Recombinant Mycoplasma mycoides subsp. mycoides SC 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial

What is gpmI and how does it differ from other phosphoglycerate mutases?

GpmI (2,3-bisphosphoglycerate-independent phosphoglycerate mutase) catalyzes the reversible conversion of 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG) during glycolysis without requiring the 2,3-bisphosphoglycerate cofactor. Unlike the 2,3-bisphosphoglycerate-dependent phosphoglycerate mutases (dPGAMs) found in mammals, gpmI utilizes a different catalytic mechanism involving a phosphohistidine intermediate. This distinction is particularly significant as it represents a potential target for antimicrobial development, given that the structural and mechanistic differences between bacterial gpmI and mammalian dPGAMs could allow for selective inhibition without affecting host enzymes .

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

Recombinant gpmI from Mmm SC is typically expressed in Escherichia coli expression systems using vectors that contain strong inducible promoters (such as T7). The general methodology involves:

• Cloning the gpmI gene into an expression vector with an appropriate tag (His-tag is commonly used)

• Transforming the construct into a suitable E. coli strain (e.g., BL21(DE3))

• Inducing protein expression with IPTG or other inducers

• Lysing cells and purifying the recombinant protein using affinity chromatography

• Confirming purity using SDS-PAGE and Western blotting

• Verifying enzymatic activity through spectrophotometric assays

Optimization of expression conditions (temperature, induction time, and concentration) is critical for obtaining active enzyme, as is the inclusion of appropriate protease inhibitors during purification to prevent degradation .

How can recombinant gpmI be utilized in vaccine development against CBPP?

Recombinant gpmI represents a potential subunit vaccine candidate against CBPP, particularly within a reverse vaccinology approach. When developing a gpmI-based vaccine component, researchers should consider:

• Immunogenicity assessment: Determine if gpmI elicits a strong immune response in cattle by measuring specific antibody titers and cellular immune responses

• Epitope mapping: Identify immunodominant regions of gpmI that could be included in subunit vaccines

• Formulation optimization: Test various adjuvants and delivery systems to enhance immunogenicity

• Protection studies: Evaluate the protective efficacy of gpmI alone or in combination with other antigens

Research has shown that using grouped recombinant Mmm proteins for immunization can provide protection against CBPP challenge. In specific trials, groups of five proteins showed significant protection, with some groups preventing the recovery of viable Mmm from lung specimens after challenge with the Mmm strain Afadé . When designing a vaccine containing gpmI, it's crucial to consider potential synergistic effects with other antigens and to evaluate both humoral and cell-mediated immune responses.

What experimental approaches can be used to investigate the role of gpmI in Mmm SC pathogenicity?

Investigating the role of gpmI in Mmm SC pathogenicity requires multi-faceted experimental approaches:

• Gene knockout/knockdown studies: Generate gpmI-deficient Mmm SC strains and assess changes in virulence in cell culture and animal models

• Site-directed mutagenesis: Create point mutations in catalytic residues to determine structure-function relationships

• In vitro cytotoxicity assays: Compare cytotoxic effects of wild-type and gpmI-modified strains on bovine epithelial cells

• Adhesion assays: Assess whether gpmI affects the ability of Mmm SC to adhere to host cells

• Metabolomic analyses: Compare metabolite profiles between wild-type and gpmI-modified strains

• Transcriptomic studies: Examine changes in gene expression patterns associated with gpmI activity

• Protein-protein interaction studies: Identify potential interaction partners of gpmI that might contribute to pathogenicity

How does gpmI activity relate to the production of virulence factors in Mmm SC?

The relationship between gpmI activity and virulence factor production in Mmm SC involves complex metabolic interconnections:

• Energy provision: GpmI's role in glycolysis ensures adequate ATP production, which is necessary for the synthesis and secretion of virulence factors

• Metabolic intermediate generation: The glycolytic intermediates produced through pathways involving gpmI may serve as precursors for virulence factor biosynthesis

• Redox balance: GpmI activity influences the NAD+/NADH ratio, potentially affecting oxidative stress responses and virulence factor regulation

• Potential regulatory functions: Beyond its catalytic role, gpmI might have moonlighting functions that influence gene expression of virulence factors

What techniques are most effective for measuring gpmI enzymatic activity in vitro?

