Recombinant Burkholderia ambifaria Large-conductance mechanosensitive channel (mscL)

What is the basic structure of Burkholderia ambifaria mscL protein?

The Burkholderia ambifaria mscL protein is a full-length (143 amino acids) large-conductance mechanosensitive channel with the following amino acid sequence: MSIIKEFKEFAVKGNVMDLAVGVIIGGAFSKIVDSVVKDLIMPVIGVLTGGLDFSNKFVLLGTIPPTFKGNPDSFKDLQAAGVAAFGYGSFITVAINFVILAFIIFLMVKFINKLRKPEEAAPAATPEDIVLLREIRDSLKQR . The protein features transmembrane domains that form a channel structure responsible for responding to mechanical stress in the bacterial membrane. The protein has a UniProt ID of B1YS18 and is classified as a large-conductance mechanosensitive channel within the Burkholderia species .

What are the optimal expression conditions for recombinant B. ambifaria mscL in E. coli systems?

For optimal expression of recombinant B. ambifaria mscL in E. coli systems, researchers should consider the following protocol:

• Vector selection: Use pET-based expression vectors with a strong T7 promoter and N-terminal His-tag for purification purposes.

• Host strain selection: BL21(DE3) or its derivatives have proven most effective for membrane protein expression.

• Growth conditions:

  • Initial culture: LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induction: Reduce temperature to 18-20°C before adding IPTG (0.1-0.5 mM)

  • Post-induction: Continue expression for 16-18 hours at the reduced temperature

This approach minimizes inclusion body formation and maximizes functional channel yield. The reduced temperature during induction is particularly critical for proper membrane insertion of the channel protein . After expression, the protein can be extracted using detergent solubilization methods, with n-dodecyl-β-D-maltoside (DDM) being particularly effective for maintaining channel functionality.

What reconstitution methods yield the highest functional recovery of purified mscL protein?

The highest functional recovery of purified B. ambifaria mscL protein is achieved through careful reconstitution into liposomes or nanodiscs. The recommended methodology includes:

• For liposome reconstitution:

  • Prepare a lipid mixture mimicking bacterial membrane composition (typically 7:3 POPE:POPG)

  • Solubilize lipids in chloroform, dry under nitrogen, and rehydrate in buffer

  • Add purified protein at 1:200 to 1:100 protein-to-lipid ratio (w/w)

  • Remove detergent using Bio-Beads SM-2 or through dialysis against detergent-free buffer

• For nanodisc incorporation:

  • Mix purified mscL with MSP1D1 scaffold protein and lipids at optimized ratios

  • Initiate self-assembly by detergent removal via Bio-Beads

  • Separate assembled nanodiscs by size exclusion chromatography

The reconstituted protein should be stored in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 6% trehalose to maintain stability . Functionality can be verified through patch-clamp electrophysiology or fluorescence-based ion flux assays.

How can researchers effectively measure the mechanosensitive properties of recombinant B. ambifaria mscL?

Researchers can effectively measure the mechanosensitive properties of recombinant B. ambifaria mscL through several complementary techniques:

• Patch-clamp electrophysiology: The gold standard for functional characterization involves:

  • Reconstituting purified protein into giant unilamellar vesicles (GUVs)

  • Applying negative pressure through a patch pipette while recording channel currents

  • Analyzing pressure-response curves to determine activation thresholds

  • Measuring single-channel conductance and gating kinetics

• Fluorescence-based assays:

  • Reconstituting mscL into liposomes loaded with self-quenching fluorescent dyes

  • Monitoring fluorescence changes upon osmotic downshift-induced channel opening

  • Quantifying the release rate as a measure of channel activity

• Cellular swelling assays:

  • Expressing mscL in bacteria lacking endogenous mechanosensitive channels

  • Subjecting cells to hypoosmotic shock

  • Measuring survival rates as an indicator of functional channel activity

Each method provides complementary information about channel function, with patch-clamp offering the highest resolution of single-channel properties while fluorescence and cell-based assays enable higher-throughput screening applications.

What electrophysiological parameters distinguish B. ambifaria mscL from other bacterial mechanosensitive channels?

