Recombinant Cronobacter sakazakii Large-conductance mechanosensitive channel (mscL)

How is recombinant C. sakazakii mscL protein expressed and purified for research applications?

The recombinant expression and purification of C. sakazakii mscL typically follows this methodological approach:

• Expression System Selection: E. coli is the preferred expression system, as confirmed by commercial sources of the protein .

• Vector Construction: The full-length coding sequence (1-135 amino acids) is cloned into an expression vector with an N-terminal His-tag for purification purposes .

• Expression Conditions: Transformed E. coli cells are typically grown to mid-log phase before induction with IPTG, followed by continued growth at lower temperatures (16-25°C) to enhance proper folding of membrane proteins.

• Cell Lysis and Membrane Fraction Isolation: Cells are harvested by centrifugation and disrupted by sonication or French press. The membrane fraction containing the overexpressed mscL is isolated by ultracentrifugation.

• Solubilization: The membrane proteins are solubilized using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG).

• Affinity Purification: The His-tagged protein is purified using immobilized metal affinity chromatography (IMAC), typically with Ni-NTA resin.

• Quality Control: The purity is assessed by SDS-PAGE, with typical preparations achieving greater than 90% purity .

• Storage: The purified protein is stored in a Tris/PBS-based buffer with 6% trehalose at pH 8.0, and for long-term storage, 50% glycerol is recommended to prevent freeze-thaw damage .

What experimental models are suitable for studying C. sakazakii mscL function in vitro?

Several experimental models are effective for investigating mscL function:

For studying mscL function, patch clamp techniques combined with reconstitution in lipid bilayers provide the most direct measurements of channel activity in response to membrane tension.

How does C. sakazakii mscL compare with mechanosensitive channels in other bacterial species?

Comparative analysis of mechanosensitive channels across bacterial species shows:

• Sequence Conservation: C. sakazakii mscL shares high sequence homology with E. coli MscL (approximately 80% identity), suggesting conserved functional mechanisms.

• Structural Features: Like other bacterial MscL proteins, C. sakazakii mscL likely forms a homopentameric structure with two transmembrane domains per subunit, though the exact oligomeric state requires experimental verification.

• Gating Properties: The specific tension threshold for C. sakazakii mscL gating has not been extensively characterized compared to E. coli MscL, representing a knowledge gap in the field.

• Physiological Role: While all bacterial MscL channels function in osmotic regulation, the specific role of C. sakazakii mscL may be adapted to the organism's environmental niche, particularly its notable desiccation resistance .

The research on C. sakazakii mechanosensitive channels is less extensive compared to model organisms like E. coli, offering opportunities for comparative studies.

How does the mscL gene contribute to C. sakazakii's desiccation resistance and pathogenicity?

Research on mechanosensitive channels in C. sakazakii indicates their complex role in desiccation resistance and pathogenicity:

While the effects of the mscM gene on desiccation resistance have been studied, showing that deletion of mscM actually enhances desiccation resistance by affecting potassium efflux , specific studies focusing on mscL's role in C. sakazakii desiccation resistance are still emerging.

Based on mechanistic studies, mscL likely contributes to desiccation resistance through:

• Osmotic Balance Regulation: During desiccation, mscL may help maintain cell viability by regulating the efflux of osmolytes in response to increasing cytoplasmic concentration.

• Membrane Integrity Preservation: By relieving excessive membrane tension during rapid environmental changes, mscL could prevent membrane damage during drying and rehydration cycles.

• Pathogenicity Connection: Research suggests mechanosensitive channels affect virulence-associated traits in C. sakazakii, as demonstrated for mscM which impacts:

  • Surface hydrophobicity (decreased by 20.52% in mscM knockout mutants)

  • Adhesion/invasion capability (reduced by 26.03% in mutants)

  • Biofilm formation (reduced by 30.19% in mutants)

These findings suggest that mechanosensitive channels, including mscL, may influence pathogenicity through multiple mechanisms, potentially explaining C. sakazakii's ability to persist in dry infant formula environments and subsequently cause infections.

What methodologies are most effective for studying mscL channel gating properties in C. sakazakii?

Studying mscL channel gating requires specialized techniques:

• Giant Spheroplast Patch Clamp Analysis:

  • Procedure: Create giant spheroplasts from C. sakazakii by lysozyme treatment in the presence of cephalexin, followed by patch-clamp recording.