Several complementary techniques can be employed to accurately measure gpmI enzymatic activity:

Spectrophotometric Coupled Assays:

• Forward reaction (3-PG → 2-PG): Couple with enolase, pyruvate kinase, and lactate dehydrogenase, monitoring NADH oxidation at 340 nm

• Reverse reaction (2-PG → 3-PG): Couple with phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase, monitoring NADH formation

Direct Assays:

• Nuclear Magnetic Resonance (NMR): Monitor the conversion between 3-PG and 2-PG in real-time

• High-Performance Liquid Chromatography (HPLC): Separate and quantify 3-PG and 2-PG directly

Kinetic Parameter Determination:
The table below summarizes typical kinetic parameters for gpmI activity measurement:

When performing these assays, it's essential to control for potential interfering activities from contaminating enzymes and to ensure substrate purity .

How can researchers address contradictory findings regarding gpmI function in different experimental systems?

Addressing contradictory findings regarding gpmI function requires systematic troubleshooting and experimental design considerations:

• Standardize experimental conditions:

  • Use consistent protein expression and purification protocols

  • Establish uniform assay conditions (pH, temperature, buffer composition)

  • Validate reagent purity and stability

• Cross-validation approaches:

  • Apply multiple independent techniques to measure the same parameter

  • Collaborate with other laboratories to replicate key findings

  • Use both in vitro and in vivo systems to validate observations

• Consider strain and genetic background differences:

  • Compare gpmI sequences and expression levels across Mmm SC strains

  • Document genetic modifications in laboratory strains

  • Assess potential compensatory mechanisms in different genetic backgrounds

• Data integration strategies:

  • Develop mathematical models to reconcile apparently conflicting data

  • Consider context-dependent effects that might explain discrepancies

  • Examine potential post-translational modifications affecting gpmI function

• Technical considerations:

  • Assess enzyme stability under different storage and assay conditions

  • Validate antibody specificity for immunological studies

  • Consider the impact of tags and fusion proteins on enzyme function

When contradictory findings persist, systematic meta-analysis and collaborative research efforts are recommended to identify variables that might account for differences in experimental outcomes .

What considerations are important when designing experiments to study gpmI in the context of host-pathogen interactions?

When studying gpmI in host-pathogen interactions, researchers should consider several key experimental design elements:

• Physiological relevance:

  • Use appropriate bovine cell types (respiratory epithelial cells, alveolar macrophages)

  • Maintain oxygen and nutrient conditions that mimic the in vivo environment

  • Consider the impact of host immune factors on experimental outcomes

• Temporal dynamics:

  • Examine multiple time points to capture both early and late host-pathogen interactions

  • Track changes in gpmI expression and activity throughout infection progression

  • Design time-course experiments to identify critical windows for intervention

• Controls and comparisons:

  • Include isogenic strains differing only in gpmI expression/activity

  • Compare virulent and attenuated Mmm SC strains

  • Include appropriate host cell controls (uninfected, treated with purified gpmI)

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate gpmI activity with global metabolic changes in both host and pathogen

  • Identify potential regulatory networks involving gpmI

• Translation to in vivo models:

  • Validate key findings in appropriate animal models

  • Consider ethical alternatives to reduce animal use when possible

  • Design experiments to specifically test hypotheses generated from in vitro studies

When studying host-pathogen interactions involving Mmm SC, it's particularly important to consider the close contact between mycoplasmas and host cells, as this proximity is necessary for the translocation of toxic compounds into host cells. Research has shown that H₂O₂ and other reactive oxygen species produced by Mmm SC require this close contact to cause cytotoxic effects in bovine epithelial cells .

How does gpmI from Mmm SC compare structurally and functionally to gpmI from other pathogenic bacteria?

The structural and functional comparison of gpmI across bacterial species reveals important insights:

• Structural conservation and divergence:

  • The catalytic core of gpmI is generally conserved across bacterial species

  • Surface-exposed regions show greater variability, potentially reflecting adaptation to different host environments

  • Mmm SC gpmI likely maintains the characteristic fold of the alkaline phosphatase superfamily

• Catalytic efficiency:

  • Kinetic parameters (Km, kcat) may vary between species, reflecting metabolic adaptations

  • Optimal pH and temperature ranges can differ significantly between mesophilic and thermophilic bacteria

  • Cofactor requirements and inhibition profiles provide insights into evolutionary adaptations

• Genomic context:

  • The organization of genes surrounding gpmI can provide insights into potential co-regulated pathways

  • In minimal genomes like those of Mycoplasma species, the conservation of gpmI underscores its essential role

  • Horizontal gene transfer events might be detected through detailed phylogenetic analysis

• Evolutionary considerations:

  • Selective pressure on gpmI might differ between environmental and host-adapted pathogens

  • The presence of paralogous genes in some bacteria but not in Mycoplasma highlights different evolutionary strategies

  • Sequence conservation patterns can identify functionally critical residues versus adaptively variable regions

Understanding these comparative aspects can inform structure-based drug design efforts targeting gpmI in Mmm SC while avoiding cross-reactivity with human phosphoglycerate mutases .

