Recombinant Burkholderia multivorans Large-conductance mechanosensitive channel (mscL)

What is Burkholderia multivorans and why is its mscL protein significant for research?

Burkholderia multivorans is a gram-negative bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of at least nine closely related species. It has emerged as the most prevalent Bcc representative in many countries, surpassing B. cenocepacia in clinical significance . The large-conductance mechanosensitive channel (mscL) in B. multivorans represents a crucial osmoregulatory protein that responds to membrane tension changes, allowing the bacterium to survive osmotic challenges in diverse environments including the cystic fibrosis lung. This protein is significant for research as it may contribute to the bacterium's environmental persistence and adaptation to the host environment during infection. While generally considered less virulent than B. cenocepacia, B. multivorans can cause chronic infections with exacerbation episodes and has been implicated in "cepacia syndrome" and epidemic outbreaks .

How does the genetic structure of B. multivorans mscL compare to other bacterial mechanosensitive channels?

The mechanosensitive channel of B. multivorans, like other bacterial mscL proteins, likely consists of a pentameric structure embedded in the cytoplasmic membrane. Although the specific genetic structure of B. multivorans mscL is not detailed in the provided literature, genomic analysis of this organism reveals considerable genetic diversity between isolates from different patients. Given that B. multivorans has shown evidence of recombination in its evolutionary history (with an Ia value of 0.852 across 13 sequence types ), its mscL gene may exhibit allelic variations that could impact channel function or regulation. These variations could be investigated through comparative genomic approaches similar to those used in multilocus sequence typing (MLST), which has proven valuable for studying Bcc epidemiology and population structure .

What expression systems are typically used for recombinant production of B. multivorans mscL?

For recombinant production of B. multivorans proteins, E. coli-based expression systems are commonly employed. Based on methodologies described for other Burkholderia proteins, a Gateway high-throughput recombinational cloning system (Invitrogen) can be utilized . The protein can be tagged with His6 for purification purposes by cloning the gene into appropriate vectors like pHIS-MBP-DEST or pTRX-HIS-DEST . For expression of membrane proteins like mscL, it may be beneficial to exclude transmembrane domains initially by expressing only the soluble portions, as demonstrated with the FixL protein where amino acids 329 to 851 (lacking transmembrane domains) were successfully expressed . Following gene amplification from B. multivorans genomic DNA, the construct should be Sanger sequenced to confirm the correct sequence before protein expression and purification.

How do genomic variations in clinical B. multivorans isolates impact mscL structure and function?

Genomic analysis of B. multivorans isolates reveals complex patterns of genomic diversity between patients, including small nucleotide polymorphisms and large structural variations . For mscL specifically, these variations could significantly impact channel structure, gating mechanism, and physiological response to osmotic stress. To investigate this relationship, researchers should consider:

• Conducting whole genome sequencing of multiple clinical isolates to identify mscL variants

• Performing site-directed mutagenesis to introduce observed clinical mutations into a reference mscL construct

• Employing patch-clamp electrophysiology to compare channel conductance and gating properties

• Using molecular dynamics simulations to predict structural changes

High-throughput approaches similar to those used for studying FixL mutations in B. multivorans could be applied, where mutations were introduced using a Q5 site-directed mutagenesis kit and confirmed by Sanger sequencing . Functional studies should assess how these variations affect bacterial survival under osmotic stress conditions relevant to the CF lung environment.

What roles does mscL play in B. multivorans adaptation during chronic cystic fibrosis infection?

During chronic cystic fibrosis infection, B. multivorans undergoes adaptive evolution within the lung environment. Evidence from genomic studies indicates limited within-patient evolution but high between-patient strain diversity, suggesting adaptation to specific host conditions . The mscL channel likely contributes to this adaptation by:

• Mediating osmotic stress responses in the hyperosmotic CF airway environment

• Potentially facilitating antibiotic resistance through efflux mechanisms

• Contributing to biofilm formation and persistence

Research approaches should include longitudinal studies of isolates from chronically infected patients to track mscL mutations over time, correlating genetic changes with phenotypic adaptations like antibiotic susceptibility profiles. Notably, B. multivorans has demonstrated collateral sensitivity to antibiotics , and the mechanosensitive channel might play a role in this phenomenon. Experimental designs should incorporate in vitro evolution experiments under CF-like conditions to observe the emergence and selection of mscL variants, potentially revealing parallel adaptations across multiple experimental lineages similar to the 30 parallel adaptations observed across multiple patients with the endemic B. multivorans strain .

