Recombinant Burkholderia cenocepacia Lipid A export ATP-binding/permease protein MsbA (msbA)

What is the role of MsbA in B. cenocepacia and why is it significant in pathogenesis?

MsbA is an essential ATP-binding cassette (ABC) transporter that functions as a lipid flippase, transporting lipid A and lipopolysaccharide (LPS) from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane in gram-negative bacteria . In B. cenocepacia, this function is particularly significant as the bacterium is an opportunistic respiratory pathogen that poses a serious threat to individuals with cystic fibrosis (CF), causing severe inflammatory responses in the lung . The proper transport of lipid A by MsbA is crucial for the formation of the outer membrane, which serves as a barrier against host antimicrobial compounds . Without functional MsbA, lipid A accumulates in the inner membrane, disrupting membrane integrity and bacterial viability, making it an attractive research target for potential therapeutic interventions .

How does B. cenocepacia MsbA differ from homologous proteins in other gram-negative bacteria?

While the core structure and function of MsbA are conserved across gram-negative bacteria, B. cenocepacia MsbA exhibits specific adaptations related to the unique composition of its lipid A. Unlike many other gram-negative bacteria, B. cenocepacia lipid A contains 4-amino-4-deoxy-L-arabinose (Ara4N) residues that confer resistance to cationic antimicrobial peptides . These modifications likely influence the substrate recognition and transport mechanism of MsbA in B. cenocepacia compared to homologs like the well-studied MsbA from Salmonella typhimurium . Structural analyses suggest that these differences may manifest in the transmembrane domains and substrate-binding pocket of the protein, although detailed comparative structural studies specific to B. cenocepacia MsbA are still emerging in the literature .

What are the basic structural features of MsbA transporters that researchers should understand?

MsbA belongs to the ABC transporter superfamily and functions as a homodimer. Each monomer consists of a transmembrane domain (TMD) containing six membrane-spanning α-helices and a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP . The protein cycles through several conformational states during the transport process: an inward-facing conformation that accepts lipid A from the cytoplasmic leaflet, an outward-facing conformation following ATP binding that releases lipid A to the periplasmic leaflet, and intermediate states during this cycle .

X-ray crystallography and cryo-electron microscopy have revealed that MsbA displays a large amplitude opening in the transmembrane portal in its inward-facing conformation, which is essential for accommodating the bulky lipid A substrate . The conformational changes during the transport cycle are driven by ATP binding and hydrolysis at the NBDs, which are transmitted to the TMDs via coupling helices . These structural features are critical considerations when designing experiments to express, purify, and study recombinant MsbA proteins.

What expression systems are most effective for producing recombinant B. cenocepacia MsbA?

For recombinant expression of B. cenocepacia MsbA, bacterial expression systems based on E. coli strains specifically designed for membrane protein expression have proven most effective. The preferred strains include C41(DE3), C43(DE3), and Lemo21(DE3), which are engineered to mitigate the toxicity often associated with overexpressing membrane proteins . Expression should be conducted under the control of the T7 promoter using vectors such as pET or pBAD series.

When establishing an expression protocol, researchers should consider the following methodological approach:

• Clone the msbA gene from B. cenocepacia (using strains like H111, K56-2, or Pc715j) with appropriate restriction sites as demonstrated for other B. cenocepacia genes .

• Transform the expression construct into the selected E. coli strain.

• Grow cultures at lower temperatures (16-20°C) after induction to slow protein production and allow proper folding.

• Induce expression with low concentrations of inducer (0.1-0.5 mM IPTG or 0.002-0.02% arabinose) to prevent formation of inclusion bodies.

• Supplement growth media with ligands or substrates that may stabilize the protein during expression.

This approach minimizes aggregation and misfolding while maximizing the yield of functional protein incorporated into the membrane.

What purification strategies yield the highest quality recombinant B. cenocepacia MsbA for structural and functional studies?

Purification of recombinant B. cenocepacia MsbA requires specialized techniques due to its membrane-embedded nature. A methodological workflow that has proven successful for similar ABC transporters includes:

• Membrane Isolation: After cell lysis, differential centrifugation is used to isolate total membranes (typically 100,000 × g for 1 hour).

• Solubilization: Carefully selected detergents are crucial. The use of facial amphiphiles like FA-3 has shown success in maintaining MsbA in an active state during crystallization studies . Alternative detergents including n-dodecyl-β-D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), or a combination of detergents may be employed depending on downstream applications.

