Recombinant Nitrosomonas europaea Holo-[acyl-carrier-protein] synthase (acpS)

What is Nitrosomonas europaea Holo-[acyl-carrier-protein] synthase and what is its fundamental function?

Nitrosomonas europaea Holo-[acyl-carrier-protein] synthase (AcpS) is an essential enzyme that catalyzes a critical post-translational modification necessary for bacterial lipid biosynthesis. Specifically, AcpS transfers the 4'-phosphopantetheine moiety from coenzyme A (CoA) to an invariant serine residue on the apo-acyl carrier protein (apoACP) . This modification converts the inactive apoACP to its active form, holo-ACP, which then serves as the central coenzyme in fatty acid biosynthesis . The reaction catalyzed can be represented as:

CoA-[4'-phosphopantetheine] + apo-acyl carrier protein → adenosine 3',5'-bisphosphate + holo-acyl carrier protein

This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4'-phosphopantetheine . In the context of N. europaea, an obligate chemolithoautotroph that derives its energy from ammonia oxidation, AcpS plays a crucial role in maintaining cell membrane integrity through lipid biosynthesis .

How is AcpS classified enzymatically and what protein family does it belong to?

AcpS (EC 2.7.8.7) belongs to the family of transferases, specifically those transferring non-standard substituted phosphate groups . It is also commonly referred to as 4'-phosphopantetheinyl transferase, reflecting its function of transferring the 4'-phosphopantetheine group . The systematic name for this enzyme class is CoA-[4'-phosphopantetheine]:apo-[acyl-carrier-protein] 4'-pantetheinephosphotransferase .

All known AcpS enzymes are evolutionarily related within a single superfamily of proteins that consists of two major subtypes :

• The trimeric ACPS type (exemplified by E. coli ACPS)

• The monomeric Sfp (PCP-synthesizing) type (exemplified by B. subtilis SFP)

These enzymes share conserved regions involved in magnesium ion binding, which is essential for their catalytic activity . In the N. europaea genome, genes necessary for energy generation, reductant generation, biosynthesis, and CO₂ and NH₃ assimilation have been identified, suggesting the presence of complete metabolic pathways including those involving AcpS .

Why is N. europaea AcpS considered a potential antibiotic target?

N. europaea AcpS represents an attractive antibiotic target for several compelling reasons. First, AcpS is a highly conserved and essential enzyme for bacterial survival as it catalyzes the first step in lipid synthesis . The essentiality of this enzyme has been demonstrated in bacterial systems like E. coli, where the acpS gene has been shown to be critical for growth .

Second, AcpS currently has no known targeted antibiotics in clinical use, making it a novel target that could potentially overcome existing resistance mechanisms . Recent research has shown that computer-aided drug design efforts targeting AcpS have resulted in the development of a structurally unique antibiotic family . This approach has yielded over 700 novel compounds targeting AcpS, with 33 of these compounds demonstrating inhibition of bacterial growth at concentrations of ≤2 μg/mL .

Third, biochemical characteristics of AcpS make it particularly suitable as a drug target. The enzyme has a high Km (approximately 40 μM) for CoA, which enables low micromolar affinity compounds to effectively inhibit its activity . Additionally, AcpS demonstrates substrate inhibition against apo-ACP, meaning that inhibition of AcpS activity is amplified as apo-ACP substrate levels increase with prolonged inhibition .

What are the key structural features of N. europaea AcpS?

While the exact crystal structure of N. europaea AcpS has not been explicitly detailed in the provided search results, we can infer its likely structural characteristics based on the conserved nature of AcpS enzymes across bacterial species. AcpS enzymes generally contain highly conserved regions involved in binding magnesium ions, which are essential for their catalytic activity .

The active site of AcpS accommodates a magnesium ion that plays a crucial role in the phosphopantetheinyl transfer reaction . Based on structural studies of AcpS from other bacterial species, the N. europaea enzyme likely exhibits a similar architecture with:

• A core domain responsible for binding CoA

• A recognition surface for interacting with the apo-ACP substrate

• Conserved residues that coordinate the magnesium ion

• A catalytic center positioned to facilitate the transfer of the 4'-phosphopantetheine moiety

The N. europaea genome consists of a single circular chromosome of 2,812,094 bp, with approximately 2,460 protein-encoding genes . The AcpS gene would be among these, likely sharing sequence homology with other bacterial AcpS genes. The gene organization and promoter elements would reflect the metabolic importance of this enzyme in the organism's lipid biosynthesis pathways.