How do post-translational modifications affect B. multivorans mscL channel gating mechanisms?

Post-translational modifications of the mscL protein in B. multivorans may significantly alter channel gating properties, potentially contributing to bacterial adaptation and survival. Research approaches to investigate this question should include:

• Mass spectrometry analysis of mscL isolated from B. multivorans grown under different environmental conditions

• Site-directed mutagenesis of key residues identified as targets for modification

• Electrophysiological characterization of modified versus unmodified channels

• Computational modeling of modification effects on protein structure and dynamics

When designing experiments, researchers should consider that B. multivorans exhibits notable genomic plasticity, including active roles for transposase IS3 and IS5 elements and prophage mobility . These genetic elements could influence gene expression patterns affecting post-translational modification pathways. Experimental conditions should mimic the CF lung environment, potentially incorporating oxidative stress factors that may drive specific modifications relevant to pathogenesis.

What are the optimal conditions for heterologous expression and purification of B. multivorans mscL?

The optimal conditions for heterologous expression and purification of B. multivorans mscL should consider the following methodological approaches:

For successful expression, the Gateway high-throughput recombinational cloning system can be employed, similar to methods used for other Burkholderia proteins . When designing the construct, consider excluding the transmembrane domains initially if expression difficulties are encountered, following the approach used for FixL protein where amino acids 329 to 851 were successfully expressed . After expression, verification of protein identity can be performed using Western blot analysis with anti-His antibodies and mass spectrometry.

How can functional assays be optimized to characterize the B. multivorans mscL channel properties?

Functional characterization of B. multivorans mscL requires multiple complementary approaches to fully understand its biophysical and physiological properties:

• Patch-clamp electrophysiology:

  • Reconstitute purified mscL into liposomes or planar lipid bilayers

  • Apply negative pressure to activate the channel

  • Record single-channel conductance at different membrane tensions

  • Compare gating threshold and kinetics with mscL from model organisms

• Fluorescence-based assays:

  • Load liposomes containing reconstituted mscL with self-quenching fluorescent dyes

  • Monitor dye release upon osmotic downshift as a measure of channel activity

  • Quantify opening probability under various conditions

• In vivo hypo-osmotic shock survival assays:

  • Create mscL knockout B. multivorans strains using pEXKm5 suicide plasmid methodology

  • Complement with wild-type or mutant mscL variants

  • Subject bacteria to rapid osmotic downshift and measure survival rates

  • Compare with survival rates in environmental versus clinical conditions

• Molecular dynamics simulations:

  • Build structural models based on homology to crystallized mscL proteins

  • Simulate membrane tension effects on channel conformation

  • Predict the impact of clinical mutations on gating properties

These methods should be calibrated using well-characterized mscL proteins from model organisms before applying to the B. multivorans channel.

What strategies can overcome challenges in crystallizing B. multivorans mscL for structural studies?

Crystallizing membrane proteins like mscL presents significant challenges due to their hydrophobic nature and conformational flexibility. For B. multivorans mscL, consider these specialized approaches:

• Construct optimization:

  • Generate multiple constructs with varying N- and C-terminal boundaries

  • Introduce mutations that stabilize specific conformational states

  • Create fusion proteins with crystallization chaperones like T4 lysozyme

• Detergent screening:

  • Systematically test a panel of at least 10-12 detergents with different properties

  • Evaluate protein stability using size-exclusion chromatography and thermal shift assays

  • Consider newer amphipathic polymers (amphipols) and nanodiscs as alternatives

• Crystallization condition optimization:

  • Employ high-throughput screening with sparse matrix conditions

  • Explore lipidic cubic phase (LCP) crystallization for membrane proteins

  • Consider addition of specific lipids from B. multivorans membranes

• Alternative structural approaches:

  • Cryo-electron microscopy for high-resolution structures without crystals

  • Nuclear magnetic resonance (NMR) for dynamics studies of isotopically labeled protein

  • X-ray free electron laser (XFEL) diffraction for microcrystals

When designing constructs, researchers should consider the genomic diversity observed in B. multivorans clinical isolates , which may provide clues to naturally occurring variants with improved stability or crystallizability.

How can genomic and proteomic data be integrated to understand mscL evolution in B. multivorans?