  • Advantages: Maintains native membrane environment; allows direct measurement of single-channel conductance.

  • Variables to Measure: Channel opening probability, conductance, tension threshold, and adaptation behaviors.

• Reconstituted Systems:

  • Procedure: Purify recombinant mscL protein and reconstitute into azolectin liposomes or defined lipid compositions, followed by patch-clamp analysis.

  • Critical Parameters: Lipid composition significantly affects channel gating; therefore, systematic variation of membrane components (PE, PG, cardiolipin ratios) is necessary to determine physiologically relevant conditions.

• Fluorescence-Based Flux Assays:

  • Implementation: Load liposomes containing reconstituted mscL with self-quenching fluorescent dyes; measure fluorescence increase upon channel opening.

  • Quantification: Rate of fluorescence change correlates with channel activity and can be calibrated to determine relative opening probabilities.

• High-Speed Atomic Force Microscopy:

  • Application: Direct visualization of conformational changes in mscL channels embedded in supported lipid bilayers.

  • Resolution: Can achieve near-atomic resolution of dynamic structural changes during gating events.

The most informative approach combines electrophysiological measurements with structural studies to correlate function with specific conformational states.

How can gene knockout studies be designed to investigate the interplay between mscL and other mechanosensitive channels in C. sakazakii?

To investigate the interplay between mscL and other mechanosensitive channels, researchers can implement the following methodological framework:

• Generation of Knockout Mutants:

  • Single Knockouts: Create individual knockout strains for mscL, mscS, and mscM genes using homologous recombination methodology.

  • Double/Triple Knockouts: Generate combinations of knockouts to assess compensatory mechanisms.

  • Technical Approach: Use the suicide plasmid pCVD442 containing homologous arm fragments as demonstrated for mscM gene deletion .

• Complementation Studies:

  • Construct complementation strains by introducing the wild-type genes on plasmids (e.g., pACYC184) to confirm phenotypic changes are due to the specific gene deletions .

• Phenotypic Characterization:

  • Osmotic Shock Survival: Measure survival rates after hypo/hyperosmotic shifts.

  • Desiccation Resistance: Determine inactivation rates after controlled drying conditions.

  • Ion Content Analysis: Measure intracellular levels of K+, Na+, Ca2+, and Mg2+ to assess ion homeostasis .

  • Membrane Permeability: Use fluorescent probes like NPN to evaluate membrane integrity changes .

• Transcriptional Analysis:

  • Perform RT-qPCR to determine whether knockout of one channel affects expression of others.

  • Design primers for all mechanosensitive channel genes and housekeeping controls.

• Electrophysiological Characterization:

  • Compare patch-clamp profiles of wild-type vs. mutant strains to identify changes in channel activity patterns.

A representative experiment for assessing desiccation resistance in channel mutants would include:

This experimental framework would provide comprehensive insights into the functional interdependence of mechanosensitive channels in C. sakazakii's stress response systems.

What role might the mscL protein play in biofilm formation of C. sakazakii?

The potential role of mscL in C. sakazakii biofilm formation can be inferred from studies on related mechanosensitive channels:

Recent research on the mscM gene in C. sakazakii demonstrated that deletion of mscM significantly reduced biofilm formation by 30.19% (p < 0.05) . This suggests mechanosensitive channels play important roles in biofilm development, with potential mechanisms including:

• Cell Adhesion Regulation: Mechanosensitive channels may influence bacterial surface properties critical for initial attachment. The mscM deletion reduced surface hydrophobicity by 20.52% (p < 0.001) and adhesion capability by 26.03% (p < 0.001) , suggesting similar effects might occur with mscL mutations.

• Osmotic Adaptation During Biofilm Development: Biofilms create microenvironments with varying osmolarity gradients. MscL may be essential for cellular adaptation to these localized conditions, affecting:

  • Cell-to-cell signaling

  • Exopolysaccharide production

  • Matrix structural integrity

• Stress Response Coordination: MscL might function as a mechanosensor that detects surface contact and triggers biofilm-associated gene expression cascades.

