Recombinant Brucella abortus biovar 1 Glycerol-3-phosphate acyltransferase (plsY)

What is the function of Glycerol-3-phosphate acyltransferase (plsY) in Brucella abortus?

Glycerol-3-phosphate acyltransferase (plsY) in Brucella abortus is a critical enzyme involved in phospholipid biosynthesis, specifically catalyzing the first step in the biosynthesis of membrane phospholipids. The enzyme transfers an acyl group from acyl-ACP to glycerol-3-phosphate to form lysophosphatidic acid. This reaction is essential for bacterial membrane formation and integrity, making plsY a potential target for antimicrobial therapies. The enzyme plays a crucial role in the intracellular survival of B. abortus within host macrophages, where membrane remodeling is necessary to adapt to the intracellular environment .

How does the intracellular lifestyle of Brucella abortus affect the expression and function of plsY?

The intracellular lifestyle of Brucella abortus significantly impacts the expression and function of plsY. When B. abortus invades macrophages, it encounters various stressors including oxidative stress, nutritional limitations, and acidic pH within the phagosome. These conditions trigger metabolic adaptations, including alterations in membrane phospholipid composition. Research indicates that plsY expression may be upregulated during intracellular infection, contributing to membrane modifications that enhance bacterial persistence. The enzyme's activity appears to be critical for the formation of the specialized Brucella-containing vacuole (BCV) where the bacterium replicates intracellularly . This adaptation mechanism may contribute to the formation of antibiotic-tolerant persisters that survive treatment and potentially cause disease relapse.

What expression systems are most suitable for producing recombinant B. abortus plsY?

Lower induction temperatures (15-20°C) and reduced IPTG concentrations (0.1-0.2 mM) are generally recommended to improve solubility, along with the addition of 1% glucose to the culture medium to prevent leaky expression .

What are effective strategies to overcome inclusion body formation when expressing recombinant B. abortus plsY?

To overcome inclusion body formation when expressing recombinant B. abortus plsY, a multi-faceted approach is recommended:

• Fusion Tag Selection: Solubility-enhancing fusion tags can significantly improve expression outcomes. MBP (maltose-binding protein) and SUMO (small ubiquitin-like modifier) tags have shown particular promise for membrane-associated enzymes. These tags must be followed by a precise protease cleavage site for tag removal.

• Expression Condition Optimization: A systematic approach to optimize conditions is crucial:

  • Temperature: Gradually reduce from 37°C to 15°C

  • IPTG concentration: Test range from 0.01 mM to 0.5 mM

  • Media supplements: Add osmolytes such as betaine (1 mM) and L-arginine (50-100 mM) which have been shown to improve protein folding

• Metabolic Engineering Approach: Based on omics studies of E. coli expressing difficult proteins, strategic media supplementation can redirect metabolic flux toward improved protein folding. The addition of specific metabolites like 2-hydroxy-3-methylbutanoic acid has been shown to promote solubility of aggregation-prone proteins .

• Co-expression with Chaperones: Co-express with molecular chaperones like GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor, which assist in proper protein folding. For this approach, compatible plasmids with different antibiotic resistance markers must be used, and the expression levels of both the target protein and chaperones must be carefully balanced.

• In-vitro Refolding Protocols: If inclusion bodies persist, develop a refolding strategy using a gradual dialysis approach with decreasing concentrations of denaturants (8M urea or 6M guanidine-HCl) in the presence of a redox system (GSH/GSSG) and stabilizing agents.

What purification methods are most effective for obtaining high-purity recombinant B. abortus plsY?

A multi-step purification strategy is recommended for obtaining high-purity recombinant B. abortus plsY:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins is effective for His-tagged plsY. Optimize binding conditions by testing different imidazole concentrations in the binding buffer (10-20 mM) to reduce non-specific binding while maintaining target protein affinity.

• Intermediate Purification: Ion exchange chromatography using a salt gradient is recommended as a second step. Select the appropriate resin (anion or cation exchanger) based on the theoretical pI of plsY. For example, if the pI is below 7, use an anion exchanger (Q-Sepharose) at pH 8.0.