Integration of genomic and proteomic data provides powerful insights into mscL evolution in B. multivorans. A comprehensive approach should include:

• Comparative genomics workflow:

  • Align mscL sequences from multiple B. multivorans isolates, particularly the ST-742 strain showing endemic infection patterns

  • Calculate nucleotide diversity (π) and identify signatures of selection (dN/dS ratios)

  • Compare with other Burkholderia species to identify conserved versus variable regions

  • Map variations to functional domains based on structural models

• Proteomic data integration:

  • Perform mass spectrometry analysis of mscL expression under different conditions

  • Identify post-translational modifications specific to particular environmental stresses

  • Correlate protein abundance with transcriptomic data to identify regulatory mechanisms

• Evolutionary analysis:

  • Construct phylogenetic trees based on mscL sequence to trace evolutionary history

  • Compare with MLST-based phylogeny to identify potential horizontal gene transfer events

  • Assess recombination effects, considering that B. multivorans shows moderate evidence of recombination (Ia value of 0.852)

• Structure-function correlation:

  • Map sequence variations to structural models to predict functional consequences

  • Identify co-evolving residues that may maintain channel function despite mutations

This integrated approach can reveal how selection pressures in different environments, particularly the CF lung, have shaped mscL evolution and function in B. multivorans.

What statistical approaches best analyze the correlation between mscL mutations and antibiotic resistance in B. multivorans?

Given that B. multivorans exhibits antibiotic collateral sensitivity , analyzing correlations between mscL mutations and antibiotic resistance profiles requires robust statistical approaches:

• Genome-wide association studies (GWAS):

  • Collect genomic data and minimum inhibitory concentration (MIC) values for multiple antibiotics across a diverse collection of B. multivorans isolates

  • Control for population structure using principal component analysis

  • Apply linear mixed models to identify significant associations between mscL variants and resistance phenotypes

• Multivariate analysis:

  • Perform principal component analysis or multidimensional scaling to visualize relationships between multiple resistance phenotypes

  • Use hierarchical clustering to identify patterns of cross-resistance or collateral sensitivity

  • Apply partial least squares regression to model complex relationships between genetic features and resistance profiles

• Bayesian network analysis:

  • Model conditional dependencies between mscL mutations, other genetic factors, and resistance phenotypes

  • Infer causal relationships and potential mechanistic links

  • Predict resistance outcomes for novel mutations

• Machine learning approaches:

  • Train supervised learning algorithms (random forests, support vector machines) to predict resistance profiles from genetic data

  • Identify feature importance to evaluate the contribution of mscL variations to resistance

  • Validate predictions with experimental testing of engineered mutants

These statistical frameworks should incorporate genomic information beyond single nucleotide polymorphisms, including the large structural genomic variations observed in B. multivorans isolates , which may influence resistance mechanisms through gene dosage effects or regulatory changes.

How can functional heterogeneity in recombinant mscL preparations be assessed and minimized?

Functional heterogeneity in recombinant mscL preparations can significantly impact experimental reproducibility and interpretation. To assess and minimize this variability:

• Analytical assessment techniques:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state distribution

  • Native mass spectrometry to identify presence of different protein species

  • Single-molecule FRET to detect conformational heterogeneity

  • Circular dichroism spectroscopy to verify proper secondary structure

• Sources of heterogeneity to control:

  • Post-translational modifications: Use mass spectrometry to identify modifications and ensure consistency

  • Lipid composition: Standardize reconstitution protocols with defined lipid mixtures

  • Detergent effects: Compare protein function in different detergents and identify optimal conditions

  • Oligomeric state: Implement additional purification steps to isolate homogeneous populations

• Experimental design considerations:

  • Include positive controls with well-characterized mscL proteins in each experimental batch

  • Prepare multiple independent protein preparations to assess batch-to-batch variability

  • Develop quantitative assays that can detect functional subpopulations

• Statistical handling of heterogeneous data:

  • Apply mixture models to identify distinct functional populations

  • Use bootstrapping approaches to estimate confidence intervals

  • Consider Bayesian analysis methods that can incorporate prior knowledge about heterogeneity

How might recombinant B. multivorans mscL be utilized to develop novel antimicrobial strategies?

