Recombinant Bordetella bronchiseptica Large-conductance mechanosensitive channel (mscL)

What is the basic structure of B. bronchiseptica mscL protein?

The B. bronchiseptica large-conductance mechanosensitive channel (mscL) is a 152-amino acid membrane protein with a molecular weight of approximately 15-17 kDa. Its amino acid sequence is: MSKATGFIKEFRDFAVKGNAIDLAVGVIIGAAFGKIVDSLVKDVVMPLVNFILGGSVDFSNKFLVLSMPDGYTGPMTYADLTKAGANVLAWGNFITIIINFVLLAFVIFWMVKAIYSARRKEEAAPEAPAAPPEDVTVLREIRDLLKDKQGS . The protein contains hydrophobic regions that span the bacterial membrane and form a channel structure responsive to mechanical tension. Similar to mechanosensitive channels in other bacteria, mscL likely assembles as a homopentamer in the native membrane, creating a central pore that opens in response to membrane tension.

What is the physiological role of mscL in B. bronchiseptica?

The mscL protein functions as an emergency release valve in B. bronchiseptica, protecting the bacterium from osmotic shock by opening in response to increased membrane tension. This channel allows the rapid efflux of water, ions, and small solutes during hypoosmotic stress, preventing cell lysis. Beyond osmotic protection, the mscL channel may play roles in the bacterium's adaptation to different environmental niches, including survival in amoeba cells and mammalian hosts . During the complex life cycle of B. bronchiseptica, which involves both amoebic and mammalian hosts, mechanosensitive channels may contribute to sensing and adapting to different mechanical environments.

How does mscL differ from other mechanosensitive channels in B. bronchiseptica?

B. bronchiseptica possesses multiple types of mechanosensitive channels, with mscL representing the large-conductance variant. Unlike smaller mechanosensitive channels (such as mscS), mscL has a higher activation threshold and larger pore size, allowing passage of molecules up to 6.5 kDa when fully open. It typically activates at higher membrane tensions than other mechanosensitive channels, serving as a last-resort protection mechanism. The mscL channel's distinctive structure and gating mechanism make it an important subject for understanding bacterial membrane dynamics and osmotic regulation during pathogenesis and environmental persistence.

What are the optimal conditions for recombinant expression of B. bronchiseptica mscL?

For optimal expression of recombinant B. bronchiseptica mscL, an E. coli expression system with a pET vector containing a 6xHis tag is typically employed. The expression protocol should include:

• Transformation into an appropriate E. coli strain (BL21(DE3) or C43(DE3))

• Culture growth at 37°C until OD600 reaches 0.6-0.8

• Induction with 0.5-1.0 mM IPTG

• Temperature reduction to 18-25°C after induction

• Expression for 4-6 hours or overnight

For membrane proteins like mscL, adding glycerol (5-10%) to the culture medium can enhance proper folding. Inclusion of membrane-mimicking environments during purification is essential for maintaining the protein's native conformation and activity.

What purification strategy yields the highest purity and activity of recombinant B. bronchiseptica mscL?

A multi-step purification approach is recommended for obtaining high-purity, functionally active recombinant B. bronchiseptica mscL:

• Membrane isolation via ultracentrifugation after cell lysis

• Solubilization with appropriate detergents (n-dodecyl-β-D-maltopyranoside (DDM) at 1-2%)

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography for removing aggregates

• Storage in a stabilizing buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% DDM, and 50% glycerol at -20°C

The purified protein should be assessed for homogeneity using SDS-PAGE and for functional activity using liposome reconstitution and patch-clamp electrophysiology or fluorescence-based assays.

How can researchers verify the functional integrity of purified recombinant mscL?

Functional verification of purified recombinant B. bronchiseptica mscL can be performed using several complementary approaches:

• Liposome reconstitution and patch-clamp analysis: Incorporate purified mscL into liposomes and measure channel activity under varying membrane tensions

• Fluorescence-based assays: Use fluorescent dyes entrapped in mscL-containing liposomes to measure release upon osmotic downshift

• In vitro translation and direct incorporation: Cell-free expression systems with direct incorporation into nanodiscs or liposomes

• Circular dichroism spectroscopy: Confirm proper secondary structure formation

• Electron microscopy: Visualize channel assembly in membrane mimetics

Functional mscL should exhibit characteristic conductance properties, including tension-dependent gating with a conductance of approximately 3 nS in standard recording conditions.