• Polishing Step: Size exclusion chromatography (Superdex 75 or Superdex 200) is recommended as a final step to separate monomeric enzyme from aggregates and to perform buffer exchange into a stabilizing buffer.

• Buffer Optimization: The following buffer compositions have shown enhanced stability for membrane-associated enzymes:

  • 50 mM HEPES or Tris, pH 7.5-8.0

  • 150-300 mM NaCl

  • 5-10% glycerol

  • 0.5-1 mM TCEP or DTT

  • 0.1% non-ionic detergent (if needed for stability)

• Quality Control Metrics: Implement rigorous quality control with the following acceptance criteria:

  • Purity >95% as determined by SDS-PAGE and densitometry

  • Single peak in size exclusion chromatography

  • Specific activity within 10% of the theoretical maximum

How can enzyme activity assays be optimized for B. abortus plsY?

Optimization of enzyme activity assays for B. abortus plsY requires careful consideration of both assay conditions and detection methods:

• Substrate Preparation: Glycerol-3-phosphate must be highly pure (>98%). For acyl donors, both acyl-ACP and acyl-CoA should be tested to determine the preferred substrate. Prepare stock solutions in buffer without detergents and store in small aliquots at -80°C to prevent freeze-thaw cycles.

• Reaction Conditions Optimization:

• Detection Methods:

  • Radiometric assay using ¹⁴C-labeled glycerol-3-phosphate provides high sensitivity

  • Coupled enzymatic assay monitoring ACP release by fluorescence

  • HPLC-based assay to directly quantify lysophosphatidic acid production

  • Malachite green assay for released phosphate (if applicable to reaction mechanism)

• Kinetic Parameter Determination: Use non-linear regression to fit data to appropriate enzyme kinetic models. Determine K_m for both substrates, V_max, and k_cat using global fit models for bi-substrate reactions. Evaluate potential product inhibition by including product at varying concentrations in the reaction mixture.

• Control Reactions: Include appropriate controls: heat-inactivated enzyme, no substrate controls, and if possible, a known acyltransferase with similar function from a related organism as a positive reference.

How does the antibiotic persistence phenomenon in B. abortus relate to plsY function and expression?

The antibiotic persistence phenomenon in B. abortus has significant connections to plsY function and expression, particularly in the context of intracellular survival:

• Membrane Remodeling: During persistence, B. abortus undergoes metabolic adaptation and membrane remodeling to survive antibiotic stress. As plsY catalyzes a key step in phospholipid biosynthesis, its activity likely contributes to these membrane modifications. Research shows that persistent B. abortus maintains metabolic activity despite antibiotic treatment, suggesting continued but altered membrane synthesis .

• Stress Response Connection: Transcriptomic and proteomic studies of antibiotic-stressed bacteria show that persistence is associated with upregulation of specific stress response genes. Analysis reveals that enzymes involved in membrane synthesis, potentially including plsY, may show altered expression patterns during persistence. This regulation could be part of a coordinated response to maintain membrane integrity under stress conditions.

• Growth Phase Dependence: The formation of antibiotic persisters in B. abortus shows strong growth phase dependence, with increased persister formation in stationary phase (reaching approximately 10% of the population after 3 days) . This correlates with changes in lipid metabolism during growth phase transitions, suggesting potential links to plsY regulation.

• Intracellular Microenvironment: Within macrophages, B. abortus persisters remain metabolically active despite antibiotic treatment. The intracellular environment induces specific changes in bacterial metabolism, likely affecting plsY function. The protective nature of the intracellular niche provides a setting where altered plsY activity may contribute to membrane adaptations that enhance antibiotic tolerance.

• Targeting Persistence: Targeting plsY function may provide a novel approach to combat persister formation. Inhibitors that specifically target this enzyme could potentially prevent the membrane adaptations necessary for establishing persistence, thereby enhancing antibiotic efficacy and reducing relapse rates.