• Affinity Chromatography: Utilizing a His-tag or other affinity tag for initial capture, followed by size exclusion chromatography to separate monomeric/dimeric protein from aggregates.

• Functional Verification: ATPase activity assays to confirm that the purified protein retains functionality.

Assessment of purity should be performed using SDS-PAGE, while proper folding can be evaluated using circular dichroism spectroscopy. Western blotting with antibodies against either MsbA or the affinity tag provides confirmation of identity .

How can researchers overcome the challenges of expressing membrane proteins like MsbA?

Membrane proteins like MsbA present significant expression challenges due to their hydrophobic nature and complex folding requirements. Researchers can implement several strategies to overcome these obstacles:

• Fusion Partners: Utilize fusion proteins such as maltose-binding protein (MBP), thioredoxin, or GFP to enhance solubility and provide a means to monitor expression and folding.

• Codon Optimization: Adapt the coding sequence to the codon usage bias of the expression host to improve translation efficiency.

• Chaperone Co-expression: Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ) to assist proper folding.

• Stabilizing Mutations: Introduce point mutations that enhance thermostability without affecting function, based on homology modeling and conservation analysis.

• Use of Nanodiscs or Amphipols: After initial purification, reconstitute the protein into nanodiscs or amphipols to provide a more native-like membrane environment.

What methodologies are most effective for studying the conformational dynamics of B. cenocepacia MsbA?

Understanding the conformational changes of MsbA during the transport cycle requires sophisticated biophysical techniques. The following approaches have proven valuable:

• X-ray Crystallography: Has successfully captured MsbA in different conformational states, as demonstrated with S. typhimurium MsbA at 2.8 Å resolution . This technique requires high-quality crystals, which for B. cenocepacia MsbA would likely benefit from the use of facial amphiphiles like FA-3 and lipid A during crystallization to stabilize specific conformations .

• Cryo-Electron Microscopy (cryo-EM): Increasingly used for membrane proteins, this technique can capture MsbA in multiple conformations without crystallization constraints. Sample preparation should focus on homogeneity and particle orientation diversity.

• Double Electron-Electron Resonance (DEER) Spectroscopy: By introducing cysteine pairs at strategic positions and labeling with spin probes, researchers can measure distances between domains during conformational changes in a near-native environment.

• Single-Molecule Förster Resonance Energy Transfer (smFRET): Allows real-time observation of conformational dynamics by measuring the energy transfer between fluorophores attached to different domains of the protein.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Provides information about protein dynamics and solvent accessibility of different regions during the transport cycle.

These methods should be used complementarily, as each provides unique insights into different aspects of MsbA's structure and dynamics. For example, crystallography and cryo-EM provide static snapshots at high resolution, while spectroscopic methods offer dynamic information in solution .

How can researchers effectively study the lipid A transport mechanism of B. cenocepacia MsbA?

Investigating the lipid A transport mechanism requires specialized assays that can monitor flippase activity. Researchers can implement the following methodological approaches:

• Reconstituted Proteoliposome Assays: MsbA should be reconstituted into liposomes with fluorescently labeled lipid A analogs. The translocation activity can be monitored by measuring fluorescence quenching or by using membrane-impermeant quenchers that only affect labeled lipids in the outer leaflet.

• ATPase Activity Coupling: Measuring ATP hydrolysis rates in the presence of different lipid substrates can provide insights into substrate specificity and transport coupling. This can be done using colorimetric assays that detect inorganic phosphate release or coupled enzyme assays .

• Binding Assays: Surface plasmon resonance (SPR) or microscale thermophoresis (MST) can quantify the binding affinity of lipid A to MsbA in different conformational states.

• Molecular Dynamics Simulations: Computational approaches can model the interactions between MsbA and lipid A during the transport process, providing hypotheses that can be tested experimentally.

The unique structure of B. cenocepacia lipid A, particularly the presence of Ara4N modifications that contribute to antimicrobial peptide resistance, makes these studies particularly relevant . Researchers should design experiments that can specifically address how these modifications affect recognition and transport by MsbA.

What structural features of B. cenocepacia MsbA contribute to substrate specificity for modified lipid A structures?

