Recombinant Yersinia pseudotuberculosis serotype O:3 Glucokinase (glk)

What is the fundamental role of glucokinase in Yersinia pseudotuberculosis metabolism?

Glucokinase (glk) in Y. pseudotuberculosis, similar to that characterized in other bacterial species like S. aureus, catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6-P), a critical initial step in glucose metabolism. This enzymatic reaction requires ATP as a phosphate donor and represents the entry point of glucose into glycolysis. Based on studies of glucokinase in S. aureus, the enzyme likely demonstrates high affinity for glucose with a Km value potentially similar to the 5.1±0.06mM observed in S. aureus . The production of G-6-P through glucokinase activity likely influences virulence factor expression in Y. pseudotuberculosis, particularly under high glucose conditions, similar to the upregulation of pathogenic factors observed in S. aureus .

How does Y. pseudotuberculosis serotype O:3 differ from other serotypes in terms of genetic expression patterns?

Although specific serotype O:3 differences in Y. pseudotuberculosis aren't directly characterized in the search results, parallels can be drawn from the closely related Y. enterocolitica serotype O:3, which shows unique expression patterns compared to other serotypes. Y. enterocolitica O:3 exhibits constitutive and enhanced invasin production compared to other serotypes, where expression is temperature-regulated and significantly reduced at 37°C . These expression differences result from specific genetic variations, including an IS1667 insertion in the promoter region and a P98S substitution in the regulatory protein RovA . Such serotype-specific variations could similarly exist in Y. pseudotuberculosis O:3 and potentially affect glk expression patterns.

What expression systems are most effective for producing recombinant proteins from Y. pseudotuberculosis?

For optimal expression of recombinant proteins from Y. pseudotuberculosis, plasmid-based expression systems have proven effective. The Asd+ plasmid pSMV13 has successfully been used for high-level synthesis of Y. pestis LcrV antigen in Y. pseudotuberculosis . For expressing recombinant glucokinase, similar plasmid systems could be employed, potentially with modifications to address the specific characteristics of the glk gene. Based on successful approaches with other bacterial enzymes, the glucokinase gene could be cloned into expression vectors like pQE 30 at appropriate restriction sites (e.g., SmaI site) and expressed in E. coli strains such as DH5α .

What are the optimal conditions for cloning and expressing Y. pseudotuberculosis glucokinase in heterologous systems?

Based on successful approaches with similar bacterial enzymes, the following methodological strategy would be recommended:

• Gene amplification: PCR-amplify the glk gene from Y. pseudotuberculosis serotype O:3 genomic DNA using high-fidelity polymerase and primers designed to incorporate appropriate restriction sites.

• Vector selection: Clone the amplified gene into an expression vector that provides:

  • An inducible promoter (e.g., T5 promoter in pQE systems)

  • An affinity tag for purification (6xHis tag has proven effective for S. aureus glucokinase)

  • Appropriate antibiotic resistance markers

• Expression host: Transform the recombinant plasmid into E. coli expression hosts like DH5α, which has been successfully used for S. aureus glucokinase expression .

• Purification strategy: Employ nickel metal chelate chromatography for His-tagged recombinant glucokinase, which has yielded pure enzyme in single-step purification of S. aureus glucokinase .

• Verification: Confirm purity by SDS-PAGE and enzymatic activity using glucose phosphorylation assays.

How does temperature affect the expression and activity of recombinant Y. pseudotuberculosis proteins?

Temperature is a critical regulatory factor for gene expression in Yersinia species. In Y. enterocolitica serotype O:3, temperature affects the expression of virulence factors differently compared to other serotypes, with constitutive invasin production rather than the temperature-dependent regulation seen in other strains . This temperature independence resulted from an IS1667 insertion into the invA promoter region and a P98S substitution in the RovA activator protein that renders it less susceptible to proteolysis at 37°C .