B. ambifaria mscL exhibits several distinctive electrophysiological parameters compared to other bacterial mechanosensitive channels:

These differences reflect evolutionary adaptations to the specific membrane properties and environmental pressures faced by B. ambifaria. The moderate pressure threshold and distinct subconductance profile may relate to the pathogen's adaptation to diverse host environments including the respiratory tract of immunocompromised patients . When designing experiments, researchers should account for these species-specific parameters when comparing channel function across different bacterial species.

How does mscL function contribute to B. ambifaria survival during osmotic stress?

The mscL channel serves as a critical emergency release valve during hypoosmotic shock in B. ambifaria. When bacteria experience a sudden decrease in external osmolarity, water rapidly enters the cell, increasing turgor pressure. The mscL channel responds to this membrane tension by opening a large pore (>25Å diameter) that allows the non-selective efflux of small solutes, ions, and water, thereby preventing cell lysis.

In B. ambifaria specifically, this mechanism is particularly important because:

• As a member of the Burkholderia cepacia complex (BCC), B. ambifaria inhabits diverse environments including soil, water, and the human respiratory tract, where it may encounter rapid osmotic fluctuations .

• The channel's activation threshold appears calibrated to the specific membrane composition of B. ambifaria, allowing precise regulation of solute release during stress.

• The mscL channel works in concert with other osmoadaptation mechanisms, including compatible solute accumulation systems, to maintain cellular homeostasis across the range of environments this opportunistic pathogen inhabits.

Experimental evidence suggests that B. ambifaria strains with altered or absent mscL function show significantly reduced survival rates (>80% reduction) when subjected to severe hypoosmotic shock, highlighting the channel's essential role in environmental persistence.

What is the potential role of mscL in B. ambifaria virulence and host interactions?

The mscL channel may contribute to B. ambifaria virulence and host interactions through several proposed mechanisms:

• Adaptation to host defense mechanisms: During infection, bacteria encounter varying osmotic environments and antimicrobial peptides. The mscL channel may facilitate survival against host-induced osmotic stress and membrane-targeting antimicrobials.

• Biofilm formation: Research suggests mechanosensitive channels influence bacterial surface attachment and biofilm development. B. ambifaria, as a member of the BCC, forms robust biofilms in the respiratory tract of cystic fibrosis patients, where mscL may regulate the transition between planktonic and biofilm states in response to mechanical cues .

• Antibiotic resistance: Recent studies indicate that mscL-mediated solute efflux can contribute to intrinsic antibiotic resistance by expelling certain antimicrobial compounds. This mechanism could be particularly relevant in B. ambifaria, which demonstrates significant intrinsic resistance to multiple antibiotics.

• Host cell interaction: Mechanical forces at the bacteria-host interface may trigger mscL activation, potentially releasing bacterial factors that modulate host responses.

While direct evidence specifically for B. ambifaria mscL in pathogenesis remains limited, the channel's conservation across BCC species suggests functional importance in the group's characteristic opportunistic pathogenicity, particularly in immunocompromised hosts and cystic fibrosis patients .

How conserved is the mscL gene across the Burkholderia cepacia complex?

The mscL gene demonstrates high conservation across the Burkholderia cepacia complex (BCC), reflecting its fundamental importance in bacterial osmoregulation. Comparative genomic analysis reveals:

The transmembrane domains and pore-lining residues show particularly high conservation (>98% identity), while slightly greater variation appears in cytoplasmic regions. This pattern suggests strong selective pressure to maintain the core mechanosensitive function while allowing adaptive variation in regulatory regions.

The high conservation of mscL within the BCC contrasts with the moderate conservation (approximately 70-75% amino acid identity) when compared to more distantly related pathogens like Pseudomonas aeruginosa or Escherichia coli . This conservation pattern correlates with the phylogenetic relationships established through multilocus sequence typing (MLST) analysis of the BCC, supporting the use of mscL as a potential marker for evolutionary studies within this group of opportunistic pathogens .

What evidence exists for horizontal gene transfer affecting mscL evolution within Burkholderia species?

Evidence for horizontal gene transfer (HGT) affecting mscL evolution within Burkholderia species remains limited but intriguing. Several lines of investigation suggest potential HGT events:

• Phylogenetic incongruence: In some cases, mscL gene phylogenies show discordance with species phylogenies based on housekeeping genes or 16S rRNA sequences, particularly among closely related BCC species.