What insights can be gained from studying gpmI in the context of Mmm SC's minimal genome?

Studying gpmI within the context of Mmm SC's minimal genome provides unique insights into essential metabolism and pathogen evolution:

• Metabolic streamlining:

  • The retention of gpmI in Mmm SC's reduced genome highlights its essential role in central metabolism

  • Analysis of gpmI conservation across Mycoplasma species can reveal the core metabolic requirements for survival

  • The absence of redundant enzymes for the PGM reaction emphasizes the critical nature of gpmI function

• Adaptations to parasitic lifestyle:

  • Comparison of gpmI sequence and regulatory elements across mycoplasmas with different host specificities

  • Evaluation of how gpmI function compensates for missing metabolic pathways in the minimal genome

  • Assessment of potential moonlighting functions that might emerge in organisms with limited protein repertoires

• Regulatory network integration:

  • Investigation of how gpmI is regulated in the context of simplified transcriptional networks

  • Analysis of potential post-transcriptional regulation mechanisms in organisms with limited regulatory proteins

  • Examination of how metabolic flux through gpmI is coordinated with host-derived nutrients

• Evolutionary implications:

  • Study of the selection pressures on gpmI in the context of genome reduction

  • Comparison with ancestral, more complex bacteria to understand the consequences of genome minimization

  • Insights into the minimum genetic requirements for glycolytic function

The study of Mmm SC and its minimal genome has revealed that despite genetic limitations, the organism maintains sophisticated virulence mechanisms, including the ability to evade host immune defenses, adhere to host cells, and release toxic metabolic products. Understanding how gpmI contributes to these capabilities despite genomic constraints provides valuable insights into both pathogen evolution and potential intervention strategies .

How might gpmI be targeted for the development of novel antimicrobials against Mmm SC?

The development of gpmI-targeted antimicrobials presents several promising avenues:

• Structure-based inhibitor design:

  • Virtual screening of chemical libraries against the gpmI active site

  • Fragment-based drug discovery approaches targeting allosteric sites

  • Rational design of transition state analogs to inhibit catalysis

• Natural product screening:

  • Evaluation of plant extracts and microbial secondary metabolites

  • Repurposing of existing compounds with known activity against related enzymes

  • Identification of molecules that selectively target bacterial gpmI over mammalian PGAM

• Peptide inhibitors:

  • Design of peptides that mimic interaction interfaces of gpmI

  • Development of cyclic peptides with enhanced stability and cell penetration

  • Exploration of peptide-small molecule conjugates for improved targeting

• Potential drug development pipeline:

  • Initial high-throughput screening against recombinant gpmI

  • Secondary validation in Mmm SC culture systems

  • Evaluation of cytotoxicity and specificity in bovine cell models

  • Pharmacokinetic and efficacy testing in appropriate animal models

Research has shown that exogenous polypeptides can inhibit phosphoglycerate mutase activity, resulting in decreased glycolytic rates and growth inhibition in certain cell lines . This suggests that peptide-based approaches might be particularly promising for targeting gpmI in Mmm SC.

What is the potential of recombinant gpmI as a diagnostic marker for CBPP?

Recombinant gpmI offers several advantages as a potential diagnostic marker for CBPP:

• Serological applications:

  • Development of ELISA assays using recombinant gpmI to detect antibodies in infected cattle

  • Lateral flow assays for field-deployable diagnostics in resource-limited settings

  • Multiplex serological panels combining gpmI with other Mmm SC antigens

• Performance characteristics:

  • Sensitivity and specificity analysis compared to existing diagnostic methods

  • Temporal dynamics of anti-gpmI antibodies during disease progression

  • Cross-reactivity assessment with antibodies against related Mycoplasma species

• Implementation considerations:

  • Stability of recombinant gpmI under field conditions

  • Cost-effectiveness compared to current diagnostic approaches

  • Potential for technology transfer to endemic regions

• Advanced applications:

  • Development of aptamer-based biosensors targeting gpmI

  • Direct detection of gpmI in clinical samples using specific antibodies

  • Combination with other biomarkers for improved diagnostic accuracy

The selection of diagnostic antigens should be informed by the presence of specific antibodies in sera from CBPP-positive animals. Research on reverse vaccinology approaches has identified antigens that elicit strong immune responses in infected cattle, which could serve as the basis for diagnostic test development .

