.1-0.5 mM IPTG)

  • Use of solubility-enhancing fusion tags (SUMO, MBP)

  • Co-expression with molecular chaperones

2. Maintaining dual enzymatic activities:
GMP synthase possesses two distinct catalytic activities (glutaminase and synthetase) that must both be preserved during purification. Research on human GMP synthase has shown that:

• Inorganic pyrophosphate inhibits the synthetase activity and uncouples the two domain functions

• Acivicin, a glutamine analog, selectively abolishes the glutaminase activity without affecting the synthetase activity when ammonia is used as the amino donor

These findings can guide the development of activity assays to verify functional integrity of both domains separately.

3. Protein stability considerations:
For optimal stability during storage:

• Store at -20°C for short-term or -80°C for extended storage

• Add glycerol (5-50% final concentration) as a cryoprotectant

• Avoid repeated freeze-thaw cycles

• Aliquot protein solutions before freezing

4. Purification strategy options:
Based on successful purification strategies for other Yersinia proteins, the following approaches may be effective for guaA:

• For denatured protein: Ni²⁺ chelation chromatography under denaturing conditions (6 M urea) followed by step dialysis

• For native protein: HPLC ion exchange followed by size-exclusion chromatography under non-denaturing conditions, as successfully applied to YopH (another Yersinia protein)

How does guaA contribute to Y. pseudotuberculosis virulence and ecological persistence?

The guaA enzyme plays multiple roles in Y. pseudotuberculosis virulence and ecological persistence:

1. Contribution to bacterial fitness:

• GMP synthesis is essential for nucleic acid production during rapid bacterial replication

• Guanine nucleotides serve as signaling molecules in bacterial stress responses

• Metabolic adaptation through regulation of guaA may help the bacterium survive in diverse environments

2. Environmental persistence mechanisms:
Recent research has revealed that Y. pseudotuberculosis can colonize entomopathogenic nematodes (EPNs) and persist through multiple infection cycles . This remarkable finding has significant implications:

• Y. pseudotuberculosis colonizes the gastrointestinal tract of EPNs and displays bright GFP fluorescence throughout the gut

• The bacteria maintain viability during long-term EPN storage, with bacterial counts remaining stable across multiple infection cycles

• This colonization represents a potential environmental reservoir for Y. pseudotuberculosis that might contribute to its persistence in endemic areas

3. Relevance to related pathogens:
The ability of Y. pseudotuberculosis to colonize nematodes has implications for other pathogens:

• Y. pestis, the causative agent of plague, evolved relatively recently from Y. pseudotuberculosis

• If Y. pestis shares this ability to colonize nematodes, it could provide new insights into the long-term persistence of plague in endemic areas worldwide

4. Methodological approaches to study these connections:

• Construction of guaA knockout or conditional mutants

• Complementation studies with wild-type or mutant recombinant guaA

• In vitro and in vivo infection models to assess virulence

• EPN colonization assays to evaluate environmental persistence

How can structural analysis of Y. pseudotuberculosis guaA inform antimicrobial drug design?

Structural analysis of Y. pseudotuberculosis guaA provides crucial insights for rational drug design targeting this pathogen:

1. Key structural features for targeting:

• The glutaminase domain contains a critical cysteine residue (equivalent to Cys104 in human GMP synthase) involved in glutamine hydrolysis

• The ATP binding site in the synthetase domain represents another potential target

• The interface between the two domains may contain allosteric sites for inhibitor binding

2. Structure-based inhibitor development approaches:

• Virtual screening of compound libraries against identified binding pockets

• Fragment-based drug discovery focusing on high-affinity binding fragments

• Structure-activity relationship studies to optimize lead compounds

• Crystallography of enzyme-inhibitor complexes to verify binding modes

3. Targeting strategies based on mechanistic insights:
Research on human GMP synthase has revealed that:

• Acivicin, a glutamine analog, irreversibly inhibits GMP synthase by covalently modifying the catalytic cysteine residue

• Enzyme inactivation is facilitated by the presence of substrates

• Inhibition of the glutaminase domain can be bypassed when ammonia is used as an alternative amino donor

These mechanistic insights can guide the development of more potent and selective inhibitors for the bacterial enzyme.