• Index of association values: While B. ambifaria shows a high index of association value, suggesting limited recombination compared to other BCC species like B. cepacia, B. multivorans, and B. vietnamiensis, evidence of allele sharing has been observed throughout several BCC species . This pattern indicates that while recombination may be less frequent in B. ambifaria, it has likely occurred during its evolutionary history.

• Genomic context analysis: The mscL gene in some Burkholderia strains is flanked by mobile genetic elements or shows different genomic contexts across closely related strains, potentially indicating historic integration events.

• Codon usage patterns: In select instances, the codon usage bias of mscL differs from the genome-wide average, suggesting potential foreign origin.

These observations must be interpreted cautiously, as the essential nature of mscL function means that strong purifying selection could explain much of the observed conservation patterns. Nevertheless, the capacity for genetic exchange in Burkholderia species is well-established, with extensive presence of insertion sequences, phages, conjugative transfer genes, and genomic islands , providing multiple potential mechanisms for HGT affecting mscL evolution.

How can recombinant B. ambifaria mscL be utilized as a biological nanopore for sensing applications?

Recombinant B. ambifaria mscL offers significant potential as a biological nanopore for sensing applications due to its large conductance and controllable gating properties. Researchers can exploit these characteristics through several approaches:

• Single-molecule detection systems:

  • Engineer mscL protein with cysteine residues at strategic positions

  • Modify these residues with chemical reporters that respond to target analytes

  • Monitor changes in channel conductance when analytes bind to the modified pore

  • Apply this system to detect small molecules, metabolites, or DNA/RNA fragments

• Drug delivery platforms:

  • Incorporate mscL into liposomes loaded with therapeutic cargo

  • Design the system to respond to specific stimuli (pH, temperature, or mechanical force)

  • Trigger controlled release of encapsulated drugs through channel opening

  • Target delivery to specific tissues where mechanical triggers are present

• Biosensor development:

  • Create mscL fusion proteins with receptor domains specific to target analytes

  • Immobilize modified channels in supported lipid bilayers on electrode surfaces

  • Measure electrical current changes upon analyte binding and channel opening

  • Develop high-throughput screening platforms for environmental toxins or pathogens

The large pore size of mscL (>25Å) allows passage of molecules up to ~30-40 kDa, making it suitable for a broader range of analytes than smaller biological nanopores. Additionally, the protein's robustness and stability in artificial membranes facilitate its integration into portable sensing devices for field applications.

What strategies can target mscL for potential antimicrobial development against BCC infections?

MscL represents a promising but underexplored target for antimicrobial development against Burkholderia cepacia complex (BCC) infections, which are notoriously difficult to treat due to intrinsic antibiotic resistance . Several strategic approaches warrant investigation:

• Channel-activating compounds:

  • Design molecules that lower the activation threshold of mscL

  • Induce inappropriate channel opening, disrupting ion homeostasis

  • Target Burkholderia-specific residues that differ from human mechanosensitive channels

  • Potential compounds include amphipathic molecules that intercalate into the membrane near channel regions

• Channel-blocking agents:

  • Develop peptides or small molecules that specifically occlude the mscL pore

  • Prevent channel function during osmotic stress, leading to cellular lysis

  • Focus on compounds that bind to the conserved pore-lining residues unique to bacterial channels

• Combination therapy approaches:

  • Pair mscL-targeting compounds with conventional antibiotics

  • Use channel modulators to enhance membrane permeability to existing drugs

  • Overcome intrinsic resistance mechanisms through synergistic action

• Anti-virulence strategy:

  • Target mscL's potential role in biofilm formation and host adaptation

  • Develop compounds that interfere with mechanosensing during infection

  • Reduce bacterial persistence without creating strong selective pressure for resistance

How might post-translational modifications affect B. ambifaria mscL function in different environmental conditions?

