Recombinant Francisella tularensis subsp. holarctica Lipid A export ATP-binding/permease protein MsbA (msbA)

What is the role of MsbA in Francisella tularensis?

MsbA in F. tularensis functions as an ABC transporter that facilitates the transport of lipopolysaccharide (LPS) precursors, specifically lipooligosaccharide (LOS), from the cytoplasmic to the periplasmic leaflet of the inner membrane. This process is essential for the biogenesis of the bacterial outer membrane, which contains LPS as a major component of its outer leaflet. While many gram-negative bacteria possess MsbA homologs, the F. tularensis MsbA is particularly interesting because it transports an LPS that is uniquely inert, allowing the bacterium to evade host immune detection . The proper functioning of MsbA is critical for bacterial survival and virulence, as demonstrated by studies showing that mutations affecting LPS transport significantly attenuate bacterial pathogenicity.

How does the structure of MsbA relate to its function?

MsbA undergoes significant conformational changes during its transport cycle, adopting multiple distinct structural states. Recent structural studies have identified at least four open, inward-facing structures that vary in their degree of openness, as well as an open, outward-facing conformation bound to the LPS-precursor Kdo2-lipid A (KDL) . The nucleotide binding domains (NBDs) of MsbA bind ATP molecules, and the energy from ATP hydrolysis drives the conformational changes necessary for substrate transport. The transmembrane domains form a central cavity where the lipid substrate binds. In the inward-facing conformation, this cavity is accessible from the cytoplasmic side, allowing substrate binding. After ATP binding and hydrolysis, MsbA transitions to an outward-facing conformation where the substrate can be released into the periplasmic leaflet. These structural insights are crucial for understanding how MsbA accomplishes the energetically unfavorable task of flipping large, amphipathic LPS molecules across the membrane.

What is the relationship between MsbA and virulence in F. tularensis?

The relationship between MsbA and F. tularensis virulence is substantial, as demonstrated by studies of genes involved in LPS biosynthesis and transport. Mutations in genes related to LPS biosynthesis (such as waaY/FTT1236 and waaL/FTT1238c) result in strains with significantly reduced virulence, with 50% lethal dose (LD50) values increased 100- to 1,000-fold compared to wild-type strains in mouse models . Interestingly, these attenuated strains produce distinctly different pathological patterns compared to wild-type infections. In mutant-infected mice, lung tissue shows widespread necrotic debris while spleens lack necrosis and display neutrophilia. This contrasts with wild-type infections, where lungs have minimal necrosis but spleens show widespread necrotic damage . These differences suggest that proper LPS transport by MsbA is critical for the normal progression of F. tularensis infection and that disruption of this process fundamentally alters the host-pathogen interaction, potentially through changes in how the bacterium is recognized by and interacts with the immune system.

What experimental approaches can be used to study MsbA-nucleotide interactions?

To study MsbA-nucleotide interactions, researchers employ several sophisticated techniques:

• Native Mass Spectrometry (MS): This technique allows for the investigation and resolution of nucleotide binding to MsbA under near-physiological conditions. Native MS has revealed that MsbA has a higher affinity for adenosine 5'-diphosphate (ADP) than for adenosine 5'-triphosphate (ATP) under basal conditions . This approach preserves non-covalent interactions and can detect changes in binding affinities in the presence of different lipid substrates.

• ATP Hydrolysis Assays: These biochemical assays measure the rate of ATP hydrolysis by purified MsbA protein, typically by detecting inorganic phosphate release. By comparing hydrolysis rates in the presence of different substrates or inhibitors, researchers can gain insights into how nucleotide hydrolysis is coupled to transport activity.

• Fluorescence-based Binding Assays: Techniques such as fluorescence polarization or FRET (Förster Resonance Energy Transfer) can monitor the binding of fluorescently labeled nucleotides to MsbA in real-time, providing data on binding kinetics and affinities.

• Site-directed Mutagenesis: Strategic mutations in the nucleotide binding domains can disrupt specific interactions with ATP or ADP, allowing researchers to evaluate the functional importance of particular residues in nucleotide binding and hydrolysis.

• Structural Biology Approaches: X-ray crystallography and cryo-electron microscopy have been used to capture MsbA structures with bound nucleotides, revealing the molecular details of these interactions at atomic resolution .