The substrate specificity of B. cenocepacia MsbA for its native lipid A, which contains unique modifications, likely resides in specific structural elements of the protein. Based on comparative analysis with MsbA structures from other organisms, researchers should focus on:

• Transmembrane Portal Region: The inward-facing conformation of MsbA displays a large amplitude opening in the transmembrane portal that accommodates bulky lipid A molecules . For B. cenocepacia MsbA, this portal likely contains specific residues that recognize the Ara4N-modified lipid A characteristic of this bacterium .

• Binding Pocket Architecture: Putative lipid A density has been observed inside the transmembrane cavity of MsbA in crystallographic studies, consistent with a "trap and flip" transport model . The binding pocket architecture in B. cenocepacia MsbA would be expected to complement the unique chemical features of its native lipid A substrate.

• Periplasmic Surface Cleft: Additional lipid A binding sites have been identified near an outer surface cleft at the periplasmic ends of the transmembrane helices, which may function as a post-transport docking site . The composition of this site in B. cenocepacia MsbA may be specialized for its modified lipid A.

To experimentally determine these structural features, site-directed mutagenesis of conserved and divergent residues in these regions, followed by functional assays measuring transport of native B. cenocepacia lipid A versus modified variants, would provide valuable insights into the molecular basis of substrate specificity.

How does B. cenocepacia MsbA contribute to antimicrobial peptide resistance?

B. cenocepacia exhibits exceptional resistance to antimicrobial peptides (AMPs), particularly polymyxins, which is crucial for its pathogenicity in cystic fibrosis patients . MsbA plays an indirect but essential role in this resistance through its function in lipid A transport. The mechanism involves several interconnected processes:

• Transport of Modified Lipid A: B. cenocepacia lipid A is uniquely modified with 4-amino-4-deoxy-L-arabinose (Ara4N) residues in both the lipid A and inner core oligosaccharide regions . These positively charged modifications reduce the net negative charge of the outer membrane, decreasing the binding affinity of cationic AMPs . MsbA must efficiently transport these modified lipid A molecules to maintain membrane integrity.

• Integration with Stress Response Systems: B. cenocepacia employs the alternative sigma factor RpoE to control the expression of genes involved in extracytoplasmic stress response . This regulatory network influences both membrane composition and the efficiency of MsbA-mediated lipid transport under antimicrobial stress conditions.

• Complementary Resistance Mechanisms: MsbA functions within a broader context of resistance mechanisms, including the zinc metalloproteases ZmpA and ZmpB, which degrade various antimicrobial peptides including β-defensins, cathelicidin LL37, and elafin . Efficient MsbA-mediated lipid A transport maintains the membrane platform required for these secreted proteases to function.

Researchers investigating this aspect should consider designing experiments that specifically probe the relationship between MsbA transport activity and the incorporation of Ara4N-modified lipid A into the outer membrane, possibly through conditional expression systems or specific transport assays with modified substrates.

What is the relationship between MsbA function and the virulence of B. cenocepacia in cystic fibrosis infections?

The functional integrity of MsbA is intrinsically linked to B. cenocepacia virulence in cystic fibrosis infections through several mechanisms:

• Maintenance of Membrane Barrier Function: Proper MsbA-mediated lipid A transport is essential for outer membrane biogenesis, which serves as the first line of defense against host immune factors in the CF lung environment .

• Support for Intracellular Survival: B. cenocepacia can survive and replicate within airway epithelial cells and macrophages, primary sentinels of the lung . This intracellular persistence depends on the structural integrity of the bacterial membrane, which is maintained through MsbA activity.

• Facilitation of Inflammatory Response Modulation: The lipid A component of LPS is a potent activator of the host inflammatory response through TLR4 signaling. The specific structure of B. cenocepacia lipid A, transported by MsbA, influences the magnitude and character of this inflammatory response, contributing to the severe inflammation observed in cystic fibrosis patients infected with this pathogen .

• Connection to Quorum-Sensing Systems: Virulence factor expression in B. cenocepacia is regulated by quorum-sensing systems including CepIR and CciIR, which control the production of proteases that degrade host antimicrobial peptides . The functional membrane established through MsbA activity provides the necessary platform for these signaling systems to operate.

How do mutations in the msbA gene affect B. cenocepacia survival and adaptation during chronic CF lung infections?