For recombinant Y. pseudotuberculosis glucokinase expression, researchers should consider:

• Optimal growth temperature for expression, which may differ from the temperature of maximal native expression

• Temperature effects on protein folding and solubility

• Temperature-dependent activity of the expressed enzyme

• Potential temperature-dependent regulatory mechanisms affecting the glk promoter

A systematic analysis of expression and activity at different temperatures (25°C, 30°C, and 37°C) would be recommended to determine optimal conditions.

What methodological approaches are most effective for characterizing the kinetic properties of Y. pseudotuberculosis glucokinase?

Based on successful approaches with other bacterial glucokinases, the following methodological workflow would be recommended:

• Substrate affinity determination:

  • Measure enzymatic activity across a range of glucose concentrations (0.1-50 mM)

  • Plot reaction velocity versus substrate concentration

  • Calculate Km and Vmax using appropriate kinetic models

  • Determine if the enzyme exhibits cooperative binding by calculating the Hill coefficient (S. aureus glucokinase showed a Hill coefficient of 1.66±0.032mM, suggesting positive cooperativity)

• Cofactor requirements:

  • Assess activity with different divalent cations (Mg2+, Mn2+, Ca2+)

  • Determine optimal ATP concentration

• Temperature and pH optima:

  • Measure activity across pH range 5.0-9.0

  • Assess activity at temperatures from 25°C to 42°C

• Inhibition studies:

  • Test product inhibition by G-6-P

  • Evaluate potential allosteric regulators

What are the challenges in distinguishing the metabolic effects of recombinant glucokinase from native Y. pseudotuberculosis responses?

This research challenge requires careful experimental design to differentiate between effects mediated by recombinant glucokinase activity versus native bacterial responses. Recommended methodological approaches include:

• Generation of clean knockouts:

  • Create a glk deletion mutant in Y. pseudotuberculosis O:3

  • Complement with wild-type or modified glk under controlled expression

• Transcriptomic analysis:

  • Compare global gene expression patterns between wild-type, glk knockout, and complemented strains

  • Identify differentially regulated pathways dependent on glucokinase activity

• Metabolomic profiling:

  • Measure intracellular concentrations of glucose-6-phosphate and downstream metabolites

  • Correlate metabolite levels with glucokinase activity

• Reporter systems:

  • Develop reporter constructs (e.g., luxCDABE fusions) to monitor expression of genes potentially regulated by glucokinase activity, similar to the approach used to study invA expression in Y. enterocolitica

How can researchers overcome the challenges in producing sufficient quantities of active recombinant Y. pseudotuberculosis glucokinase?

Producing adequate yields of active recombinant glucokinase from Y. pseudotuberculosis may present several challenges. Based on successful approaches with other bacterial enzymes, the following strategies are recommended:

• Optimization of expression conditions:

  • Test multiple expression vectors and promoter systems

  • Evaluate different E. coli expression strains (BL21, Rosetta, Arctic Express)

  • Optimize induction parameters (inducer concentration, temperature, duration)

• Solubility enhancement:

  • Co-express with molecular chaperones if inclusion bodies form

  • Test expression as fusion proteins with solubility tags (MBP, SUMO, TrxA)

  • Optimize buffer compositions during purification

• Purification strategy:

  • Implement a multi-step purification process including:
    a) Affinity chromatography (Ni-NTA for His-tagged protein)
    b) Ion exchange chromatography
    c) Size exclusion chromatography for highest purity

• Activity preservation:

  • Include stabilizing agents (glycerol, reducing agents) in storage buffers

  • Determine optimal storage conditions (temperature, buffer composition)

How does glucokinase activity in Y. pseudotuberculosis potentially influence virulence factor expression?