How does the catalytic mechanism of AcpS function at the molecular level?

The catalytic mechanism of AcpS involves several coordinated steps that facilitate the transfer of the 4'-phosphopantetheine moiety from CoA to apo-ACP. Based on studies of AcpS enzymes, the likely mechanism includes:

• Substrate binding: CoA and apo-ACP bind to their respective binding sites on the enzyme.

• Magnesium coordination: A magnesium ion in the active site coordinates with the phosphate groups of CoA, properly positioning the molecule for the reaction .

• Nucleophilic attack: The hydroxyl group of the conserved serine residue (typically serine 36) in apo-ACP performs a nucleophilic attack on the phosphate group linking the 4'-phosphopantetheine moiety to CoA .

• Transfer and release: The 4'-phosphopantetheine group is transferred to the serine residue, forming holo-ACP, while adenosine 3',5'-bisphosphate is released as a byproduct .

• Product dissociation: The holo-ACP product dissociates from the enzyme, completing the catalytic cycle.

This mechanism is supported by the observation that mutations in AcpS can significantly reduce its catalytic efficiency. For example, in E. coli, the G4D mutation in AcpS1 resulted in approximately 5-fold reduction in catalytic efficiency compared to wild-type AcpS .

What are the optimal conditions for expressing recombinant N. europaea AcpS?

Based on general protocols for recombinant protein expression and the specific characteristics of N. europaea proteins, the following methodological approach is recommended for expressing recombinant N. europaea AcpS:

Expression System Selection:

• E. coli BL21(DE3) or similar strains are typically suitable hosts for AcpS expression

• Consider codon optimization for N. europaea genes if expression yields are low

Vector Construction:

• Clone the N. europaea acpS gene into an expression vector with an inducible promoter (T7 or tac)

• Include a purification tag (6xHis, GST, or MBP) preferably at the N-terminus to avoid interfering with the active site

• Consider including a protease cleavage site for tag removal if native protein is required

Culture Conditions:

• Growth medium: LB or 2xYT supplemented with appropriate antibiotics

• Temperature: Initial growth at 37°C until OD₆₀₀ reaches 0.6-0.8

• Induction: Reduce temperature to 16-25°C before induction with 0.1-0.5 mM IPTG

• Post-induction incubation: 16-18 hours at reduced temperature (improving protein folding)

Considerations for N. europaea Proteins:

• N. europaea has a GC content of approximately 50.7%, which is relatively high and may affect expression efficiency

• The obligate chemolithoautotrophic nature of N. europaea suggests its proteins may have specific folding requirements

• Supplementing growth medium with magnesium (1-5 mM MgCl₂) may improve AcpS folding and stability, given the enzyme's requirement for this cofactor

What purification strategies are most effective for obtaining active recombinant N. europaea AcpS?

A comprehensive purification strategy for obtaining high-yield, active recombinant N. europaea AcpS would involve the following methodological approach:

Cell Lysis and Initial Clarification:

• Resuspend cell pellet in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM MgCl₂, and 10% glycerol

• Include protease inhibitor cocktail to prevent protein degradation

• Lyse cells via sonication or high-pressure homogenization

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

Affinity Chromatography:

• For His-tagged AcpS: Apply clarified lysate to Ni-NTA resin

• Wash with buffer containing 20-30 mM imidazole to remove non-specifically bound proteins

• Elute with buffer containing 250 mM imidazole

Secondary Purification:

• Apply affinity-purified protein to size exclusion chromatography column (e.g., Superdex 75 or 200)

• Use buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM MgCl₂

• Analyze fractions by SDS-PAGE to identify pure AcpS protein

Activity Preservation Considerations:

• Include 5-10% glycerol in all buffers to enhance protein stability

• Maintain 1-2 mM MgCl₂ throughout purification as it's essential for AcpS activity

• Consider adding 1 mM DTT to prevent oxidation of cysteine residues

• Store purified protein at -80°C in small aliquots to avoid freeze-thaw cycles

Validation of Active Enzyme:

• Confirm enzyme activity using an in vitro assay measuring the conversion of apo-ACP to holo-ACP

• Monitor the reaction by detecting the release of adenosine 3',5'-bisphosphate or by using a fluorescently labeled apo-ACP substrate

How can the activity of recombinant N. europaea AcpS be measured in vitro?