During chronic cystic fibrosis infections, B. cenocepacia undergoes evolutionary adaptation to the lung environment, with implications for MsbA function:

• Selective Pressure on Lipid Transport Systems: The constant exposure to antimicrobial peptides and other host defense mechanisms in the CF lung creates selective pressure that may drive adaptive mutations in msbA and related genes involved in lipid A biosynthesis and transport .

• Observed Clonal Diversification: Studies have demonstrated that chronic B. cenocepacia infections often result from colonization by a few clonal bacterial strains that subsequently diversify . An example is the epidemic B. cenocepacia clone ST856 prevalent in Serbian CF populations, which exhibits variations in virulence and genotype as a consequence of lung adaptation .

• Potential for Compensatory Mutations: If mutations occur in msbA that compromise transport efficiency, compensatory mutations may arise in genes involved in lipid A biosynthesis or modification, potentially altering the substrate for MsbA and adapting the transport system to the chronic infection environment.

• Stress-Induced Mutagenesis: The extracytoplasmic stress response controlled by RpoE, which influences MsbA function, may itself promote mutagenesis under the stress conditions present in the CF lung, creating a feedback loop of adaptation .

Researchers investigating this aspect should consider longitudinal studies of clinical isolates from CF patients to track changes in the msbA gene sequence and expression levels over time, correlating these changes with alterations in lipid A structure, antimicrobial resistance, and virulence characteristics.

How can structural information about MsbA be leveraged to develop inhibitors targeting B. cenocepacia infections?

The development of MsbA inhibitors represents a promising therapeutic approach for B. cenocepacia infections, particularly given the intrinsic antibiotic resistance of this pathogen. Researchers can leverage structural information through the following methodological framework:

• Structure-Based Drug Design: Using high-resolution structures of MsbA in different conformational states , researchers can identify potential binding pockets for small molecule inhibitors. Key targets include:

  • The nucleotide-binding domains, particularly the ATP-binding site

  • The transmembrane domains at the lipid A binding site

  • Interfaces between domains that are critical for conformational changes

• Virtual Screening Approaches: Molecular docking of compound libraries against identified binding sites can prioritize candidates for experimental validation. Docking should account for the dynamic nature of MsbA by using multiple conformational states from crystallography or molecular dynamics simulations .

• Fragment-Based Drug Discovery: This approach involves screening small molecular fragments that bind with low affinity but high ligand efficiency, which can then be elaborated into more potent inhibitors. For membrane proteins like MsbA, biophysical methods such as surface plasmon resonance or thermal shift assays can be adapted for fragment screening.

• Allosteric Inhibitor Development: Beyond the active site, allosteric sites that influence conformational dynamics can be targeted. The intermediate inward-facing conformation observed in structural studies of MsbA suggests potential allosteric sites that could lock the transporter in an inactive state .

• Lipid A Analogs as Competitive Inhibitors: Designing molecular mimics of lipid A that bind MsbA but cannot be transported represents another strategy. These would need to account for the unique Ara4N modifications found in B. cenocepacia lipid A .

Validation of potential inhibitors should include ATPase activity assays, lipid A transport assays in reconstituted systems, and ultimately assessment of antibacterial activity against B. cenocepacia with particular attention to efficacy in relevant infection models.

What advanced genetic tools can be used to study the function of MsbA in B. cenocepacia?

Investigating MsbA function in B. cenocepacia requires sophisticated genetic approaches due to the essential nature of this gene and the genetic complexity of this organism. Researchers can employ the following methodologies:

• Conditional Expression Systems: Since msbA is essential, conditional expression systems like those based on rhamnose-inducible promoters or tetracycline-responsive elements allow for controlled depletion of MsbA to study the effects on cell viability and membrane integrity.

• CRISPR-Cas9 Genome Editing: Adapted for use in Burkholderia species, this technology enables precise genetic modifications, including:

  • Introduction of point mutations to study structure-function relationships

  • Creation of fluorescent protein fusions to visualize MsbA localization

  • Engineering of specific lipid A modifications to study substrate specificity

• Suicide Vector-Based Mutagenesis: As demonstrated for other B. cenocepacia genes, suicide vectors like pSHAFT2 can be used for targeted gene replacement or interruption . The methodology involves:

  • Amplification of gene fragments (~1,200 bp)

  • Cloning into vectors like pBBR1MCS or pBluescriptII

  • Transfer to B. cenocepacia using conjugation with E. coli donor strains

  • Selection of double crossover recombinants by antibiotic sensitivity

• Transposon Mutagenesis Libraries: For identifying genetic interactions with msbA, transposon libraries can reveal synthetic lethal or suppressor relationships that provide insights into the wider genetic network affecting lipid A transport.