Based on the understanding that metabolic enzymes can significantly impact virulence in pathogenic bacteria, the following relationships may exist:

• Metabolic regulation of virulence:

  • Glucose-6-phosphate produced by glucokinase activity may serve as a metabolic signal that influences virulence gene expression, similar to how G-6-P formation in S. aureus leads to upregulation of various pathogenic factors

  • Changes in intracellular energy status (ATP/ADP ratio) resulting from glucokinase activity may affect global regulatory networks

• Host environment adaptation:

  • Glucokinase activity may be crucial for bacterial survival in glucose-limited environments within the host

  • The ability to efficiently utilize available glucose through glucokinase activity could enhance Y. pseudotuberculosis persistence during infection

• Potential interaction with virulence mechanisms:

  • Y. pseudotuberculosis implements various virulence strategies, including outer membrane vesicle (OMV) production, which has been demonstrated in recombinant Y. pseudotuberculosis expressing Y. pestis antigens

  • Metabolic status influenced by glucokinase activity may affect OMV production or composition

What experimental models are most appropriate for studying the role of Y. pseudotuberculosis glucokinase in pathogenesis?

To effectively investigate the relationship between Y. pseudotuberculosis glucokinase and pathogenesis, several experimental models would be valuable:

• Cellular infection models:

  • Epithelial cell invasion assays to assess how glucokinase activity affects bacterial entry and survival

  • Macrophage infection models to evaluate intracellular persistence

  • These cellular models could build on approaches used to study Y. enterocolitica adhesion and invasion properties

• Animal infection models:

  • Murine infection models have successfully been used to study Y. enterocolitica colonization factors and protective efficacy of Y. pseudotuberculosis-derived vaccines against Y. pestis

  • Compare colonization, persistence, and disease progression between wild-type and glk mutant strains

  • Assess tissue-specific effects, particularly focusing on splenic and hepatic involvement, which are common sites for Y. pseudotuberculosis abscesses

• Ex vivo tissue models:

  • Human intestinal tissue explants to evaluate tissue tropism and invasion in a more physiologically relevant context

  • Spleen tissue models to investigate mechanisms of abscess formation

How might understanding Y. pseudotuberculosis glucokinase contribute to identifying new therapeutic targets?

Investigations into Y. pseudotuberculosis glucokinase could reveal potential therapeutic targets through several mechanisms:

• Metabolic vulnerability:

  • If glucokinase is essential for Y. pseudotuberculosis survival or virulence in host tissues, it may represent a viable drug target

  • Selective inhibitors of bacterial glucokinase that spare human hexokinases could have therapeutic potential

• Virulence attenuation:

  • Understanding how glucose metabolism through glucokinase affects virulence factor expression could identify regulatory pathways amenable to therapeutic intervention

  • Targeting the connection between metabolism and virulence rather than directly targeting the enzyme might provide alternative approaches

• Biomarker development:

  • Glucokinase activity or expression levels might serve as biomarkers for active Y. pseudotuberculosis infection

  • This could be particularly valuable for identifying serious invasive infections that can lead to bacteremia and splenic abscesses, which carry high mortality rates when untreated

How could recombinant Y. pseudotuberculosis expressing modified glucokinase be utilized in vaccine development?

Building on the successful use of recombinant Y. pseudotuberculosis for vaccine development against Y. pestis , modified glucokinase could potentially be incorporated into vaccine strategies:

• Metabolic attenuation:

  • Engineered Y. pseudotuberculosis with modified glucokinase activity could serve as live attenuated vaccine strains with controlled growth and immunogenicity

  • Such strains might retain immunogenicity while demonstrating reduced virulence

• Antigen delivery system:

  • Recombinant Y. pseudotuberculosis strains engineered to overproduce outer membrane vesicles (OMVs) through metabolic modifications involving glucokinase could serve as efficient antigen delivery vehicles

  • This approach builds on the demonstrated success of Y. pseudotuberculosis OMVs containing Y. pestis LcrV antigen, which afforded complete protection against both pulmonary and subcutaneous Y. pestis infection

• Combination approaches:

  • Integration of metabolic engineering (through glucokinase modification) with antigen expression could generate multivalent vaccine candidates

  • A systematic approach similar to that used in developing the recombinant Y. pseudotuberculosis vaccine against Y. pestis could be implemented