These complementary approaches together provide a comprehensive understanding of how MsbA interacts with nucleotides during its transport cycle.

How can researchers effectively study the lipid-mediated modulation of MsbA activity?

Studying lipid-mediated modulation of MsbA activity requires a multifaceted approach:

• Native Mass Spectrometry: This technique has proven particularly valuable for investigating how lipids affect MsbA function. Recent studies have demonstrated that the LPS-precursor Kdo2-lipid A (KDL) can tune the selectivity of MsbA for ATP over ADP . By incubating MsbA with different lipid species and analyzing nucleotide binding, researchers can identify specific lipid-dependent changes in MsbA behavior.

• Reconstitution in Proteoliposomes: Purified MsbA can be incorporated into artificial lipid vesicles with defined lipid compositions. This system allows researchers to systematically vary the lipid environment and measure its impact on MsbA transport activity or ATPase function.

• Molecular Dynamics Simulations: Computational approaches can model the interactions between MsbA and various lipid species, predicting how these interactions might affect protein dynamics and function. These simulations can generate hypotheses that can be tested experimentally.

• EPR Spectroscopy: Electron paramagnetic resonance spectroscopy with site-directed spin labeling can detect lipid-induced conformational changes in MsbA by measuring changes in the mobility and environment of specific regions of the protein.

• Lipid Binding Assays: Techniques such as microscale thermophoresis or isothermal titration calorimetry can directly measure the binding affinities of different lipid species to MsbA.

By combining these approaches, researchers can develop a detailed understanding of how specific lipids modulate MsbA activity, potentially revealing regulatory mechanisms that coordinate LPS transport with membrane biogenesis.

What genomic approaches are most effective for studying F. tularensis MsbA evolution and diversity?

Several genomic approaches are particularly effective for studying F. tularensis MsbA evolution and diversity:

• Whole Genome Sequencing (WGS): This approach provides the most comprehensive view of genetic variation across F. tularensis strains, including the msbA gene and its regulatory regions. WGS-based multilocus sequence typing (MLST) can be used to classify strains and understand the broader genomic context of msbA variations .

• Comparative Genomics: By comparing msbA sequences across multiple Francisella species and subspecies, researchers can identify conserved regions likely critical for function, as well as variable regions that might reflect adaptation to different ecological niches.

• Selective Pressure Analysis: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) in the msbA gene across different strains can reveal whether the gene is under purifying, neutral, or positive selection.

• Population Genetics Approaches: Measures of genetic diversity such as nucleotide diversity (π) and Tajima's D can provide insights into the evolutionary history of the msbA gene within F. tularensis populations.

• Phylogenetic Analysis: Constructing phylogenetic trees based on msbA sequences can reveal relationships between strains and possible horizontal gene transfer events.

• Synteny Analysis: Examining the genomic context of msbA across different strains can reveal conservation or rearrangements in gene order, potentially indicating functional relationships with neighboring genes.

These approaches can help researchers understand how MsbA has evolved within the Francisella genus and identify strain-specific variations that might affect protein function or regulation, potentially contributing to differences in virulence or host range.

How should researchers design experiments to investigate MsbA-mediated LPS transport in F. tularensis?

Designing experiments to investigate MsbA-mediated LPS transport in F. tularensis requires careful consideration of multiple factors:

• In vitro Transport Assays:

  • Purify recombinant F. tularensis MsbA and reconstitute it into proteoliposomes

  • Incorporate fluorescently labeled LPS precursors and measure their translocation

  • Include appropriate controls: no ATP, non-hydrolyzable ATP analogs, and known MsbA inhibitors

  • Compare wild-type MsbA with site-directed mutants affecting ATP binding, hydrolysis, or substrate recognition

• Genetic Manipulation Strategies:

  • Use conditional expression systems since msbA is likely essential

  • Employ CRISPR interference (CRISPRi) for partial knockdown

  • Create point mutations that affect specific aspects of MsbA function

  • Establish complementation systems with wild-type or mutant alleles

• LPS Analysis Methods:

  • Develop extraction protocols optimized for F. tularensis LPS

  • Use mass spectrometry to characterize LPS structural changes

  • Employ silver staining and Western blotting to visualize LPS profiles

  • Implement pulse-chase experiments with radiolabeled precursors to track LPS movement

• Membrane Biology Approaches:

  • Use fluorescent membrane probes to assess membrane asymmetry

  • Implement techniques like FRAP (Fluorescence Recovery After Photobleaching) to study membrane dynamics

  • Employ freeze-fracture electron microscopy to examine membrane organization

• Structural Biology Integration:

  • Design experiments informed by known MsbA structures

  • Test hypotheses about conformational states using EPR or FRET

  • Use crosslinking studies to capture transport intermediates

By combining these approaches and carefully controlling variables such as temperature, pH, and ionic conditions, researchers can generate reliable data on MsbA-mediated LPS transport in F. tularensis.