• RNA-Seq and ChIP-Seq Analyses: These approaches can identify genes co-regulated with msbA under different stress conditions, particularly those involved in the RpoE-mediated extracytoplasmic stress response that influences antimicrobial peptide resistance .

When applying these genetic tools, researchers should be mindful of the potential polar effects on neighboring genes and the complex regulation of lipid A biosynthesis and transport pathways.

How can researchers effectively study the interactions between B. cenocepacia MsbA and the host immune system in cystic fibrosis?

Investigating the complex interplay between B. cenocepacia MsbA-transported lipid A and the host immune response requires integrated experimental approaches spanning multiple biological scales:

• Cell Culture Models of CF Airway Epithelium:

  • Air-liquid interface cultures of primary CF bronchial epithelial cells or CFTR-mutated cell lines

  • Co-culture systems incorporating epithelial cells and macrophages to recapitulate the complexity of host-pathogen interactions

  • Measurement of inflammatory mediators (IL-8, IL-1β, TNF-α) and antimicrobial peptide production in response to bacterial challenge

• Ex Vivo Infection Models:

  • Precision-cut lung slices from CF mouse models or explanted human CF lungs

  • Organoid cultures derived from CF patient cells

  • These systems preserve tissue architecture and cellular diversity while allowing controlled infection experiments

• Advanced Microscopy Techniques:

  • Live cell imaging to track bacterial trafficking in host cells

  • Super-resolution microscopy to visualize MsbA localization during infection

  • Correlative light and electron microscopy to link MsbA function with ultrastructural changes in bacterial and host cell membranes

• Immunological Assays:

  • Neutrophil extracellular trap (NET) formation assays to assess the interaction between B. cenocepacia and a key CF lung defense mechanism

  • Macrophage phagocytosis and killing assays to determine how MsbA-dependent membrane properties affect intracellular survival

  • Inflammasome activation studies, as B. cenocepacia has been shown to activate the NLRP3 inflammasome through mechanisms potentially linked to LPS recognition

• Systems Biology Approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) of both pathogen and host during infection

  • Network analysis to identify key nodes in host-pathogen interaction networks that depend on MsbA function

  • Computational modeling of infection dynamics incorporating MsbA transport kinetics and host immune responses

These methodologies should be applied using both wild-type B. cenocepacia and strains with modified msbA expression or function to directly assess the contribution of this transporter to host-pathogen interactions in the CF lung environment.

How can understanding B. cenocepacia MsbA function contribute to developing combination therapies for CF patients?

The strategic targeting of MsbA function in B. cenocepacia could enhance the efficacy of existing antibiotics and novel therapeutics through several mechanisms:

• Membrane Permeabilization Strategies: Partial inhibition of MsbA could compromise outer membrane integrity, potentially increasing the penetration of antibiotics that normally have limited efficacy against B. cenocepacia. This approach would target the fundamental role of MsbA in maintaining the lipid A component of the outer membrane barrier .

• Sensitization to Antimicrobial Peptides: By interfering with MsbA-mediated transport of Ara4N-modified lipid A, researchers could potentially restore B. cenocepacia sensitivity to endogenous antimicrobial peptides and polymyxin antibiotics . This represents a form of antibiotic adjuvant therapy that leverages the host's innate defense mechanisms.

• Anti-Virulence Approach: Rather than directly killing bacteria, modulating MsbA function could attenuate virulence by altering the immunostimulatory properties of lipid A and reducing inflammatory damage in the CF lung . This represents a precision-based approach to disrupting pathogenesis while minimizing selection pressure for resistance.

• Multi-Target Inhibition Strategies: Combination therapies targeting both MsbA and complementary resistance mechanisms, such as the zinc metalloproteases ZmpA and ZmpB that degrade antimicrobial peptides , could provide synergistic effects by simultaneously compromising multiple defense systems.

For clinical translation, researchers should focus on developing assays that can rapidly assess the effects of potential MsbA-targeting compounds on membrane permeability, antimicrobial peptide sensitivity, and inflammatory responses in relevant CF infection models. This multifaceted approach acknowledges the complex role of MsbA in both bacterial survival and host-pathogen interactions.