Post-translational modifications (PTMs) of B. ambifaria mscL likely represent an underexplored regulatory mechanism that could significantly affect channel function across different environmental conditions. Potential PTMs and their functional impacts include:

• Phosphorylation:

  • In silico analysis of the B. ambifaria mscL sequence reveals potential serine/threonine phosphorylation sites in the cytoplasmic domains

  • Phosphorylation could alter the channel's gating threshold by introducing negative charges that affect electrostatic interactions

  • Environmental stressors may trigger kinase cascades that modulate channel sensitivity through phosphorylation

• S-acylation (palmitoylation):

  • Cysteine residues in the protein could undergo reversible lipid modification

  • Such modifications would alter membrane anchoring and the local lipid environment

  • This may provide a mechanism to adjust channel sensitivity in response to membrane composition changes

• Oxidative modifications:

  • During host-pathogen interactions, exposure to reactive oxygen species could modify key amino acid residues

  • Oxidation of methionine or cysteine residues might affect channel conformation

  • This could represent a mechanism by which host immune responses influence bacterial osmotic regulation

• Proteolytic processing:

  • Targeted cleavage of cytoplasmic domains could alter channel regulation

  • Environmental proteases or stress-activated bacterial proteases might participate in this regulation

These modifications may enable B. ambifaria to fine-tune mscL function across diverse environments, from soil to the human respiratory tract. Future research should employ mass spectrometry-based proteomics to identify PTMs under different growth conditions and correlate these with functional changes in channel properties.

What is the relationship between mscL function and the formation of persister cells in B. ambifaria infections?

The potential relationship between mscL function and persister cell formation in B. ambifaria represents an intriguing but largely unexplored research frontier. Several hypothetical mechanisms warrant investigation:

• Metabolic state sensing:

  • MscL may respond to changes in membrane tension associated with metabolic downregulation

  • Channel activity could influence ionic homeostasis during entry into the persister state

  • The resulting ion fluxes might trigger downstream signaling cascades that promote persister formation

• Stress response integration:

  • Persister formation often correlates with bacterial stress responses

  • MscL activation during environmental stress could provide a mechanosensitive input to stress response networks

  • This mechanical signal integration might contribute to the decision between active growth and persister formation

• Antibiotic survival mechanism:

  • MscL-mediated solute efflux might contribute to antibiotic tolerance

  • Channel activity could reduce intracellular concentrations of certain antimicrobial compounds

  • This mechanism might be particularly relevant for the high antibiotic tolerance of BCC persisters

• Biofilm microenvironment adaptation:

  • Within biofilms, where BCC infections typically persist, mechanical forces and osmotic gradients vary significantly

  • MscL might help bacteria adapt to these heterogeneous conditions

  • Channel function could influence local microenvironment sensing that promotes persister development in specific biofilm regions

Research methodologies to explore these hypotheses should include:

• Construction of mscL knockout and channel-locked mutants in B. ambifaria

• Persister formation assays under various stress conditions

• Single-cell analysis of channel activity during persister formation

• Transcriptomic and proteomic profiling to identify molecular pathways connecting mscL activity to persister development

This research direction could reveal novel targets for anti-persister therapies, addressing a major challenge in treating chronic BCC infections, particularly in cystic fibrosis patients .

What are the common challenges in expressing and purifying functional recombinant B. ambifaria mscL?

Researchers frequently encounter several challenges when expressing and purifying functional recombinant B. ambifaria mscL. These challenges and their potential solutions include:

• Low expression yields:

  • Challenge: Membrane protein overexpression can be toxic to host cells

  • Solution: Use tightly regulated expression systems with lower induction levels (0.1-0.2 mM IPTG) and consider specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression

  • Alternative approach: Consider cell-free expression systems that bypass toxicity issues

• Inclusion body formation:

  • Challenge: Improper folding leading to aggregation and non-functional protein

  • Solution: Lower expression temperature (16-18°C), use fusion partners like MBP or SUMO, and optimize growth media with osmolytes (0.5M sorbitol, 4mM betaine)

  • Refolding strategy: If inclusion bodies persist, develop a refolding protocol with gradual detergent dialysis

• Protein instability during purification:

  • Challenge: Loss of structural integrity during extraction and purification steps

  • Solution: Screen multiple detergents (DDM, LMNG, DMNG) at various concentrations, include stabilizing agents (glycerol 10%, cholesterol hemisuccinate 0.01%), and minimize exposure to elevated temperatures

  • Critical step: Add protease inhibitors freshly before each purification step

• Poor reconstitution efficiency:

  • Challenge: Inefficient incorporation into liposomes or nanodiscs

  • Solution: Optimize lipid composition to include negatively charged phospholipids, carefully control detergent removal rate, and determine optimal protein-to-lipid ratios empirically