Several complementary methodological approaches can be employed to measure the activity of recombinant N. europaea AcpS in vitro:

1. Spectrophotometric Assay:

• Principle: Coupling the release of adenosine 3',5'-bisphosphate to enzymatic reactions that produce a chromogenic product

• Method: Include auxiliary enzymes that utilize adenosine 3',5'-bisphosphate and monitor absorbance changes at appropriate wavelengths

• Advantages: Continuous monitoring, easily scalable for high-throughput screening

• Limitations: Potential interference from auxiliary enzymes or buffer components

2. HPLC/LC-MS Based Methods:

• Principle: Direct detection of reaction products (holo-ACP) or substrates (apo-ACP, CoA)

• Method: Analyze reaction mixtures at various time points using HPLC or LC-MS

• Advantages: High specificity, direct quantification of substrates and products

• Limitations: Requires specialized equipment, not suitable for continuous monitoring

3. Gel-Shift Assay:

• Principle: Differential migration of apo-ACP versus holo-ACP on non-denaturing PAGE

• Method: Incubate AcpS with apo-ACP and CoA, then analyze by native PAGE

• Advantages: Visual confirmation of ACP modification, minimal equipment needed

• Limitations: Semi-quantitative, requires optimization of gel conditions

4. Radioactive Assay:

• Principle: Use of [³H] or [¹⁴C]-labeled CoA to track 4'-phosphopantetheine transfer

• Method: Incubate AcpS with labeled CoA and apo-ACP, then quantify radioactivity in purified holo-ACP

• Advantages: High sensitivity, direct quantification of transfer

• Limitations: Requires radioactive materials handling, specialized equipment

Recommended Reaction Conditions:

• Buffer: 50 mM Tris-HCl (pH 7.5)

• Salts: 10 mM MgCl₂ (critical for activity)

• Substrates: 5-50 μM apo-ACP, 5-50 μM CoA (considering the Km for CoA is approximately 40 μM)

• Temperature: 30°C (optimal for most bacterial enzymatic assays)

• Time: 15-30 minutes (initial velocity conditions)

What is known about inhibitors of N. europaea AcpS and their mechanisms?

Recent research has yielded significant advances in developing inhibitors specific to bacterial AcpS, including approaches that may be applicable to N. europaea AcpS. The most comprehensive data comes from computer-aided drug design efforts that have generated a unique antibiotic family targeting AcpS .

Novel Thienyltetrazole Inhibitor Family:
A series of tri-substituted, penta-atomic aromatic heterocyclic compounds have been developed specifically as AcpS inhibitors . These compounds are characterized by:

• Core structure: Containing a central thiophene ring that can be functionalized at up to three additional points

• Functional groups: Incorporating at least one negatively charged carboxylic acid bioisostere (tetrazole)

• Synthesis accessibility: Developed to be synthesized in fewer than six steps from readily available materials

• Library size: Over 700 compounds with established structure-function-activity relationships

Inhibition Characteristics:
The lead compound DNM0547 demonstrates competitive inhibition against AcpS substrates, consistent with binding to the enzyme's active site . The inhibitory potency of these compounds generally correlates with:

• IC₅₀ values in the range of 1-15 μM for AcpS enzyme inhibition

• MIC values of ≤2 μg/mL against various Gram-positive bacteria, including MRSA

Inhibition Mechanism Considerations:
The effectiveness of AcpS inhibitors is enhanced by two key factors:

• The high Km for CoA (approximately 40 μM) enables compounds with low micromolar affinity to effectively inhibit activity

• AcpS demonstrates substrate inhibition against apo-ACP, which amplifies inhibition as apo-ACP substrate levels increase during prolonged AcpS inhibition

Table 1: Representative AcpS Inhibitor Data from Computer-Aided Drug Design Studies

How is the acpS gene organized in the N. europaea genome?