How does B. bronchiseptica mscL contribute to bacterial survival during host infection?

B. bronchiseptica mscL likely plays multiple roles during infection of mammalian hosts. During respiratory tract colonization, the bacterium encounters various osmotic and mechanical stresses, including those imposed by respiratory secretions and host immune responses. The mscL channel may help B. bronchiseptica adapt to these changing conditions by:

• Responding to membrane tension during phagocytosis by immune cells

• Facilitating survival within intracellular compartments, similar to its role in amoeba cells

• Contributing to bacterial persistence during environmental transitions

• Potentially serving as a sensory mechanism for detecting host microenvironments

Research demonstrates that B. bronchiseptica can survive intracellularly in phagocytic cells, a trait potentially facilitated by mechanosensitive channels that help the bacteria adapt to intracellular osmotic conditions . This adaptation mechanism may be particularly important during the establishment of chronic infections.

What role might mscL play in B. bronchiseptica's amoeba-mammalian transmission cycle?

The mscL protein may be crucial for B. bronchiseptica's remarkable ability to transition between amoeba and mammalian hosts. Studies have shown that B. bronchiseptica can survive within amoeba cells and subsequently efficiently colonize mammalian respiratory tracts . This dual-host life cycle likely requires specialized adaptation mechanisms:

• During amoeba invasion and intracellular survival, mscL may respond to mechanical stresses imposed by phagosomal trafficking

• In the amoeba sorus (fruiting body), where B. bronchiseptica has been shown to localize and replicate, mscL might facilitate adaptation to the structural constraints of this environment

• During transmission from amoeba to mammals, mscL could help the bacteria withstand rapid osmotic changes

• The mechanosensing properties of mscL might contribute to bacterial detection of the transition between hosts

Experiments have demonstrated that B. bronchiseptica can persist in amoeba cells for extended periods and remain fully capable of colonizing the mammalian respiratory tract afterward, suggesting sophisticated adaptation mechanisms that may involve mechanosensitive channels .

How can recombinant mscL be used to study B. bronchiseptica pathogenesis mechanisms?

Recombinant B. bronchiseptica mscL serves as a valuable tool for investigating bacterial pathogenesis through several research approaches:

• Structure-function studies: Site-directed mutagenesis of recombinant mscL can reveal regions critical for channel gating and bacterial survival under stress

• Reconstitution experiments: Purified mscL incorporated into model membranes allows precise characterization of channel properties under controlled conditions

• Inhibitor screening: Recombinant mscL can be used to identify compounds that modulate channel function, potentially leading to novel antimicrobials

• Host-pathogen interaction studies: Antibodies raised against recombinant mscL can track protein expression and localization during infection

• Vaccine development: As a membrane protein, mscL could be evaluated as a potential vaccine candidate, similar to other B. bronchiseptica outer membrane proteins that have shown protective potential

These applications contribute to a more comprehensive understanding of how B. bronchiseptica adapts to different hosts and environments, potentially revealing new targets for therapeutic intervention.

What electrophysiological methods are most suitable for characterizing recombinant B. bronchiseptica mscL function?

Advanced electrophysiological characterization of recombinant B. bronchiseptica mscL requires specialized techniques:

• Spheroplast patch-clamp: The most direct method involves expressing recombinant mscL in E. coli, creating spheroplasts, and performing patch-clamp recordings under precisely controlled membrane tension

• Planar lipid bilayer recordings: Purified recombinant mscL reconstituted into planar lipid bilayers allows detailed conductance measurements and pharmacological studies

• Pressure-clamp systems: Specialized equipment that applies defined pressure gradients while simultaneously recording channel activity

• Combined fluorescence-electrophysiology: Coupling electrophysiological recordings with fluorescence measurements to correlate structural changes with functional states

The most informative approach is to characterize mscL at multiple levels of membrane tension, recording the channel's conductance, open probability, and kinetics. These properties can then be compared with those of mscL channels from other bacterial species to identify unique features of the B. bronchiseptica channel.

How can researchers investigate the structural dynamics of mscL during gating?