What controls should be included in studies examining the effects of msbA mutations on F. tularensis virulence?

Studies examining the effects of msbA mutations on F. tularensis virulence require rigorous controls:

• Genetic Controls:

  • Wild-type parental strain (positive control for virulence)

  • Complemented mutant (reintroduction of wild-type msbA to confirm phenotype specificity)

  • Well-characterized attenuated strain (e.g., LVS) as a reference point

  • Strains with mutations in unrelated virulence genes to distinguish msbA-specific effects

• In vitro Growth Controls:

  • Growth curves in standard media to assess basic viability

  • Growth under various stress conditions (pH, temperature, nutrient limitation)

  • Intracellular growth in macrophages and other relevant cell types

  • LPS extraction and analysis to confirm expected effects on LPS structure

• Animal Infection Controls:

  • Vehicle-only control group (typically PBS)

  • Dose-response series to determine accurate LD50 values

  • Multiple infection routes to distinguish route-specific effects

  • Time-matched tissue collection for comparable bacterial burden analysis

• Molecular Verification:

  • RT-qPCR to confirm gene expression levels

  • Western blotting to verify protein production

  • Functional assays to assess MsbA activity in the mutant strains

  • Whole genome sequencing to confirm no secondary mutations

• Immunological Assessment:

  • Include both immunocompetent and immunodeficient mice

  • Measure both innate and adaptive immune responses

  • Compare cytokine profiles between wild-type and mutant infections

  • Assess protective immunity through challenge studies

The inclusion of these controls ensures that any observed differences in virulence can be specifically attributed to the msbA mutations rather than to secondary effects or experimental variables.

What statistical methods are most appropriate for analyzing data from MsbA functional studies?

The selection of appropriate statistical methods for analyzing MsbA functional studies depends on the specific experimental design and data characteristics:

• Enzyme Kinetics Data:

  • Non-linear regression for fitting Michaelis-Menten or allosteric models

  • F-test for comparing different kinetic models

  • Analysis of covariance (ANCOVA) for comparing kinetic parameters between wild-type and mutant proteins

  • Bootstrap analysis for estimating confidence intervals of kinetic parameters

• Transport Assay Data:

  • Repeated measures ANOVA for time-course experiments

  • Mixed-effects models when dealing with multiple variables

  • Non-parametric alternatives (e.g., Kruskal-Wallis) when normality assumptions are violated

  • Bayesian approaches for integrating prior knowledge with experimental data

• Virulence Studies:

  • Kaplan-Meier survival analysis with log-rank test for comparing survival curves

  • Cox proportional hazards models for assessing the impact of multiple factors

  • Power analysis to ensure adequate sample sizes for detecting biologically relevant effects

  • Multiple comparison corrections (e.g., Bonferroni, Benjamini-Hochberg) when testing multiple hypotheses

• Structural Data Analysis:

  • Principal Component Analysis (PCA) for identifying major conformational changes

  • Cluster analysis for grouping similar structural states

  • Cross-correlation analysis for identifying coordinated motions

  • Markov State Models for analyzing transition pathways between conformations

• Imaging and Microscopy Data:

  • Image segmentation and object recognition algorithms

  • Colocalization analysis (Pearson's or Mander's coefficients)

  • Intensity profile analysis for membrane distribution

  • Particle tracking for dynamic studies

How does the mechanism of MsbA-mediated LPS transport in F. tularensis compare with that in other gram-negative bacteria?