The Nitrosomonas europaea genome consists of a single circular chromosome of 2,812,094 bp . Based on the genomic analysis data, we can infer several aspects about the organization of the acpS gene within this genome:

The N. europaea genome contains a total of 2,460 protein-encoding genes with an average length of 1,011 bp and intergenic regions averaging 117 bp . The genes are distributed relatively evenly around the genome, with approximately 47% transcribed from one strand and 53% from the complementary strand .

While the exact position of the acpS gene is not specified in the search results, we can make informed inferences about its likely genomic context based on its function and the organization of related metabolic genes:

• Genomic Context: The acpS gene in N. europaea is likely positioned in proximity to other genes involved in fatty acid biosynthesis and lipid metabolism, forming a functional cluster.

• Essential Nature: Given that the acpS gene has been shown to be essential for growth in other bacteria such as E. coli , it is very likely located in a core region of the N. europaea chromosome that demonstrates high conservation across ammonia-oxidizing bacteria.

• Regulatory Elements: The promoter region of acpS in N. europaea might contain regulatory elements similar to those observed in E. coli, where the acpS gene expression is tightly regulated to ensure proper levels of holo-ACP for fatty acid biosynthesis .

It's worth noting that the N. europaea genome has several notable features that may influence the organization and expression of genes like acpS:

• Complex repetitive elements constitute approximately 5% of the genome, including 85 predicted insertion sequence elements in eight different families

• The GC skew analysis indicates that the genome is divided into two unequal replichores

• The genome has limited genes for catabolism of organic compounds but plentiful genes encoding transporters for inorganic ions

What genetic approaches can be used to study N. europaea AcpS function?

Several complementary genetic approaches can be employed to study the function of AcpS in Nitrosomonas europaea:

1. Conditional Knockout/Knockdown Systems:

• Methodology: Create conditional expression systems where acpS expression can be regulated by inducible promoters or degradation tags

• Application: Similar to studies in E. coli where a conditional acpS mutant was shown to accumulate apoACP under nonpermissive conditions

• Expected outcome: Under depletion conditions, observe accumulation of apoACP and disruption of lipid biosynthesis

• Technical considerations: Given the essential nature of acpS, complete knockout is likely lethal, necessitating conditional approaches

2. Site-Directed Mutagenesis:

• Methodology: Introduce specific mutations in conserved residues of AcpS based on structural studies

• Application: Similar to the G4D mutation in E. coli AcpS1 that resulted in approximately 5-fold reduction in catalytic efficiency

• Expected outcome: Mutations in catalytic residues should result in varying degrees of enzymatic impairment

• Technical approach: Generate mutant constructs, express in heterologous systems, and characterize biochemically

3. Heterologous Complementation:

• Methodology: Express N. europaea acpS in E. coli conditional acpS mutants

• Application: Test if N. europaea AcpS can functionally replace E. coli AcpS

• Expected outcome: Successful complementation would indicate functional conservation

• Comparative analysis: Compare complementation efficiency with acpS from other bacteria

4. Suppressor Studies:

• Methodology: Investigate alternative pathways for phosphopantetheinyl transfer

• Application: Similar to how YhhU in E. coli was shown to suppress the acpS conditional lethal phenotype when overexpressed

• Expected outcome: Identify potential backup systems or compensatory pathways

• Genomic approach: Search for YhhU homologs in the N. europaea genome

5. Transcriptomic Analysis:

• Methodology: RNA-Seq under different growth conditions to monitor acpS expression patterns

• Application: Understand regulatory networks controlling acpS expression

• Expected outcome: Identify co-regulated genes and potential regulatory factors

• Integration: Correlate expression profiles with metabolic pathways and growth conditions

How can N. europaea AcpS be utilized in screening for novel antimicrobial agents?

Nitrosomonas europaea AcpS represents an attractive target for antimicrobial development, particularly because AcpS inhibitors could potentially address the growing concern of antibiotic resistance. The following methodological approaches can be employed for screening novel antimicrobial agents targeting N. europaea AcpS:

1. High-Throughput Biochemical Screening:

• Methodology: Develop miniaturized assays measuring AcpS enzymatic activity in the presence of compound libraries

• Technical approach: Utilize spectrophotometric, fluorescence-based, or coupled enzyme assays adaptable to 384 or 1536-well formats

• Advantage: Can screen large chemical libraries (10⁴-10⁶ compounds) rapidly

• Considerations: Requires purified recombinant N. europaea AcpS and appropriate detection systems

