Recombinant Staphylococcus aureus 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial

What is phosphoglycerate mutase and what is its role in S. aureus metabolism?

Phosphoglycerate mutase (PGAM) catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic pathway. In S. aureus, this enzyme exists in two forms: GpmI (manganese-dependent) and GpmA (metal-independent, using 2,3-bisphosphoglycerate as a cofactor). This reaction represents a critical step in glycolysis, allowing the bacterium to metabolize glucose for energy production and biosynthetic precursors. GpmI appears to be the primary phosphoglycerate mutase used by S. aureus under normal growth conditions, while GpmA becomes critical during manganese limitation .

How does S. aureus benefit from having two different phosphoglycerate mutase isozymes?

S. aureus benefits from this isozyme redundancy through metabolic flexibility during infection. The metal-dependent GpmI is the primary enzyme under normal conditions, but when faced with host-imposed manganese limitation (a form of nutritional immunity), S. aureus can rely on the metal-independent GpmA. This adaptation is crucial for bacterial survival, as glucose consumption is essential during infection but increases cellular manganese demand. By expressing a metal-independent variant, S. aureus can continue glycolysis while mitigating the increased manganese requirements. Research has shown that loss of GpmA reduces the ability of S. aureus to cause invasive disease in wild-type mice but not in calprotectin-deficient mice, indicating that GpmA specifically helps overcome manganese limitation during infection .

What is the genomic organization of gpmI and gpmA in S. aureus?

GpmI is encoded within the glycolytic operon that contains several other glycolytic enzymes, including gapR, gapA, pgk, tpiA, and eno. This genomic organization suggests coordinated expression with other glycolytic enzymes. In contrast, gpmA is not part of an operon and is expressed independently of other glycolytic enzymes. This independent expression may allow for specific regulation of GpmA in response to environmental conditions, particularly metal limitation .

What is the difference between GpmI and GpmA in terms of structure and function?

The fundamental difference between these isozymes lies in their cofactor requirements. GpmI is manganese-dependent, requiring this metal ion for catalytic activity. In contrast, GpmA is metal-independent and uses 2,3-bisphosphoglycerate as a catalytic cofactor. Despite catalyzing the same reaction, these enzymes likely have distinct structural features that account for their different cofactor dependencies. Both enzymes catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate, but their expression patterns and importance differ depending on environmental conditions .

How is the expression of gpmI and gpmA regulated in S. aureus?

Research indicates differential regulation of these isozymes, particularly in response to manganese availability. In manganese-limited conditions, such as during S. aureus infection in the presence of calprotectin (a host manganese-binding protein), gpmA expression is upregulated. Studies have shown that expression of gpmA increases approximately four-fold in a S. aureus mutant lacking the manganese transporters MntABC and MntH (ΔmntCΔmntH), similar to the increase observed in the presence of calprotectin. In contrast, gpmI expression remains unchanged in the manganese transport mutant, suggesting that GpmI remains the primary phosphoglycerate mutase under normal conditions while GpmA expression is specifically induced during manganese limitation .

What alternative methods exist for studying phosphoglycerate mutase activity in S. aureus?

Several approaches can be used to study phosphoglycerate mutase activity:

• Enzymatic assays: Measuring the interconversion of 2-phosphoglycerate and 3-phosphoglycerate using coupled enzyme assays.

• Growth curve analysis: Comparing growth of wild-type and isozyme-deficient mutants under various conditions.

• Metabolite feeding experiments: Supplementing growth media with metabolites that bypass specific glycolytic steps to identify the affected pathway.

• Metal depletion studies: Using calprotectin or metal chelators to impose manganese limitation.

• In vivo infection models: Using wild-type and calprotectin-deficient mice to assess the role of GpmI and GpmA during infection.

Metabolite feeding experiments have been particularly valuable, demonstrating that the growth defect of ΔgpmA mutants under manganese-limited conditions can be rescued by providing pyruvate, which bypasses the phosphoglycerate mutase step in glycolysis .

What experimental approaches can be used to study GpmI function in metal-limited conditions?