To investigate mscL's specific role in biofilm formation, researchers should consider these methodological approaches:

• Crystal Violet Assays: Quantify biofilm formation in wild-type vs. mscL knockout strains under various environmental conditions

• Confocal Microscopy: Analyze biofilm architecture using fluorescent reporters

• Transcriptomic Analysis: Identify differentially expressed genes in biofilm-growing cells lacking mscL

• Flow Cell Systems: Evaluate biofilm development under continuous flow conditions that create mechanical forces

Understanding mscL's role in biofilm formation has significant implications for developing strategies to control C. sakazakii persistence in food production environments and medical settings.

How do sequence variations in mscL correlate with virulence in different C. sakazakii sequence types?

C. sakazakii strains exhibit genetic diversity that can be classified into multiple sequence types (STs) using multilocus sequence typing (MLST) . The correlation between mscL sequence variations and virulence requires a multifaceted analysis:

• Sequence Comparison Across STs:

  • Studies using the seven-locus MLST scheme (atpD, fusA, glnS, gltB, gyrB, infB, and pps) have identified at least 12 sequence types in C. sakazakii .

  • ST4 represents approximately one-third (22/60) of C. sakazakii strains and contains almost equal numbers of clinical and infant formula isolates .

  • ST8 may represent a particularly virulent grouping as 7/8 strains were clinical in origin .

• Methodological Approach to Correlation Analysis:

  • Extract and sequence mscL genes from representative strains of each sequence type

  • Perform multiple sequence alignment to identify amino acid substitutions

  • Map variations to functional domains of the mscL protein

  • Correlate specific variations with clinical outcomes and virulence factors

• Functional Validation:

  • Express variants in isogenic backgrounds and compare:

    • Channel conductance properties

    • Osmotic stress response

    • Desiccation survival rates

    • Adhesion to human intestinal cell lines (such as HCT-8 used for C. sakazakii studies)

• Potential Correlations:

  • Variations in the N-terminal domain might affect sensitivity to membrane tension

  • Transmembrane region mutations could alter channel conductance

  • C-terminal variations might influence interactions with other cellular components

A comprehensive analysis should include core genome MLST using 2831 target genes for higher resolution of strain relationships, combined with functional studies of mscL variants to establish causal relationships rather than mere correlations.

What are the current challenges and solutions in expressing and purifying functional recombinant C. sakazakii mscL for structural studies?

Membrane proteins like mscL present significant challenges for structural studies, requiring specialized approaches:

Major Challenges:

• Low Expression Yields: Membrane proteins often express poorly in heterologous systems.

  • Solution: Optimization of expression using specialized strains (C41/C43) and tunable promoters for controlled expression.

• Protein Aggregation/Misfolding: Overexpression can lead to inclusion body formation.

  • Solution: Lower induction temperatures (16-20°C), use of mild detergents, and fusion partners that enhance solubility.

• Detergent Selection: Identifying detergents that maintain protein stability while extracting from membranes.

  • Solution: Systematic screening of detergent panels (maltoside series, glucoside series) for optimal solubilization.

• Maintaining Functionality: Ensuring the purified protein retains native conformation and activity.

  • Solution: Functional validation using liposome reconstitution and patch-clamp analysis after each purification step.

• Protein Stability: Preventing degradation during purification and storage.

  • Solution: Addition of protease inhibitors, optimized buffer conditions (50% glycerol, 6% trehalose) , and avoiding freeze-thaw cycles.

Advanced Methodological Approaches:

• Amphipol Stabilization: Replacing conventional detergents with amphipathic polymers that stabilize membrane proteins for structural studies.

• Lipid Cubic Phase Crystallization: For X-ray crystallography studies, this method provides a membrane-like environment conducive to crystal formation.

• Nanodiscs: Reconstituting mscL into nanoscale phospholipid bilayers supported by membrane scaffold proteins for enhanced stability.

• Cryo-EM Sample Preparation: Recent advances allow structural determination of smaller membrane proteins like mscL using single-particle cryo-EM.

Current commercial preparations of recombinant C. sakazakii mscL typically achieve greater than 90% purity , but structural biology applications may require further optimization of these protocols to achieve the conformational homogeneity necessary for high-resolution structural studies.

How can the mscL channel be targeted for developing novel antimicrobial strategies against C. sakazakii?

The essential nature of mechanosensitive channels for bacterial survival under osmotic stress makes mscL a potential target for novel antimicrobial development:

• Target Validation Methodology:

  • Essentiality Assessment: While mscL deletion may not be lethal under standard growth conditions , its critical role during osmotic stress suggests it could be targeted in combination therapies or environment-specific treatments.