What are the most promising approaches for developing specific inhibitors of B. cenocepacia MsbA?

Developing specific inhibitors of B. cenocepacia MsbA presents unique challenges but also opportunities for therapeutic innovation:

• Targeting Species-Specific Structural Features: Comparative analysis of MsbA structures from different bacterial species can identify unique structural elements in B. cenocepacia MsbA that could be targeted for selective inhibition . Key regions to focus on include:

  • The transmembrane domains that interact with the Ara4N-modified lipid A specific to B. cenocepacia

  • Regions involved in conformational changes during the transport cycle

  • Interfaces between protein subunits critical for dimerization and function

• Nucleotide-Binding Domain Inhibitors: ATP hydrolysis is essential for MsbA function, making the nucleotide-binding domains attractive targets. While ATP-binding sites are conserved across ABC transporters, adjacent residues that differ between species can be exploited to develop selective inhibitors that interact with both the conserved ATP-binding pocket and species-specific residues.

• Lipid A Competitive Analogues: Developing structural mimics of B. cenocepacia lipid A that competitively bind to MsbA but cannot be transported represents another approach. These would need to incorporate key recognition elements including the Ara4N modifications characteristic of B. cenocepacia lipid A .

• Allosteric Inhibitors: Targeting non-conserved allosteric sites that influence conformational dynamics could provide selectivity while avoiding the challenges of competing with ATP or lipid A binding. The intermediate inward-facing conformation observed in structural studies suggests potential binding sites that could lock the transporter in an inactive state .

• Covalent Inhibitors: Identifying non-conserved cysteine residues in B. cenocepacia MsbA that could be targeted by covalent inhibitors offers another strategy for achieving selectivity.

For all these approaches, researchers should implement screening cascades that progressively assess compounds for: (1) binding to recombinant B. cenocepacia MsbA, (2) inhibition of ATPase activity, (3) disruption of lipid A transport, (4) specificity against other ABC transporters, and (5) antibacterial activity against B. cenocepacia with limited toxicity to human cells.

How can researchers evaluate the efficacy of MsbA-targeting compounds in relevant models of CF lung infection?

Evaluating MsbA-targeting therapeutic candidates requires a systematic approach using progressively complex and clinically relevant models:

• In Vitro Bacterial Assays:

  • Minimum inhibitory concentration (MIC) determinations in standard media and in artificial CF sputum medium to account for the unique chemical environment of CF airways

  • Time-kill studies to assess bactericidal versus bacteriostatic effects

  • Biofilm inhibition and disruption assays, as B. cenocepacia forms biofilms in the CF lung

  • Selection for resistance studies to determine the genetic barrier to resistance

• Cell-Based Infection Models:

  • Intracellular survival assays in macrophages and airway epithelial cells, as B. cenocepacia can persist within these cell types

  • Transmigration assays to assess effects on bacterial ability to cross epithelial barriers

  • Co-culture systems incorporating both epithelial and immune cells to model complex interactions

  • Measurement of inflammatory mediators to determine if compounds reduce the hyperinflammatory response characteristic of B. cenocepacia infections

• Ex Vivo Models:

  • Precision-cut lung slices from CF mouse models or explanted human CF lungs treated with MsbA inhibitors

  • CF patient-derived bronchial organoids to assess drug efficacy in patient-specific contexts

  • Human CF sputum samples to test antimicrobial activity in authentic host material

• In Vivo Models:

  • Acute and chronic infection models in CFTR-deficient mice

  • Galleria mellonella infection model as a rapid screening tool

  • Rat models, which have been validated for studying B. cenocepacia pathogenesis

• Combination Therapy Assessment:

  • Checkerboard assays to determine synergy with conventional antibiotics

  • Combination studies with CFTR modulators to assess efficacy in the context of restored CFTR function

  • Evaluation of effects on polymyxin sensitivity, given the role of MsbA in transporting Ara4N-modified lipid A that contributes to polymyxin resistance

These evaluations should include appropriate controls including strains with altered msbA expression to confirm that observed effects are mediated through the intended target. Pharmacokinetic and pharmacodynamic studies in relevant models are also essential to determine whether sufficient drug concentrations can be achieved at the site of infection.