4. Selective targeting strategies:

• Identify structural differences between bacterial and human GMP synthases

• Design inhibitors that selectively target the bacterial enzyme

• Exploit bacterial-specific binding pockets or conformational states

• Develop compounds with limited penetration into mammalian cells

How can multi-omics data be integrated to understand guaA expression and regulation?

The recently developed Yersiniomics database provides a valuable resource for multi-omics analysis of Y. pseudotuberculosis guaA:

1. Yersiniomics database resources:
This comprehensive database contains:

• 200 genomic datasets

• 317 transcriptomic datasets

• 62 proteomic datasets for Yersinia species

The platform includes several integrated tools for data analysis:

• Omics browsers ("genomics," "transcriptomics," and "proteomics")

• Genome viewer and heatmap viewer for transcriptomics and proteomics results

• Gene viewer for accessing associated omics data

2. Integration methodologies:
To effectively integrate multi-omics data for guaA analysis:

a) Comparative genomics approach:

• Extract guaA sequences across Yersinia species

• Analyze sequence conservation and polymorphisms

• Identify regulatory elements in promoter regions

b) Transcriptome analysis:

• Compare guaA expression levels across different growth conditions

• Identify co-expressed genes that may function in related pathways

• Determine environmental triggers that alter guaA expression

c) Proteome analysis:

• Quantify GuaA protein abundance across conditions

• Identify post-translational modifications

• Map protein-protein interactions involving GuaA

d) Correlation analysis:

• Calculate correlation coefficients between transcriptomic and proteomic data

• Identify conditions where transcript and protein levels are discordant

• Infer potential post-transcriptional regulatory mechanisms

3. Research applications:
This integrated approach can address several key questions:

• How does guaA expression change during infection versus environmental persistence?

• Are there differences in guaA regulation between pathogenic and non-pathogenic Yersinia species?

• What environmental signals trigger changes in guaA expression?

• How does guaA expression correlate with virulence factor expression?

What experimental approaches can be used to study the interaction between Y. pseudotuberculosis guaA and host immune responses?

Investigating the interaction between Y. pseudotuberculosis guaA and host immune responses requires sophisticated experimental approaches:

1. Immunogenicity assessment:

• Stimulate dendritic cells or macrophages with purified recombinant guaA

• Measure cytokine production using ELISA or multiplex assays

• Analyze immune cell activation markers by flow cytometry

• Identify pattern recognition receptors engaged by guaA

2. Adaptive immune response characterization:

• Conduct T cell proliferation assays using recombinant guaA as antigen

• Analyze T cell polarization through cytokine profiling

• Measure antibody responses in infection models

• Map immunodominant epitopes using peptide arrays

3. Protection studies:
Research with recombinant Yersinia outer proteins (Yops) has demonstrated that:

• Recombinant proteins can be used to vaccinate mice

• Protection levels can be assessed following challenge with virulent Y. pestis

• Mouse hyperimmune serum generated with recombinant Yersinia proteins reacts with native bacterial proteins

Similar approaches could be applied to determine whether guaA can elicit protective immune responses against Y. pseudotuberculosis infection.

4. Functional immunomodulation studies:

• Assess the impact of recombinant guaA on phagocytosis capacity

• Determine effects on antimicrobial responses (ROS, NO production)

• Investigate potential interference with host signaling pathways

• Evaluate impact on antigen presentation mechanisms

5. Methodological considerations:

• Ensure recombinant protein preparations are free of endotoxin contamination

• Include proper controls for non-specific protein effects

• Verify that recombinant protein retains native conformation

• Consider using both human and murine immune cells for cross-species comparison