  • Vulnerability Analysis: Determining conditions where mscL function becomes essential:

    • During osmotic transitions in food processing environments

    • In desiccated and rehydrated states relevant to powdered infant formula

    • During host invasion processes where osmotic conditions change

• Drug Development Strategies:

  Approach	Methodology	Advantages	Challenges
  Small molecule gating modifiers	High-throughput screening of compounds that lock channels in open state	Potential specificity for bacterial channels	Delivery across bacterial membrane
  Peptide inhibitors	Design of peptides that mimic channel domains	Higher specificity; potentially lower toxicity	Stability and delivery issues
  Antisense technologies	PNA/PMO oligomers targeting mscL mRNA	Highly specific; adjustable for resistance	Cellular uptake limitations
  CRISPR-Cas delivery systems	Phage-delivered gene editing to disrupt mscL	High specificity; potential for environmental application	Delivery challenges; regulatory concerns

• Screening Methodologies:

  • Fluorescence-Based Assays: Bacterial cells loaded with calcium-sensitive fluorophores to detect channel modulation

  • Growth Inhibition Assays: Under cycling osmotic conditions to identify compounds that interfere with adaptation

  • Patch-Clamp Validation: Direct measurement of channel activity in presence of lead compounds

• Translational Applications:

  • Biofilm Prevention: Compounds targeting mscL could prevent biofilm formation in food production environments

  • Food Preservation: Targeted treatments for infant formula production that specifically inhibit C. sakazakii survival

  • Medical Device Coatings: Surface treatments that modulate mscL function to prevent bacterial colonization

This strategy is particularly relevant given C. sakazakii's concerning resistance profiles, with studies showing 75% resistance to ampicillin and multiresistance patterns in certain sequence types .

What is the relationship between mscL expression and temperature adaptation in C. sakazakii?

The relationship between mscL expression and temperature adaptation in C. sakazakii represents an important research area given the bacterium's ability to survive in diverse environments:

• Temperature Ranges Relevant to C. sakazakii:

  • C. sakazakii exhibits greater pigment production at temperatures below 36.8°C, with optimal pigment production at 25.8°C .

  • The bacterium can survive in stock cultures stored at 17-30.8°C without transfer for up to 8 years .

  • Survival through food processing temperatures and subsequent rehydration at consumption temperatures (approximately 40-45°C) is critical for pathogenicity.

• Methodological Approaches to Study Temperature-Dependent Expression:

  • qRT-PCR Analysis: Monitor mscL transcript levels at different growth temperatures (4°C, 25°C, 37°C, 45°C) during:

    • Exponential growth phase

    • Stationary phase

    • During temperature shifts

  • Western Blot Analysis: Quantify protein levels using antibodies against recombinant mscL protein

  • Transcriptional Fusions: Construct mscL promoter-reporter fusions to visualize expression patterns

  • RNA-Seq Analysis: Perform global transcriptome analysis to position mscL within temperature-responsive gene networks

• Functional Implications:

  • Cell Membrane Fluidity: Temperature affects membrane fluidity, which directly impacts mechanosensitive channel tension sensitivity and gating properties.

  • Thermal Stress Response: MscL may participate in the general stress response network, interacting with temperature-sensitive regulatory systems.

  • Adaptation to Host Environment: Regulation of mscL expression may facilitate transition from environmental temperatures to human body temperature (37°C).

• Research Design Parameters:

  Temperature Condition	Experimental Setup	Key Measurements	Expected Outcomes
  Cold shock (25°C to 4°C)	Exponential phase culture subjected to rapid temperature drop	mscL expression; membrane fluidity; survival rates	Potential upregulation to compensate for decreased membrane fluidity
  Heat shock (25°C to 45°C)	Exponential phase culture subjected to rapid temperature increase	mscL expression; protein aggregation; membrane integrity	Possible role in preventing membrane damage during heat stress
  Growth at different constant temperatures	Continuous culture at 25°C, 37°C, and 42°C	Growth rates; mscL expression profiles; channel activity	Temperature-dependent expression patterns
  Temperature cycling	Repetitive shifts between 25°C and 37°C	Adaptation rates; expression stability	Insights into environmental persistence mechanisms

Understanding the temperature-dependence of mscL expression could provide valuable insights into C. sakazakii's remarkable environmental persistence and contribute to developing targeted control strategies in food production settings.