The mechanosensitive channel of B. multivorans represents a potential target for novel antimicrobial strategies, particularly for cystic fibrosis patients chronically infected with this opportunistic pathogen. Future research directions should explore:

• Channel-targeting antimicrobials:

  • Design small molecules that lock the channel in open conformation, disrupting osmotic balance

  • Develop peptide inhibitors based on structural analyses that block channel function

  • Create antibody-based approaches targeting extracellular epitopes of the channel

• Combination therapy approaches:

  • Exploit collateral sensitivity patterns observed in B. multivorans by combining mscL inhibitors with antibiotics

  • Test synergistic effects with membrane-active antibiotics that may enhance mscL activation

  • Investigate potential for reducing antibiotic resistance development through combined targeting

• Delivery strategies for CF lung environment:

  • Design inhaled formulations with appropriate particle size for deep lung delivery

  • Develop strategies to penetrate B. multivorans biofilms, which may involve osmotic challenge

  • Consider interaction with CF mucus and impact on drug availability

• Resistance prevention strategies:

  • Map potential resistance mutations using laboratory evolution experiments

  • Target highly conserved regions identified through comparative genomics

  • Develop multiple simultaneous targeting approaches to reduce resistance emergence

This research direction should be informed by the genomic analysis of clinical isolates, which has revealed patterns of genomic diversity and evidence for parallel adaptations across multiple patients , suggesting predictable evolutionary trajectories that might be anticipated in drug development.

What is the relationship between mscL function and biofilm formation in B. multivorans infections?

The relationship between mechanosensitive channel function and biofilm formation represents an important area for future investigation, particularly given the significance of biofilms in chronic CF infections. Research should explore:

• Mechanosensing in biofilm development:

  • Compare biofilm formation between wild-type and mscL mutant strains under various osmotic conditions

  • Investigate mscL expression patterns during different stages of biofilm development

  • Examine how mechanical forces within biofilms may influence mscL activation

• mscL role in biofilm stress responses:

  • Characterize how osmotic fluctuations in the CF airway affect biofilm integrity through mscL function

  • Investigate antibiotic penetration and efficacy in wild-type versus mscL-mutant biofilms

  • Examine potential protective effects of mscL during antibiotic treatment of biofilms

• Signaling pathways connecting mechanosensing and biofilm regulation:

  • Identify downstream molecular events following mscL activation that influence biofilm-related gene expression

  • Investigate potential overlap with two-component regulatory systems known to influence virulence, such as the FixLJ system studied in B. multivorans

  • Explore ionic flux through mscL as a potential signal for biofilm regulatory networks

• In vivo biofilm dynamics:

  • Develop animal models to study B. multivorans biofilm formation with various mscL mutations

  • Examine the spatial distribution of mscL expression within biofilm structures using reporter constructs

  • Investigate biofilm architecture differences between clinical isolates with different mscL variants

These studies should consider the genomic plasticity observed in B. multivorans , which may facilitate adaptation to biofilm growth conditions through mutations in mscL or its regulatory elements.

How does the host immune response affect B. multivorans mscL expression and function during infection?

Understanding the interplay between host immunity and bacterial mechanosensing is critical for comprehending B. multivorans pathogenesis in cystic fibrosis. Future research should address:

• Immune effector impacts on mscL expression:

  • Investigate how exposure to antimicrobial peptides affects mscL gene expression and protein function

  • Examine effects of pro-inflammatory cytokines on osmotic conditions in the CF airway and consequent mscL activation

  • Study mscL expression changes during macrophage engulfment and phagolysosomal processing

• mscL-dependent survival mechanisms:

  • Compare survival of wild-type and mscL mutants within THP-1-derived macrophages, similar to studies performed with FixL mutants

  • Investigate whether mscL contributes to B. multivorans persistence within macrophages through osmotic adaptation

  • Examine the role of mscL in bacterial response to oxidative burst and other immune killing mechanisms

• Evolutionary adaptation of mscL during chronic infection:

  • Analyze mscL sequences from longitudinal clinical isolates to identify adaptive mutations

  • Correlate mscL variants with changes in inflammatory markers in patient samples

  • Test whether mscL variants from late-stage chronic infections show altered responses to immune effectors

• Translational implications:

  • Investigate whether mscL function could be modulated to enhance immune clearance

  • Explore the potential for mscL as a biomarker for B. multivorans adaptation during chronic infection

  • Develop therapeutic approaches that synergize with host immune mechanisms

This research direction should consider the observed parallel adaptations across multiple patients infected with B. multivorans , which may include convergent evolution in response to common immune pressures.