Several sophisticated experimental approaches can be employed:

Research has successfully used calprotectin binding site mutants with altered metal-binding properties to demonstrate that the increased sensitivity of ΔgpmA mutants is specifically due to manganese limitation. The ΔS2 mutant, which can bind either manganese or zinc, increased sensitivity of ΔgpmA, while the ΔS1 mutant, which cannot bind manganese, abrogated this sensitivity .

How does the host's nutritional immunity affect GpmI function during infection?

Host nutritional immunity, particularly the sequestration of manganese by calprotectin, directly impacts GpmI function. During infection, host cells produce calprotectin, which binds manganese with high affinity, creating a manganese-limited environment. Since GpmI is manganese-dependent, its activity is compromised under these conditions. This creates selective pressure for S. aureus to utilize GpmA, the metal-independent isozyme. Research has demonstrated that S. aureus relies on GpmA for continued glycolysis during infection, as evidenced by the attenuated virulence of ΔgpmA mutants in wild-type mice but not in calprotectin-deficient mice. This suggests that the interplay between host nutritional immunity and bacterial metabolic adaptation is a critical aspect of S. aureus pathogenesis .

What structural differences between GpmI and GpmA account for their different metal dependencies?

While the specific structural differences remain an area of active research, key distinctions likely exist in their active sites and catalytic mechanisms. GpmI belongs to the family of metal-dependent phosphoglycerate mutases that utilize a metal ion (typically manganese) to coordinate the phosphate group during catalysis. In contrast, GpmA belongs to the 2,3-bisphosphoglycerate-dependent family, which uses 2,3-bisphosphoglycerate as a cofactor and follows a different catalytic mechanism involving a phosphohistidine intermediate. These fundamental differences in catalytic mechanism necessitate distinct active site architectures. Structural biology approaches, including X-ray crystallography and cryo-electron microscopy, would be valuable for elucidating these differences and potentially informing selective inhibitor design .

What are the best methods for recombinant expression and purification of GpmI?

For recombinant expression and purification of S. aureus GpmI, a systematic approach is recommended:

• Expression system selection: E. coli BL21(DE3) is typically used for heterologous expression of S. aureus proteins. Consider using a strain optimized for expression of proteins from low-GC organisms.

• Vector construction: Insert the gpmI gene into an expression vector with an appropriate tag (His-tag or GST-tag) for purification. The pET system is commonly used for high-level expression.

• Induction conditions: Optimize temperature (often 16-25°C for improved solubility), IPTG concentration (typically 0.1-1 mM), and induction time (4-16 hours).

• Lysis and initial purification: Use buffer containing:

  • 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 150-300 mM NaCl

  • 10% glycerol as a stabilizer

  • Protease inhibitors

  • Critical: Include 1-5 mM MnCl₂ to ensure proper folding and stability of GpmI

• Affinity chromatography: Use Ni-NTA for His-tagged proteins or glutathione sepharose for GST-tagged proteins.

• Additional purification: Size exclusion chromatography to remove aggregates and obtain pure, homogeneous protein.

• Storage: Store with 10% glycerol at -80°C; avoid repeated freeze-thaw cycles.

Maintaining manganese in buffers throughout purification is essential for preserving GpmI activity .

How can GpmI activity be measured in vitro?

GpmI activity can be measured using several established methods:

• Direct assay: Measure the conversion of 3-phosphoglycerate to 2-phosphoglycerate using ion-exchange chromatography or mass spectrometry.

• Coupled enzymatic assay: Link GpmI activity to subsequent reactions in glycolysis:

  • 3-phosphoglycerate → 2-phosphoglycerate (GpmI)

  • 2-phosphoglycerate → phosphoenolpyruvate (enolase)

  • Phosphoenolpyruvate → pyruvate (pyruvate kinase) + ATP

  • ATP + glucose → glucose-6-phosphate + ADP (hexokinase)

  • Glucose-6-phosphate + NADP⁺ → 6-phosphogluconolactone + NADPH (G6PDH)

  • Monitor NADPH formation spectrophotometrically at 340 nm

• Metal dependency characterization: Perform activity assays with varying concentrations of manganese and other divalent metals to determine specificity.