How can the study of gpmI contribute to understanding the evolution of metabolic pathways in minimal genome organisms?

The study of gpmI in Mmm SC provides a unique window into metabolic evolution in minimal genome organisms:

• Evolutionary retention patterns:

  • Analysis of why gpmI is conserved despite extensive genome reduction

  • Comparison with other glycolytic enzymes to identify differential selection pressures

  • Examination of gpmI sequence conservation relative to the whole proteome

• Functional adaptation:

  • Investigation of potential kinetic optimizations in minimal genome organisms

  • Assessment of substrate specificity changes compared to ancestral enzymes

  • Evaluation of protein stability adaptations in the context of limited chaperone systems

• Metabolic network integration:

  • Analysis of how reduced metabolic networks maintain functionality despite fewer components

  • Study of metabolic flux distribution in simplified pathways

  • Identification of critical control points in streamlined metabolic systems

• Implications for synthetic biology:

  • Insights into the minimum requirements for functional glycolysis

  • Design principles for engineered minimal cells

  • Understanding of essential enzyme characteristics for minimal metabolism

The study of Mmm SC has shown that despite having a small genome, it can efficiently exploit its limited genetic information to fulfill basic replication functions while also damaging host cells to acquire necessary bio-molecules. This strategy represents a fascinating evolutionary adaptation that allows the organism to thrive despite genomic constraints .

What emerging technologies might advance our understanding of gpmI in Mmm SC?

Several cutting-edge technologies hold promise for enhancing our understanding of gpmI:

• CRISPR-Cas9 gene editing:

  • Precise modification of gpmI in Mmm SC to study structure-function relationships

  • Creation of conditional knockdown systems for essential genes

  • Development of CRISPR interference approaches for temporal control of gpmI expression

• Single-cell technologies:

  • Analysis of gpmI expression heterogeneity in Mmm SC populations

  • Single-cell metabolomics to correlate gpmI activity with metabolic states

  • Spatial transcriptomics to study gpmI expression in the context of host tissue infection

• Advanced structural biology:

  • Cryo-electron microscopy to resolve gpmI structure at near-atomic resolution

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

  • Time-resolved X-ray crystallography to capture catalytic intermediates

• Systems biology approaches:

  • Genome-scale metabolic modeling to predict the impact of gpmI modulation

  • Multi-omics data integration to understand gpmI in the context of global cellular processes

  • Network analysis to identify non-obvious interactions involving gpmI

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize gpmI localization during infection

  • Live-cell imaging with activity-based probes to track gpmI function in real-time

  • Correlative light and electron microscopy to link gpmI activity with cellular ultrastructure

These technologies will enable researchers to address complex questions about gpmI function in ways that were previously impossible, potentially revealing new aspects of Mmm SC pathogenicity and metabolism .

What are the key unanswered questions regarding gpmI's role in Mmm SC pathogenicity?

Despite progress in understanding Mmm SC, several critical questions about gpmI remain unanswered:

• Regulatory mechanisms:

  • How is gpmI expression regulated during different stages of infection?

  • What environmental signals modulate gpmI activity?

  • Is there evidence for post-translational modification of gpmI affecting its function?

• Metabolic integration:

  • How does gpmI activity coordinate with other metabolic pathways, particularly those involved in virulence?

  • What is the relationship between gpmI function and the production of toxic metabolites like H₂O₂?

  • How does gpmI activity respond to changes in host metabolic environment?

• Host interaction:

  • Does gpmI have additional non-catalytic roles in host-pathogen interactions?

  • Can gpmI or its metabolic products trigger specific host immune responses?

  • Is there evidence for direct interaction between gpmI and host cell components?

• Therapeutic targeting:

  • What are the most promising approaches for selective inhibition of gpmI?

  • How can we predict and prevent the development of resistance to gpmI inhibitors?

  • What would be the consequences of partial versus complete inhibition of gpmI activity?

• Strain variation:

  • How does gpmI sequence and expression vary among different Mmm SC strains?

  • Do these variations correlate with differences in virulence or host range?

  • Are there specific gpmI variants associated with outbreaks or treatment resistance?

Addressing these questions will require integrated approaches combining molecular genetics, biochemistry, structural biology, and in vivo infection models .