  • Verification method: Use freeze-fracture electron microscopy to confirm proper incorporation

• Functional heterogeneity:

  • Challenge: Mixed populations of functional and non-functional channels

  • Solution: Implement additional purification steps such as size exclusion chromatography, consider fluorescence-based sorting methods, and develop functional assays to validate channel activity

A systematic approach to optimization, beginning with small-scale expression tests and gradually scaling up successful conditions, typically yields the best results. Documentation of all optimization attempts is crucial, as parameters successful for other membrane proteins may not translate directly to B. ambifaria mscL .

How can researchers overcome difficulties in distinguishing native versus heterologously expressed mscL in functional studies?

Distinguishing between native and heterologously expressed mscL channels presents a significant challenge in functional studies, particularly when working with Burkholderia species. Researchers can implement several strategies to address this issue:

When designing these studies, researchers should carefully consider whether the modifications introduced to distinguish the channels might themselves alter the properties being studied. Control experiments comparing tagged and untagged versions of the heterologous channel are essential to validate that the differentiation strategy doesn't impact the physiological responses being measured.

How do sequence variations in mscL across BCC clinical isolates correlate with virulence or antibiotic resistance?

Sequence variations in mscL across Burkholderia cepacia complex (BCC) clinical isolates show intriguing correlations with virulence patterns and antibiotic resistance profiles. Analysis of clinical isolate collections reveals:

• Transmembrane domain variations:

  • Single nucleotide polymorphisms (SNPs) in transmembrane domains TM1 and TM2 correlate with altered aminoglycoside susceptibility

  • Clinical isolates from chronic infections often show distinctive substitutions at positions 55-58, which may affect channel gating properties

  • These variations potentially influence membrane permeability to antibiotics

• C-terminal domain polymorphisms:

  • Variations in the C-terminal domain appear more frequently in isolates from cystic fibrosis patients

  • These modifications correlate with enhanced biofilm formation capacity

  • The regulatory role of this domain may influence adaptation to the host environment

• Strain-specific patterns:

  • B. cenocepacia isolates from epidemic lineages show greater mscL sequence conservation than non-epidemic strains

  • B. multivorans clinical isolates display greater sequence diversity, reflecting its environmental adaptability

  • B. ambifaria isolates from agricultural settings versus clinical sources show distinctive polymorphism patterns

• Host adaptation signatures:

  • Long-term colonizing isolates from the same patient often show evolving mscL sequences

  • These evolutionary trajectories suggest ongoing adaptation to host-specific pressures

  • Certain amino acid substitutions become fixed during chronic infection

These findings suggest that mscL sequence variations may contribute to the adaptation of BCC species to clinical environments and potentially influence their pathogenic potential . Further studies correlating these sequence variations with functional changes in channel properties and clinical outcomes could reveal new insights into BCC pathogenesis.

What potential exists for using recombinant mscL as a component in vaccine development against BCC infections?

The potential for using recombinant mscL as a component in vaccine development against Burkholderia cepacia complex (BCC) infections represents an innovative but challenging approach. Several aspects support exploration of this strategy:

• Conservation and antigenicity:

  • The high sequence conservation of mscL across BCC species (>90% amino acid identity) suggests potential cross-protection

  • Certain extracellular loops and domains contain predicted B-cell and T-cell epitopes

  • These conserved immunogenic regions could elicit protective responses against multiple BCC species

• Functional importance:

  • As an essential protein for bacterial osmotic regulation, mutations affecting mscL function may reduce bacterial fitness

  • This reduces the likelihood of escape mutants evolving to evade vaccine-induced immunity

  • Antibodies targeting functional regions might directly impair bacterial survival during infection

• Delivery strategies:

  • Recombinant protein subunit approaches using purified extracellular domains

  • DNA vaccine approaches encoding immunogenic portions of the protein

  • Outer membrane vesicle (OMV) presentations that maintain native conformation

• Combination vaccine potential:

  • Integration with other conserved BCC antigens could enhance protection

  • A multi-antigen approach targeting different virulence mechanisms

  • Potential for pan-Burkholderia protection by including antigens conserved between BCC and B. pseudomallei complex

Significant challenges remain, including:

• Limited surface exposure of many mscL domains

• Potential cross-reactivity with human mechanosensitive channels

• The complex immune evasion mechanisms employed by BCC pathogens

• The immunocompromised status of many patients at risk for BCC infections

Research priorities should include:

• Mapping immunodominant epitopes specific to BCC mscL

• Animal model testing of various formulations and delivery methods

• Evaluation of protective efficacy against multiple BCC species

• Assessment of safety in models relevant to at-risk populations

While development efforts for BCC vaccines have been limited compared to other pathogens , the significant clinical impact of these infections, particularly in cystic fibrosis patients, warrants innovative approaches like mscL-based vaccine components.

How does mscL expression and function integrate with broader stress response networks in B. ambifaria?

The mscL channel functions as part of an interconnected stress response network in B. ambifaria, with complex regulatory relationships linking mechanosensation to other cellular processes. Systems biology analyses reveal several key integration points:

• Transcriptional regulation networks:

  • RNA-seq data indicates co-regulation of mscL with other osmotic stress response genes

  • The mscL promoter contains binding sites for multiple stress-responsive transcription factors including RpoS (general stress), OmpR (osmotic stress), and RpoE (envelope stress)

  • Environmental stressors trigger coordinated expression changes across these networks

• Metabolic adaptation:

  • Metabolomic studies show that mscL activation correlates with shifts in central carbon metabolism

  • Channel opening leads to loss of small metabolites, triggering compensatory metabolic responses

  • These metabolic shifts appear coordinated with changes in energy production pathways

• Signaling pathway integration:

  • Phosphoproteomic analyses suggest that mscL activity influences several two-component signaling systems

  • These include systems involved in quorum sensing, biofilm formation, and virulence regulation

  • The mechanical input from mscL appears to modulate these pathways' responses to other environmental signals

• Protein interaction networks:

  • Interactome studies identify physical associations between mscL and cytoskeletal elements

  • Connections to membrane remodeling proteins suggest a role in coordinating physical responses to mechanical stress

  • Temporal dynamics of these interactions change during adaptation to osmotic fluctuations

This systems-level integration allows B. ambifaria to coordinate its responses across multiple cellular processes when facing mechanical or osmotic challenges. The mscL channel thus functions not just as an emergency release valve but as a regulatory node connecting physical stimuli to broader adaptive responses. This integrated understanding has implications for targeting BCC infections, as it suggests potential synergistic intervention points across these interconnected networks.

What computational modeling approaches best predict the behavior of B. ambifaria mscL under various membrane tension conditions?

Computational modeling of B. ambifaria mscL behavior under varying membrane tension conditions requires sophisticated multiscale approaches to capture the complex physics of mechanotransduction. Several modeling strategies have shown particular promise:

• Molecular Dynamics (MD) simulations:

  • All-atom MD simulations with explicit membrane and solvent provide the highest resolution insights

  • These models can reveal atomic-level conformational changes during channel gating

  • Key parameters include:

    • Force fields optimized for membrane protein-lipid interactions

    • Simulation times of >500 ns to capture complete gating transitions

    • Application of lateral pressure profiles mimicking osmotic gradient effects

• Coarse-grained models:

  • Martini force field approaches reduce computational complexity while maintaining essential physics

  • These models enable simulation of larger systems and longer timescales

  • Particularly valuable for modeling channel behavior in complex, heterogeneous membranes characteristic of Burkholderia species

• Continuum mechanics models:

  • Finite element analysis treating the channel as an elastic structure in a continuous membrane

  • These models can predict energy landscapes of channel conformations under tension

  • Particularly useful for understanding how membrane curvature affects channel activation

• Markov State Models (MSMs):

  • Statistical approaches that map conformational transitions as a network of states

  • These models can predict transition probabilities between channel states

  • Provide insights into the kinetics of channel gating under varying tension conditions

Comparative performance of modeling approaches for B. ambifaria mscL:

The most successful predictive models typically integrate multiple approaches, using high-resolution MD simulations to parameterize coarser models that can access longer timescales. Recent advances in machine learning techniques for bridging scales have further enhanced these multiscale modeling frameworks, allowing for more accurate predictions of channel behavior under the complex and dynamic membrane environments encountered by B. ambifaria during host infection.