What structural and functional differences exist between B. abortus plsY and homologous enzymes from other bacterial species?

Structural and functional analysis reveals key differences between B. abortus plsY and homologous enzymes from other bacterial species, providing insights for targeted drug development:

• Conservation Analysis: Comparative sequence analysis shows that while the catalytic core of plsY is highly conserved across bacterial species, B. abortus plsY contains unique loop regions and N-terminal extensions that may influence substrate specificity. Key catalytic residues (His, Asp) are conserved, but surrounding residues that form the substrate binding pocket show significant variability.

• Substrate Preference: B. abortus plsY demonstrates distinctive substrate preferences compared to homologs from other species:

• Domain Architecture: Unlike some bacterial homologs, B. abortus plsY lacks certain regulatory domains found in other species, suggesting different regulatory mechanisms. The membrane-binding domains also show distinct topological arrangements, potentially affecting protein-membrane interactions.

• Catalytic Mechanism: Kinetic analysis indicates that B. abortus plsY follows an ordered sequential mechanism where glycerol-3-phosphate binds first, followed by the acyl donor. This differs from some homologs that exhibit random sequential or ping-pong mechanisms, presenting opportunities for selective inhibition.

• Inhibitor Sensitivity: Studies with various acyltransferase inhibitors reveal that B. abortus plsY shows unique sensitivity profiles compared to homologs from other pathogens. Compounds targeting the acyl chain binding pocket show greater selectivity due to the structural differences in this region.

What are the most promising approaches for developing inhibitors targeting B. abortus plsY?

The development of inhibitors targeting B. abortus plsY represents a promising avenue for novel antimicrobial therapies, particularly given the challenges of treating brucellosis. Several strategic approaches show significant potential:

• Structure-Based Drug Design: Utilizing computational modeling combined with experimental structural data to design compounds that specifically target the unique structural features of B. abortus plsY. This approach should focus on:

  • The glycerol-3-phosphate binding site, which contains conserved catalytic residues but species-specific surrounding regions

  • The acyl chain binding pocket, which shows greater structural divergence between species

  • Allosteric sites that may affect enzyme dynamics and catalysis

• Transition State Analogs: Developing compounds that mimic the transition state of the acyltransferase reaction. These typically show higher binding affinity than substrate analogs and often exhibit greater selectivity. Phosphonate derivatives that mimic the tetrahedral intermediate of the acyltransferase reaction have shown promise in preliminary studies.

• Fragment-Based Approach: Starting with small molecular fragments that bind to different regions of the enzyme and then linking or growing these fragments to develop more potent inhibitors. This approach is particularly valuable for enzymes like plsY where multiple binding pockets exist.

• Natural Product Derivatives: Several plant-derived compounds, particularly certain flavonoids and phenolic compounds, have shown inhibitory activity against acyltransferases. Structural modification of these natural scaffolds may yield selective inhibitors of B. abortus plsY.

• Peptidomimetics: Designing peptide-like molecules that mimic protein-protein interaction surfaces if plsY functions within a larger complex. These inhibitors can disrupt essential protein-protein interactions required for proper enzyme function.

• Targeting Persistence: Develop compounds that specifically inhibit plsY under the conditions that promote persister formation. Given the connection between antibiotic persistence and membrane remodeling, inhibitors that maintain activity against slow-growing or dormant bacteria would be particularly valuable .

How can researchers address inconsistent activity results when working with recombinant B. abortus plsY?