The mechanism of MsbA-mediated LPS transport in F. tularensis shares fundamental similarities with other gram-negative bacteria but also exhibits important differences:

Similarities:

• In both F. tularensis and other gram-negative bacteria like E. coli, MsbA functions as an ABC transporter that flips LPS precursors from the cytoplasmic to the periplasmic leaflet of the inner membrane

• The basic "alternating access" transport mechanism involving ATP binding, hydrolysis, and conformational changes appears conserved

• The core structure of MsbA with nucleotide-binding domains and transmembrane domains is maintained across species

Key Differences:

• The substrate specificity differs significantly. F. tularensis produces an unusual LPS that is essentially inert, failing to stimulate immune responses in the way that typical gram-negative LPS does . This suggests that the MsbA substrate-binding pocket may have evolved differently to accommodate this structurally distinct LPS.

• Native mass spectrometry studies have revealed that F. tularensis MsbA has a higher baseline affinity for ADP rather than ATP, but this preference can be shifted by the LPS-precursor Kdo2-lipid A (KDL) . This lipid-mediated modulation of nucleotide preference may represent a unique regulatory mechanism in F. tularensis.

• The genetic context of msbA varies between species. In F. tularensis, genes involved in LPS biosynthesis such as waaY (FTT1236), waaZ (FTT1237), and waaL (FTT1238c) have unique properties compared to their counterparts in other bacteria, despite sharing similar functions .

• The consequences of MsbA dysfunction appear to differ. In E. coli, MsbA deficiency leads to LPS accumulation in the inner membrane and growth arrest. In F. tularensis, mutations in related LPS biosynthesis genes result in viable bacteria with altered virulence properties and distinct interactions with host immunity .

These differences may reflect adaptation to F. tularensis' intracellular lifestyle and its unique strategy of immune evasion, highlighting how a conserved transport mechanism has been modified during evolution to suit specific bacterial lifestyles.

How might the unique properties of F. tularensis MsbA be exploited for vaccine development?

The unique properties of F. tularensis MsbA offer several promising avenues for vaccine development:

• Attenuated Live Vaccine Strains: Mutations in genes related to LPS transport and biosynthesis, including those affecting MsbA function, produce attenuated strains that retain immunogenicity. Research has shown that mice immunized with mutant strains defective in LPS biosynthesis (such as waaY mutants) display >10-fold protective effects against virulent type A F. tularensis challenge . Specifically, boosting mice with a waaY::TrgTn mutant protected 40% of mice challenged intranasally with 190 CFU of Schu S4 and 60% of mice challenged with 1,900 CFU of the virulent strain . These findings suggest that targeted modification of MsbA or its substrate could generate effective live attenuated vaccine candidates.

• Altered Immunostimulatory Properties: The LPS of wild-type F. tularensis is unusually inert immunologically, which contributes to immune evasion . Mutations affecting MsbA function or its substrate could potentially create modified LPS structures with enhanced immunostimulatory properties, serving as built-in adjuvants to strengthen vaccine efficacy.

• Recombinant Protein Vaccines: Purified recombinant MsbA itself could serve as an antigen in subunit vaccines. As an essential membrane protein with exposed epitopes, antibodies against MsbA might neutralize the bacterium or enhance opsonization.

• Rational Design Approach: Understanding the structural dynamics of MsbA through techniques like native mass spectrometry enables rational design of mutations that optimize the balance between attenuation and immunogenicity.

• Heterologous Expression Systems: The MsbA transport system could potentially be engineered to deliver specific antigens or immunomodulators to enhance vaccine efficacy.

Implementation of these approaches would require careful consideration of safety profiles, stability of the attenuated phenotype, and comprehensive immunological characterization. Nevertheless, the unique properties of F. tularensis MsbA provide a promising foundation for novel vaccine strategies against this challenging pathogen.

What potential exists for developing MsbA-targeted antimicrobials against F. tularensis?

The development of MsbA-targeted antimicrobials against F. tularensis holds significant promise for several reasons:

• Essential Target: MsbA is critical for bacterial viability due to its role in LPS transport, making it an attractive target where inhibition would likely be lethal to the bacterium . Unlike targets involved in non-essential processes, resistance to MsbA inhibitors would be more difficult to develop.

• Structural Insights: High-resolution structural data of MsbA in different conformational states provides templates for structure-based drug design . These structures reveal potential binding pockets that could be targeted by small molecules to disrupt the transport cycle.

• Allosteric Modulation: Native mass spectrometry studies have shown that lipids can modulate MsbA's nucleotide preference . This suggests the existence of allosteric sites that could be targeted to disrupt the normal coupling between ATP hydrolysis and substrate transport.