Investigating the structural dynamics of B. bronchiseptica mscL during its tension-induced gating requires sophisticated biophysical techniques:

• Site-directed spin labeling and electron paramagnetic resonance (EPR): Introducing spin labels at specific residues allows tracking of protein movement during gating

• Förster resonance energy transfer (FRET): Strategic placement of fluorescent pairs can reveal conformational changes during channel opening

• Molecular dynamics simulations: Computational modeling of mscL embedded in lipid bilayers under varying tension

• Hydrogen-deuterium exchange mass spectrometry: Identifies regions of the protein that become exposed during conformational changes

• Cryo-electron microscopy: Capturing different conformational states of the channel

These techniques can reveal how the B. bronchiseptica mscL transitions from closed to open states in response to membrane tension, potentially identifying unique adaptations that contribute to this pathogen's environmental versatility.

What approaches can be used to identify small molecule modulators of B. bronchiseptica mscL?

Identification of small molecule modulators of B. bronchiseptica mscL can employ several complementary screening approaches:

• High-throughput fluorescence-based assays: Using fluorescent dye release from mscL-containing liposomes to identify compounds that affect channel gating

• Patch-clamp electrophysiology: Direct measurement of channel activity in the presence of candidate compounds

• In silico molecular docking: Computational screening of compound libraries against structural models of B. bronchiseptica mscL

• Bacterial growth assays: Testing compounds for their ability to compromise bacterial osmotic regulation under controlled stress conditions

• Thermal shift assays: Measuring changes in protein stability upon compound binding

Compounds that specifically modulate mscL function could serve as research tools for investigating channel properties and potentially as leads for novel antimicrobial agents targeting B. bronchiseptica's ability to adapt to osmotic stress.

How does B. bronchiseptica mscL compare structurally and functionally to mscL from other bacterial pathogens?

Comparative analysis of B. bronchiseptica mscL with those from other bacterial pathogens reveals insights into evolutionary adaptations:

These differences likely reflect adaptations to different environmental niches and stress conditions encountered by these pathogens. B. bronchiseptica's mscL properties may be specifically tuned to its lifecycle that spans both environmental (including amoeba) and mammalian host settings .

What can B. bronchiseptica mscL research reveal about bacterial adaptation to different hosts?

Research on B. bronchiseptica mscL provides important insights into bacterial adaptation mechanisms:

• The conservation of mscL across B. bronchiseptica strains suggests its importance in the bacterium's life cycle

• Given B. bronchiseptica's ability to colonize both amoebae and mammals, mscL may represent an adaptation that facilitates survival across dramatically different host environments

• The channel's properties may reflect specific adaptations to respiratory tract conditions, where osmotic fluctuations occur

• Comparison with mechanosensitive channels from strictly host-restricted pathogens (like B. pertussis) can reveal how these proteins contribute to host range determination

Studies showing B. bronchiseptica's ability to survive in amoeba sori and subsequently infect mammalian hosts highlight the remarkable adaptability of this pathogen , potentially involving mechanosensitive channels like mscL that respond to physical aspects of different cellular environments.

How does the expression of mscL vary during different phases of B. bronchiseptica's life cycle?

The expression of mscL in B. bronchiseptica likely varies according to the bacterium's life cycle phase and environmental conditions:

• During the Bvg- phase (associated with environmental persistence and amoeba colonization), mscL expression may be upregulated to help manage osmotic challenges in diverse environments

• In the Bvg+ phase (associated with mammalian infection), expression patterns may shift to accommodate the specific osmotic environment of the respiratory tract

• During transition between hosts, temporary upregulation might occur to protect against osmotic shock

• Within amoeba cells, expression levels may adapt to the intracellular environment, potentially contributing to the bacterium's remarkable ability to survive and replicate in the amoeba sorus

Research has demonstrated that B. bronchiseptica modulates between Bvg- and Bvg+ phases during its complex life cycle, with Bvg- phase genes being particularly important for amoeba colonization . As a membrane channel involved in environmental adaptation, mscL may be regulated as part of this phase variation system.

Can B. bronchiseptica mscL serve as a target for novel antibacterial compounds?

B. bronchiseptica mscL presents several characteristics that make it an attractive potential target for novel antibacterial development:

• As a membrane protein essential for osmotic protection, inhibitors forcing the channel into an open state could compromise bacterial viability under physiological conditions

• The structural conservation of mscL across Bordetella species suggests that targeting this protein could provide broad activity against multiple pathogens

• The differences between bacterial and eukaryotic mechanosensitive channels could allow for selective targeting

• Compounds that lock mscL in specific conformational states could disrupt B. bronchiseptica's ability to transition between different hosts and environments

Researchers can investigate this potential through screening compound libraries against purified recombinant mscL in functional assays, followed by validation in bacterial cultures and infection models.