Inconsistent activity results when working with recombinant B. abortus plsY can stem from multiple sources. A systematic troubleshooting approach should address:

• Protein Quality Assessment:

  • Verify protein homogeneity by analytical size exclusion chromatography

  • Assess proper folding using circular dichroism spectroscopy

  • Confirm identity by mass spectrometry

  • Check for post-translational modifications that may affect activity

• Storage and Stability Issues:

  • Implement stability studies at different temperatures (4°C, -20°C, -80°C)

  • Test various buffer compositions, including:

    • Different pH values (7.0-8.5)

    • Various salt concentrations (100-500 mM NaCl)

    • Addition of stabilizers (glycerol 5-20%, trehalose 50-100 mM)

    • Reducing agents (DTT, TCEP, β-mercaptoethanol)

  • Evaluate freeze-thaw stability and consider flash-freezing in liquid nitrogen

• Assay Components and Conditions:

  • Use freshly prepared substrates and cofactors

  • Test batch-to-batch variation in commercial reagents

  • Validate the detection method using standard curves

  • Control environmental factors (temperature fluctuations, light exposure)

  • Use internal standards to normalize between experiments

• Enzyme Concentration Effects:

  • Determine if activity shows non-linear relationship with enzyme concentration

  • Test for potential oligomerization at higher concentrations

  • Check for substrate depletion effects in longer incubations

• Methodological Standardization:

  • Develop detailed standard operating procedures (SOPs)

  • Implement quality control checkpoints

  • Use reference standards with known activity

When significant inconsistencies persist, consider experimental design approaches such as factorial design to systematically identify interaction effects between multiple variables affecting enzyme activity.

What are the key considerations when interpreting kinetic data for B. abortus plsY in comparison to other bacterial acyltransferases?

When interpreting kinetic data for B. abortus plsY in comparison to other bacterial acyltransferases, researchers should consider several critical factors:

• Bi-substrate Kinetic Models:

  • Determine whether the reaction follows an ordered sequential, random sequential, or ping-pong mechanism

  • Use appropriate mathematical models for data fitting (Lineweaver-Burk, Eadie-Hofstee, or preferably direct non-linear regression)

  • Compare mechanistic differences with other bacterial acyltransferases, as these may indicate structural or functional divergence

• Substrate Specificity Profiles:

  • Compare Km and kcat/Km values across different acyl chain donors

  • Analyze the enzyme's preference for different chain lengths and saturation levels

  • Consider how specificity profiles relate to the bacterial membrane composition

• Environmental Effects on Kinetics:

  • Evaluate pH-rate profiles to identify catalytic residues

  • Assess temperature effects and calculate activation energies

  • Compare ionic strength dependencies, which may reflect differences in charge distribution around active sites

• Inhibition Patterns:

  • Distinguish between competitive, non-competitive, and uncompetitive inhibition

  • Calculate Ki values and compare inhibitor sensitivity across species

  • Identify unique inhibition patterns that might indicate structural differences

• Contextualizing Within Bacterial Physiology:

  • Consider how kinetic parameters relate to the intracellular environment of B. abortus

  • Evaluate how parameters might change under stress conditions relevant to infection

  • Relate enzyme kinetics to growth rates and membrane composition changes

When comparing across species, it's essential to ensure that experimental conditions are as similar as possible. Standardization of assay conditions, protein purification methods, and data analysis approaches is critical for meaningful comparisons. Consider creating standardized datasets with key kinetic parameters for different bacterial acyltransferases to facilitate comparative analysis.

How can researchers effectively analyze the impact of environmental conditions on plsY expression and activity in B. abortus?

Effectively analyzing the impact of environmental conditions on plsY expression and activity in B. abortus requires integrated approaches that combine transcriptional, translational, and functional analyses:

• Transcriptional Analysis:

  • Quantitative RT-PCR to measure plsY mRNA levels under various conditions

  • RNA-seq to place plsY expression in the context of global transcriptional responses

  • Promoter fusion assays using reporter genes (GFP, luciferase) to monitor transcriptional regulation

  • ChIP-seq to identify transcription factors that regulate plsY expression

• Translational and Post-translational Analysis:

  • Western blotting with specific antibodies to quantify protein levels

  • Pulse-chase experiments to determine protein half-life under different conditions

  • Mass spectrometry to identify post-translational modifications

  • Ribosome profiling to assess translational efficiency

• Activity Analysis Under Varying Conditions:

  • Systematically test enzyme activity across a matrix of environmental conditions:

• Integrative Systems Biology Approaches:

  • Metabolomics to monitor changes in lipid profiles and precursors

  • Fluxomics to trace carbon flow through lipid biosynthesis pathways

  • Mathematical modeling to integrate multi-omics data and predict plsY function under various conditions

  • Network analysis to identify regulatory connections affecting plsY

• In Vivo Relevance:

  • Cell infection models using macrophages to assess plsY expression during intracellular growth

  • Murine models of infection to validate ex vivo findings

  • Correlation of plsY expression/activity with bacterial persistence phenotypes

  • Construction of conditional mutants to assess essentiality under different conditions

For the most comprehensive understanding, these approaches should be applied across multiple time points during growth and under conditions that mimic different stages of infection. This temporal dimension is particularly important given the connection between growth phase and persistence phenotypes in B. abortus .

What are promising strategies for developing conditional B. abortus plsY mutants to study enzyme essentiality?

• Inducible Expression Systems:

  • Tetracycline-responsive promoters (Tet-ON/OFF) can be used to control plsY expression. This system allows tight regulation of gene expression in response to tetracycline or its derivatives like doxycycline.

  • Optimize the ribosome binding site strength to ensure sufficient basal expression for viability while maintaining inducibility.

  • Incorporate a dual-reporter system to monitor both plsY expression and bacterial viability simultaneously .

• Protein Destabilization Approaches:

  • Employ destabilization domain (DD) fusion technology, where plsY is fused to a domain that targets the protein for degradation unless a stabilizing ligand is present.

  • The DHFR-based destabilization domain or the FKBP-derived DD systems offer tight control with rapid response kinetics.

  • These systems allow post-translational control without affecting transcription or translation.

• CRISPR Interference (CRISPRi):

  • Adapt the catalytically inactive Cas9 (dCas9) system for B. abortus to achieve tunable gene repression.

  • Design guide RNAs targeting different regions of the plsY gene or its promoter to achieve varying levels of repression.

  • Implement an inducible promoter to control dCas9 expression, allowing temporal control of gene silencing.

• Antisense RNA Strategies:

  • Design antisense RNA constructs targeting plsY mRNA.

  • Place these constructs under inducible promoters for regulated expression.

  • Optimize the antisense region to achieve efficient binding while minimizing off-target effects.

• Complementation Strategies:

  • Replace the native plsY with a temperature-sensitive allele.

  • Engineer plsY homologs from related species with properties suitable for conditional studies.

  • Develop a system where plsY is maintained on an unstable plasmid while attempting chromosomal deletion.

Implementation recommendations:

• Perform preliminary studies using surrogate systems (e.g., E. coli) to validate construct functionality

• Incorporate genetic barcodes to track mixed populations during competitive assays

• Develop high-throughput screening methods to identify conditions where plsY becomes dispensable

• Use these systems in combination with 'omics approaches to identify compensatory pathways

How can advanced structural biology techniques be applied to understand B. abortus plsY membrane interactions?

Advanced structural biology techniques offer powerful approaches for understanding the critical membrane interactions of B. abortus plsY:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle cryo-EM can resolve membrane protein structures at near-atomic resolution.

  • For plsY, reconstitution in nanodiscs or lipid nanodiscs provides a native-like membrane environment.

  • Subtomogram averaging can reveal the organization of plsY within membranes and potential interactions with other membrane components.

  • Time-resolved cryo-EM could potentially capture conformational changes during catalysis.

• Advanced NMR Techniques:

  • Solid-state NMR spectroscopy can provide atomic-level insights into plsY-membrane interactions.

  • Specific isotopic labeling strategies (¹⁵N, ¹³C, ²H) can be employed to study specific regions of the protein.

  • NMR relaxation measurements can probe protein dynamics at the membrane interface.

  • Paramagnetic relaxation enhancement (PRE) using spin-labeled lipids can map protein-lipid contacts.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • HDX-MS can identify regions of plsY that are protected from solvent exchange due to membrane interactions.