• Kinetic analysis: Determine Km and Vmax values for both forward and reverse reactions under various conditions.

• Inhibition studies: Assess the effect of potential inhibitors on enzyme activity.

All assays should include appropriate controls, including heat-inactivated enzyme and reactions lacking substrate .

What experimental models are appropriate for studying the role of GpmI in S. aureus pathogenesis?

Several complementary experimental models can be used:

• In vitro cellular models:

  • Human neutrophil co-culture to assess bacterial survival during phagocytosis

  • Macrophage infection models to study intracellular survival

  • Epithelial cell adhesion and invasion assays

• Ex vivo tissue models:

  • Human skin explants for studying skin infection

  • Whole blood survival assays to assess resistance to innate immunity

• In vivo animal models:

  • Murine systemic infection: Intravenous injection to assess dissemination and organ colonization

  • Skin abscess model: Subcutaneous injection to study localized infection

  • Specialized models comparing wild-type and calprotectin-deficient mice to specifically assess the impact of metal limitation

• Genetic approaches:

  • Isogenic mutants lacking gpmI, gpmA, or both

  • Complemented strains expressing wild-type or modified versions of these enzymes

  • Conditional expression systems to control enzyme levels during different infection stages

Studies have successfully used wild-type and calprotectin-deficient mice to demonstrate that GpmA specifically contributes to overcoming manganese limitation during infection, providing a powerful model for studying the interplay between bacterial metabolism and host nutritional immunity .

How should kinetic data for GpmI be analyzed?

Kinetic data for GpmI should be analyzed using rigorous enzyme kinetics approaches:

• Initial rate determination: Ensure measurements are made during the linear phase of the reaction.

• Michaelis-Menten kinetics:

  • Plot initial velocity versus substrate concentration

  • Fit data to the Michaelis-Menten equation: v = (Vmax × [S]) / (Km + [S])

  • Determine Km and Vmax using non-linear regression

  • Consider using linearization methods (Lineweaver-Burk, Eadie-Hofstee) as complementary approaches

• Metal dependency analysis:

  • Plot activity versus metal concentration

  • Determine EC50 for manganese and other relevant metals

  • Consider cooperative binding effects using the Hill equation if appropriate

• pH and temperature optima:

  • Plot activity versus pH or temperature

  • Determine optimal conditions and stability ranges

• Statistical validation:

  • Perform experiments in triplicate at minimum

  • Calculate standard error/deviation

  • Use appropriate statistical tests to compare conditions

• Data visualization:

  • Create clear, labeled graphs with appropriate scales

  • Include error bars representing variation

  • Consider using heat maps for complex datasets with multiple variables

Proper controls should include reactions without enzyme, without substrate, and with heat-inactivated enzyme to account for background reactions .

What are common pitfalls in interpreting GpmI activity data?

Researchers should be aware of several potential pitfalls:

• Overlooking metal contamination: Trace amounts of manganese in buffers or from labware can affect results, especially in metal dependency studies. Use high-purity reagents and consider treating buffers with chelators followed by thorough rinsing.

• Ignoring the reversible nature: Phosphoglycerate mutase catalyzes a reversible reaction, so the direction of the reaction depends on substrate concentrations. Ensure experimental conditions favor the desired direction.

• Misinterpreting in vivo phenotypes: Growth defects in mutants may result from polar effects on adjacent genes rather than the specific function of GpmI. Always confirm with complementation studies.

• Neglecting metabolic context: GpmI functions within a metabolic network, so altered activity may affect upstream and downstream reactions. Consider metabolomics approaches to gain a comprehensive view.

• Disregarding the dual-isozyme system: When studying GpmI, consider the potential compensatory role of GpmA, especially under metal-limited conditions.

• Enzyme stability issues: Manganese-dependent enzymes may lose activity during purification or storage. Include fresh manganese in reaction buffers and verify enzyme activity regularly.

• Overlooking environmental variables: Temperature, pH, and ionic strength can significantly affect enzyme activity and should be carefully controlled and reported .