2. Structure-Based Virtual Screening:

• Methodology: Use computational approaches similar to those employed in developing the thienyltetrazole inhibitor family

• Technical approach: Generate homology models of N. europaea AcpS if crystal structure is unavailable; dock virtual compound libraries

• Critical considerations: Focus on the active site and substrate binding regions, considering the lipophilic and cationic nature of the binding pocket

• Validation: Test top-scoring compounds in biochemical assays to confirm predicted binding

3. Fragment-Based Drug Discovery:

• Methodology: Screen libraries of low-molecular-weight compounds (fragments) against AcpS

• Technical approach: Use biophysical methods like thermal shift assays, NMR, or surface plasmon resonance

• Advantage: Identifies core scaffolds with efficient binding properties that can be elaborated into more potent inhibitors

• Follow-up: Link or grow fragments to develop compounds with improved potency and specificity

4. Whole-Cell Screening with Target Validation:

• Methodology: Screen compounds for growth inhibition of N. europaea or suitable model organisms

• Technical approach: Use growth-based assays followed by target engagement studies to confirm AcpS inhibition

• Advantage: Identifies compounds with good cell penetration and whole-cell activity

• Validation methods: Genetic approaches (AcpS overexpression should increase resistance) or biochemical confirmation

5. Rational Design Based on Known Inhibitors:

• Methodology: Modify existing AcpS inhibitors like the thienyltetrazole family to optimize for N. europaea AcpS

• Technical approach: Systematic structure-activity relationship studies with focused compound libraries

• Key considerations: Maintain the essential pharmacophore (e.g., the tetrazole bioisostere) while modifying other positions to improve potency or specificity

What are the current challenges in developing AcpS inhibitors as antibiotics?

The development of AcpS inhibitors as effective antibiotics faces several significant challenges that researchers must address:

1. Selective Toxicity:

• Challenge: Ensuring inhibitors target bacterial AcpS without affecting human phosphopantetheinyl transferases

• Complexity: Humans possess phosphopantetheinyl transferases with similar functions but different structures

• Research approach: Detailed structural comparisons between bacterial and human enzymes to identify exploitable differences

• Importance: Critical for minimizing potential side effects in therapeutic applications

2. Cellular Penetration:

• Challenge: Many in vitro active compounds fail to achieve sufficient intracellular concentrations in bacteria

• Specific considerations for N. europaea: As a Gram-negative bacterium, it has an outer membrane that presents an additional permeability barrier

• Research direction: Incorporation of structural features that facilitate transport across bacterial membranes

• Evidence: The thienyltetrazole compounds designed for AcpS inhibition required optimization for cellular activity beyond enzyme inhibitory potency

3. Resistance Development:

• Challenge: Potential for bacteria to develop resistance through mutations in AcpS or activation of alternative pathways

• Evidence base: In E. coli, YhhU can suppress the acpS conditional lethal phenotype when overexpressed

• Research strategy: Identify and characterize potential bypass mechanisms or resistance pathways

• Mitigation approach: Combination therapies or dual-targeting compounds

4. Spectrum of Activity:

• Challenge: Ensuring broad-spectrum activity across clinically relevant pathogens

• Complexity: Structural differences in AcpS between bacterial species may affect inhibitor binding

• Current evidence: Some AcpS inhibitors show activity against Gram-positive bacteria but require synergy with other agents (like colistin) for Gram-negative coverage

• Research need: Comprehensive comparative analysis of AcpS across bacterial species

5. Pharmacokinetic and Safety Profiles:

• Challenge: Developing compounds with suitable drug-like properties (absorption, distribution, metabolism, excretion)

• Specific concern: The charged nature of effective AcpS inhibitors (e.g., tetrazole groups) may present bioavailability challenges

• Research direction: Medicinal chemistry optimization beyond target potency to address drug-likeness

• Evidence: Lead optimization of the thienyltetrazole family required balancing multiple parameters beyond simple enzyme inhibition

What are promising areas for future research on N. europaea AcpS?