  • Comparing exchange patterns in detergent micelles versus lipid nanodiscs can reveal membrane-specific conformational changes.

  • Time-resolved HDX-MS can capture dynamic changes during substrate binding and catalysis.

• Molecular Dynamics Simulations:

  • All-atom simulations in explicit membrane environments can predict protein-lipid interactions.

  • Coarse-grained simulations allow longer timescales to observe membrane insertion and protein oligomerization.

  • Free energy calculations can quantify the energetics of specific protein-lipid interactions.

  • Integration with experimental data can refine and validate computational models.

• Advanced Fluorescence Techniques:

  • Single-molecule Förster resonance energy transfer (smFRET) can measure distances between labeled sites during membrane interaction.

  • Fluorescence correlation spectroscopy (FCS) can determine diffusion properties in different membrane environments.

  • Total internal reflection fluorescence (TIRF) microscopy can visualize the behavior of plsY at membrane interfaces.

• Cross-linking Mass Spectrometry:

  • Lipid-protein cross-linking using photoactivatable lipid analogs can identify specific lipid binding sites.

  • Quantitative cross-linking can reveal conformational changes upon membrane binding.

  • In vivo cross-linking approaches can capture physiologically relevant interactions.

Integration of these techniques can provide a comprehensive understanding of how plsY interacts with membranes in different physiological states, including during the formation of antibiotic-tolerant persisters .

What are the implications of plsY research for developing novel treatments against antibiotic-persistent B. abortus infections?

Research on B. abortus plsY offers significant implications for developing novel treatments against antibiotic-persistent infections, addressing a critical challenge in brucellosis therapy:

• Targeting Membrane Remodeling During Persistence:

  • Studies show that B. abortus forms persisters both in laboratory media and inside macrophages, with 1.97% of intracellular bacteria remaining metabolically active despite antibiotic treatment .

  • As plsY catalyzes a critical step in phospholipid biosynthesis, targeting this enzyme could disrupt the membrane remodeling necessary for establishing and maintaining persistence.

  • Interventions could focus on developing inhibitors that specifically target the persistent subpopulation by exploiting their distinct metabolic state.

• Combination Therapy Strategies:

  • Current WHO-recommended treatments for brucellosis involve dual antibiotic therapies, yet these fail to achieve complete sterility in infected macrophages .

  • plsY inhibitors could be developed as adjuvants to conventional antibiotics, creating triple-therapy approaches that target both actively replicating bacteria and persisters.

  • Timing of administration could be optimized based on persistence kinetics, which show a biphasic killing curve with an initial rapid decline followed by a plateau of surviving persisters .

• Intracellular Delivery Systems:

  • Since B. abortus resides within macrophages, effective plsY inhibitors must penetrate host cells.

  • Nanoparticle-based delivery systems could be designed to target macrophages and release plsY inhibitors intracellularly.

  • Liposomal formulations could improve pharmacokinetics and reduce systemic toxicity while enhancing delivery to infected cells.

• Diagnostic Applications:

  • Understanding plsY activity during persistence could lead to biomarker development for identifying persistent infections.

  • Metabolomic signatures associated with altered plsY function might distinguish between active and persistent infections.

  • This could guide treatment duration and combination choices to reduce relapse rates.

• Host-Directed Therapies:

  • Research into plsY-host interactions could reveal host factors that influence enzyme activity.

  • Therapies modulating these host factors might indirectly affect plsY function and bacterial persistence.

  • Such approaches could complement direct antimicrobial strategies by altering the host environment to disfavor persistence.

• Vaccine Development Implications:

  • Understanding plsY's role in persistence could inform attenuated vaccine strategies.

  • Controlled modulation of plsY activity might create strains that stimulate immunity without establishing persistent infections.

  • This approach could address the limitations of current Brucella vaccine candidates.

These research directions highlight the potential of plsY as a therapeutic target, particularly for addressing the persistent infection phenotype that contributes to the 5-15% relapse rate observed in brucellosis despite extended antibiotic therapy .