How can contradictory findings about GpmI function be reconciled?

Contradictory findings regarding GpmI function can be reconciled through several approaches:

• Strain-specific differences: Different S. aureus strains may show varying dependency on GpmI versus GpmA. Always report the specific strain used and consider testing key findings in multiple strains.

• Experimental condition variations: Small differences in metal availability, growth media, oxygen levels, or growth phase can lead to contradictory results. Standardize conditions and report them in detail.

• Methodology differences: Different assay methods may yield conflicting results. Use multiple complementary approaches to verify key findings.

• Genetic background effects: Mutations in one gene may have different phenotypes depending on the genetic background. Consider whole-genome sequencing to identify potential modifiers.

• Compensatory adaptations: Bacteria may adapt to gene deletions through compensatory mutations or metabolic rewiring. Use acute gene inhibition or conditional expression systems to minimize adaptation.

• Verification through multiple approaches:

  • In vitro biochemical assays

  • Genetic complementation

  • Metabolomics analysis

  • In vivo infection models

• Meta-analysis: When possible, perform systematic reviews or meta-analyses of published data to identify patterns and sources of variation .

What are the major gaps in our understanding of GpmI function in S. aureus?

Despite significant advances, several knowledge gaps remain:

• Structural basis of metal specificity: Detailed structural information on GpmI and how it binds manganese remains limited. Structural studies would enhance our understanding of metal dependency.

• Regulatory mechanisms: How S. aureus coordinates the expression of GpmI and GpmA in response to changing metal availability requires further investigation.

• Metabolic integration: The broader metabolic consequences of shifting between GpmI and GpmA during infection are not fully understood.

• Host-pathogen interface: The spatial and temporal dynamics of manganese availability during different stages of infection need further characterization.

• Therapeutic targeting: The potential of GpmI as a target for antimicrobial development remains largely unexplored.

Future research should address these gaps through interdisciplinary approaches combining structural biology, genetics, metabolomics, and infection models .

How might GpmI research contribute to addressing antibiotic resistance in S. aureus?

Research on GpmI has significant potential to address the growing challenge of antibiotic resistance in S. aureus:

• Novel therapeutic targets: As a critical metabolic enzyme with a different cofactor requirement than human phosphoglycerate mutase, GpmI represents a potential target for selective inhibition.

• Combination therapies: Inhibitors targeting GpmI could be combined with conventional antibiotics to enhance efficacy or overcome resistance mechanisms.

• Host-directed therapeutics: Understanding how S. aureus adapts to host-imposed manganese limitation could lead to strategies that enhance this aspect of nutritional immunity.

• Metabolic vulnerabilities: Research on GpmI and GpmA reveals metabolic adaptations that could be exploited to create new vulnerabilities in drug-resistant strains.

• Biomarker development: Knowledge of how S. aureus regulates these isozymes could lead to biomarkers for monitoring infection progression or treatment efficacy.

With the increasing prevalence of multidrug-resistant S. aureus strains, including MRSA, VRSA, and MDRSA, novel approaches based on metabolic targeting rather than conventional mechanisms could help address this urgent public health challenge .

What technologies will advance our understanding of phosphoglycerate mutase isozymes?

Emerging technologies that will drive future advances include:

• CRISPR-Cas9 genome editing: Precise genetic manipulation to create isozyme variants with altered properties or regulated expression.

• Single-cell techniques: Analyzing isozyme expression and activity at the single-cell level to understand population heterogeneity during infection.

• Advanced imaging: Using fluorescent reporters and microscopy to visualize metal availability and enzyme activity in real-time during infection.

• Structural biology advances: Cryo-electron microscopy and X-ray crystallography to determine high-resolution structures of GpmI and GpmA.

• Systems biology approaches: Integrating transcriptomics, proteomics, and metabolomics data to understand the system-wide impact of isozyme switching.

• Microfluidics: Creating controlled environments to study bacterial adaptation to dynamic changes in metal availability.

• Computational modeling: Predicting the metabolic consequences of altered isozyme expression and identifying potential vulnerabilities.