Several promising research directions could significantly advance our understanding of N. europaea AcpS and its potential applications:

1. Structural Biology:

• Research goal: Determine high-resolution crystal structures of N. europaea AcpS in complex with substrates and inhibitors

• Methodological approach: X-ray crystallography or cryo-electron microscopy of the enzyme in different functional states

• Expected impact: Provide crucial insights for structure-based drug design and understanding of species-specific features

• Comparative analysis: Structural comparison with AcpS from other organisms to identify unique features of N. europaea AcpS

2. Systems Biology Integration:

• Research goal: Understand how AcpS functions within the broader metabolic network of N. europaea

• Methodological approach: Combine metabolomics, transcriptomics, and computational modeling

• Expected impact: Identify metabolic vulnerabilities and potential synergistic targets

• Relevance: Particularly important given N. europaea's unique position as an obligate chemolithoautotroph with a specialized metabolism

3. Environmental and Ecological Implications:

• Research goal: Elucidate the role of AcpS in N. europaea's environmental adaptation and nitrification processes

• Methodological approach: Field studies combined with laboratory experiments under various environmental stressors

• Expected impact: Better understanding of how inhibiting AcpS might affect nitrogen cycling in environmental settings

• Context: N. europaea plays a crucial role in the biogeochemical N cycle through nitrification

4. Synthetic Biology Applications:

• Research goal: Engineer N. europaea AcpS and carrier proteins for production of novel bioactive compounds

• Methodological approach: Modify substrate specificity through protein engineering

• Expected impact: Create new biocatalysts for synthesis of valuable compounds

• Innovation potential: Leverage the flexibility of carrier protein-based biosynthetic systems

5. Comparative Analysis Across Nitrifying Bacteria:

• Research goal: Compare AcpS structure, function, and regulation across different ammonia-oxidizing bacteria

• Methodological approach: Phylogenetic analysis combined with biochemical characterization

• Expected impact: Identify conserved features and species-specific adaptations

• Significance: Provide insights into evolutionary adaptation of fundamental metabolic processes in specialized bacteria

How might advances in computational methods enhance our understanding of N. europaea AcpS?

Computational methods are rapidly evolving and offer powerful approaches to advance our understanding of N. europaea AcpS at multiple levels:

1. Advanced Molecular Dynamics Simulations:

• Methodological approach: Employ long-timescale simulations using specialized computing architectures

• Specific applications:

  • Simulate the complete catalytic cycle of AcpS

  • Investigate protein-protein interactions between AcpS and carrier proteins

  • Model the effects of mutations on enzyme dynamics and function

• Expected insights: Reveal transient states and conformational changes not captured in static structural studies

• Technical advancements: Enhanced sampling methods and polarizable force fields will improve simulation accuracy

2. Machine Learning for Inhibitor Design:

• Methodological approach: Apply deep learning models to predict AcpS-inhibitor interactions

• Specific implementations:

  • Graph neural networks to model molecular interactions

  • Generative models to design novel inhibitor scaffolds

  • Quantitative structure-activity relationship (QSAR) models to predict potency

• Practical advantage: The existing dataset of over 700 synthesized AcpS inhibitors provides valuable training data

• Expected impact: Accelerate the discovery of potent, selective inhibitors with optimized properties

3. Quantum Mechanics/Molecular Mechanics (QM/MM) Studies:

• Methodological approach: Apply hybrid QM/MM methods to study the reaction mechanism in atomic detail

• Specific focus: Model the electron transfer process during the phosphopantetheinyl transfer reaction

• Expected insights: Detailed understanding of transition states and energy barriers

• Impact: Guide the design of transition-state analogs as potent inhibitors

4. Genomic Context Analysis and Gene Regulatory Networks:

• Methodological approach: Apply comparative genomics and network analysis to understand acpS regulation

• Implementation: Predict transcription factor binding sites and regulatory elements controlling acpS expression

• Context: Analyze the positioning of acpS within the N. europaea genome in relation to other metabolic genes

• Expected outcome: Better understanding of how acpS expression is coordinated with other aspects of bacterial metabolism

5. Multi-scale Modeling:

• Methodological approach: Integrate models across scales from molecular to cellular

• Implementation: Connect enzyme-level inhibition to whole-cell metabolic effects using genome-scale metabolic models

• Specific application: Predict system-wide effects of AcpS inhibition on N. europaea metabolism

• Expected impact: Identify synergistic targets and better understand resistance mechanisms