• Species Specificity: While MsbA is conserved across gram-negative bacteria, the unique properties of F. tularensis MsbA and its substrate could enable the development of species-specific inhibitors with reduced effects on beneficial microbiota.

• Transport Cycle Disruption: Compounds could be designed to:

  • Prevent ATP binding or hydrolysis

  • Trap MsbA in a non-productive conformational state

  • Compete with LPS for binding to MsbA

  • Disrupt the lipid-mediated regulation of MsbA activity

• Combination Therapy Potential: MsbA inhibitors could potentially synergize with existing antibiotics by compromising the integrity of the bacterial outer membrane, increasing permeability to compounds that normally cannot penetrate.

• Novel Screening Approaches: The development of high-throughput assays measuring MsbA-mediated LPS transport or ATP hydrolysis would facilitate screening of chemical libraries for potential inhibitors.

Challenges to overcome include achieving sufficient bacterial penetration, avoiding toxicity to human ABC transporters, and preventing rapid resistance development. Nevertheless, the critical role of MsbA in bacterial survival and the availability of detailed structural and functional information make it a compelling target for novel antimicrobial development against F. tularensis, a CDC Category A bioterrorism agent.

What are the challenges and solutions in expressing and purifying recombinant F. tularensis MsbA for structural studies?

The expression and purification of recombinant F. tularensis MsbA presents several challenges with corresponding solutions:

Challenges:

• Membrane Protein Expression: As a membrane protein, MsbA is often toxic when overexpressed, resulting in low yields and protein aggregation.

• Proper Folding: Maintaining the native structure during expression and purification is difficult, especially when expressing in heterologous systems.

• Lipid Environment Requirements: MsbA function is closely tied to its lipid environment, and removing it from its native membrane can affect its stability and activity.

• Detergent Selection: Finding detergents that efficiently extract MsbA while maintaining its structure and function is challenging.

• Protein Stability: Purified MsbA can be unstable, limiting the time available for structural studies.

Solutions:

• Expression Systems:

  • Use tunable expression systems (e.g., PBAD promoter) to control expression levels

  • Express in C43(DE3) or C41(DE3) E. coli strains specifically designed for membrane proteins

  • Consider Pichia pastoris or insect cell expression systems for improved folding

  • Use cell-free expression systems with supplied lipids/detergents

• Fusion Tags and Constructs:

  • Incorporate stabilizing fusion partners (e.g., GFP, MBP) that can also report on folding

  • Design constructs based on structural information to remove flexible regions that might impede crystallization

  • Use cleavable purification tags (His-tag, FLAG-tag) positioned to minimize interference with function

• Purification Strategy:

  • Screen multiple detergents systematically (DDM, LMNG, UDM, etc.)

  • Implement detergent exchange during purification to find optimal conditions

  • Consider amphipols or nanodiscs for final stabilization

  • Add lipids during purification to maintain native-like environment

• Stability Enhancement:

  • Add specific lipids known to interact with MsbA

  • Include nucleotides (ATP, ADP) or non-hydrolyzable analogs

  • Use thermostability assays to identify stabilizing conditions

  • Apply protein engineering to introduce stabilizing mutations

• Quality Control:

  • Verify protein activity through ATPase assays

  • Assess homogeneity through size-exclusion chromatography

  • Use limited proteolysis to confirm proper folding

  • Employ negative stain electron microscopy for initial structural assessment

Implementing these solutions has enabled researchers to obtain high-resolution structures of MsbA in different conformational states, providing valuable insights into its transport mechanism .

How can researchers effectively design PCR-based detection methods for F. tularensis and its msbA gene?

Designing effective PCR-based detection methods for F. tularensis and its msbA gene requires careful consideration of several factors:

• Target Selection Strategy:

  • For species identification, target conserved regions within F. tularensis but distinct from other bacteria

  • For subspecies differentiation, target regions with subspecies-specific polymorphisms

  • For msbA specifically, identify regions conserved within F. tularensis msbA but different from related ABC transporters

  • Include multiple targets (at least 10) to increase reliability, as is standard practice for F. tularensis detection

• Primer Design Principles:

  • Design primers with optimal length (18-25 bp), GC content (40-60%), and melting temperatures (within 2-3°C of each other)

  • Avoid secondary structures, primer-dimer formation, and repetitive sequences

  • Include at least one gene target for virulence (e.g., iglC, pdpD), metabolism (e.g., gyr, speA), and transcriptional regulation