What is the potential of recombinant B. bronchiseptica mscL as a vaccine component?

Evaluating recombinant B. bronchiseptica mscL as a potential vaccine component requires consideration of several factors:

• As a membrane protein, mscL could potentially elicit protective antibody responses, similar to other B. bronchiseptica outer membrane proteins that have shown vaccine potential

• Its conservation across B. bronchiseptica strains suggests it could provide broad protection

• The immune response to mscL would need careful characterization, as studies with other B. bronchiseptica proteins have shown varying efficacy

• Combination with other antigens might be necessary for complete protection

Research has identified other B. bronchiseptica surface proteins with protective potential, including porin protein (PPP) and lipoprotein (PL), which showed protection ratios of 62.5% and 50%, respectively, in challenge studies . Similar methodical evaluation of recombinant mscL would be required to assess its vaccine potential.

How might recombinant mscL research contribute to reducing endotoxicity in B. bronchiseptica vaccines?

Recombinant protein approaches offer potential advantages for developing safer B. bronchiseptica vaccines with reduced endotoxicity:

• Purified recombinant proteins like mscL lack the lipopolysaccharide (LPS) associated with whole-cell vaccine reactogenicity

• Expression systems can be optimized to produce endotoxin-free recombinant proteins for vaccine development

• Structural studies of mscL could inform the design of non-toxic mimetics that induce protective immunity

• Combining recombinant mscL with other carefully selected antigens could provide comprehensive protection while avoiding the reactogenicity of whole-cell preparations

Current research on B. bronchiseptica vaccines has highlighted the need to reduce endotoxicity while maintaining protective efficacy . The investigation of defined recombinant proteins represents one promising approach to address this challenge.

How can recombinant B. bronchiseptica mscL be utilized in biosensor development?

The mechanosensitive properties of recombinant B. bronchiseptica mscL make it valuable for various biosensor applications:

• Tension-sensing applications: Reconstituted mscL in artificial membranes can detect mechanical forces in engineered systems

• Controlled-release technologies: mscL channels can be engineered to open in response to specific stimuli, releasing encapsulated compounds

• Environmental stress detection: Bacteria expressing modified mscL variants can serve as reporters for environmental conditions

• Nanofluidic devices: Incorporation of mscL into synthetic membranes enables creation of tension-controlled nanovalves

Implementation requires careful optimization of protein reconstitution conditions and appropriate detection systems for channel opening events. The large conductance of mscL makes it particularly suitable for applications requiring high-throughput molecular passage upon activation.

What modifications can enhance the utility of recombinant B. bronchiseptica mscL for research applications?

Strategic modifications of recombinant B. bronchiseptica mscL can expand its research utility:

• Affinity tags: Addition of His-tags, FLAG-tags, or biotin acceptor peptides facilitates purification and immobilization

• Fluorescent protein fusions: Creating GFP or mCherry fusions enables visualization of expression and localization

• Site-directed mutations: Introducing mutations at key residues can alter channel gating properties for specialized applications

• Cysteine substitutions: Strategic placement of cysteines enables site-specific labeling with fluorophores or spin labels

• Chimeric constructs: Creating chimeras with portions from other bacterial mscL proteins for comparative structure-function analysis

Each modification should be validated to ensure retention of proper folding and function, typically through a combination of biochemical and electrophysiological approaches.

How can high-throughput approaches accelerate B. bronchiseptica mscL research?

Modern high-throughput technologies can significantly advance B. bronchiseptica mscL research:

• Automated patch-clamp platforms: Enable rapid functional screening of mscL variants or potential modulators

• Microfluidic devices: Allow parallel testing of mscL function under various conditions

• Droplet-based screening: Encapsulation of mscL-containing proteoliposomes in droplets for massively parallel functional assays

• Deep mutational scanning: Comprehensive analysis of how mutations throughout mscL affect channel function

• AI-assisted molecular dynamics: Computational prediction of mscL structural dynamics to guide experimental design

These approaches can generate comprehensive datasets on mscL structure-function relationships, potentially revealing novel insights into how this channel contributes to B. bronchiseptica's remarkable adaptability across diverse environments and hosts.