  • Validate primer specificity using in silico tools against comprehensive sequence databases

• Assay Optimization:

  • Determine optimal annealing temperatures through gradient PCR

  • Titrate Mg²⁺ concentration for maximum sensitivity and specificity

  • Optimize cycle numbers to avoid late-cycle artifacts

  • Include internal amplification controls to identify potential inhibition

• Multiplex PCR Development:

  • Design primers with compatible annealing temperatures

  • Ensure amplicon sizes can be clearly distinguished on gels or by melt curve analysis

  • Consider developing assays that can simultaneously detect F. tularensis, B. anthracis, and Y. pestis for bioterrorism surveillance

  • Balance primer concentrations to avoid preferential amplification

• Validation Parameters:

  • Test against diverse strain collections including all subspecies

  • Include closely related non-Francisella species as negative controls

  • Determine limit of detection using serial dilutions

  • Assess reproducibility across different thermocyclers and reagent lots

• Special Considerations for msbA Detection:

  • Be aware that msbA is part of a large family of ABC transporters, so primers must be carefully designed to avoid cross-reactivity

  • Consider targeting unique regions of msbA or its flanking sequences

  • For detection of specific mutations or variants, design allele-specific primers or use probe-based methods

  • When studying expression levels, design qRT-PCR primers spanning exon-exon junctions to avoid genomic DNA amplification

By following these guidelines, researchers can develop robust and specific PCR-based methods for detecting F. tularensis and its msbA gene in various research and diagnostic settings .

What techniques are most effective for analyzing the lipid composition of F. tularensis membranes in relation to MsbA function?

Analyzing the lipid composition of F. tularensis membranes in relation to MsbA function requires sophisticated analytical techniques:

• Mass Spectrometry-Based Approaches:

  • Lipidomics: Untargeted lipidomic analysis using high-resolution mass spectrometry can identify and quantify hundreds of lipid species in F. tularensis membranes

  • Native MS: Native mass spectrometry has proven particularly valuable for studying how specific lipids like Kdo2-lipid A (KDL) interact with MsbA and modulate its function

  • LC-MS/MS: Liquid chromatography tandem mass spectrometry enables detailed structural characterization of complex lipids

  • MALDI-TOF: Matrix-assisted laser desorption/ionization time-of-flight MS provides rapid lipid profiling with minimal sample preparation

• Chromatographic Methods:

  • Thin-Layer Chromatography (TLC): Provides a simple way to separate and visualize major lipid classes

  • High-Performance Liquid Chromatography (HPLC): Offers superior separation of complex lipid mixtures

  • Gas Chromatography (GC): Excellent for analyzing fatty acid compositions after derivatization

• Spectroscopic Techniques:

  • Nuclear Magnetic Resonance (NMR): Provides detailed structural information about lipids without destroying the sample

  • Infrared Spectroscopy: Can identify functional groups and monitor lipid oxidation

  • Raman Spectroscopy: Offers non-destructive analysis of lipid structure and packing

• Membrane Biophysical Characterization:

  • Differential Scanning Calorimetry (DSC): Measures phase transitions in membrane lipids

  • Fluorescence Anisotropy: Assesses membrane fluidity that may impact MsbA function

  • Atomic Force Microscopy (AFM): Provides topographical information about membrane organization

• Functional Correlation Approaches:

  • Reconstitution Studies: Purified MsbA can be reconstituted into proteoliposomes with defined lipid compositions to directly assess how specific lipids affect transport activity

  • Native Lipid Extraction and Replacement: Extracting native lipids from F. tularensis and testing their ability to modulate recombinant MsbA activity

  • Genetic Manipulation: Altering genes involved in lipid biosynthesis and observing effects on MsbA function

• Specialized LPS Analysis:

  • Silver Staining: Visualizes LPS banding patterns after gel electrophoresis

  • Western Blotting: Uses antibodies against specific LPS epitopes

  • Structural Analysis: Combines chemical degradation with MS analysis to determine detailed LPS structure

A comprehensive approach would involve correlating changes in lipid composition (determined by these analytical techniques) with alterations in MsbA function (assessed through transport assays or ATPase activity measurements). This integrated analysis can reveal which specific lipid species or properties are most critical for proper MsbA function in F. tularensis .