Recombinant Edwardsiella ictaluri Small-conductance mechanosensitive channel (mscS)

How does E. ictaluri mscS function in bacterial physiology?

E. ictaluri mscS, similar to other bacterial mechanosensitive channels, primarily functions as an emergency valve that responds to mechanical stretch of the membrane. When bacterial cells encounter hypo-osmotic environments, these channels allow efflux of solutes to the external environment, preventing excessive turgor pressure that could lead to cell lysis .

The channel's open probability increases dramatically (by several orders of magnitude) in response to membrane tension conveyed via the lipid bilayer. This mechanism represents one of the simplest and most direct forms of mechanosensation in biological systems, where the mechanical force is directly sensed by the channel protein without requiring additional signaling components .

What expression systems are optimal for producing recombinant E. ictaluri mscS?

The most effective expression system for recombinant E. ictaluri mscS production is E. coli. Based on available research protocols, the recombinant full-length protein (amino acids 1-286) can be successfully expressed with an N-terminal His tag in E. coli expression systems . This approach allows for:

• High protein yield suitable for structural and functional studies

• Proper folding of the mechanosensitive channel

• Efficient purification using affinity chromatography

When designing expression systems, researchers should consider codon optimization for E. coli if expression efficiency becomes problematic. The protein's membrane-associated nature may require specialized strains optimized for membrane protein expression.

What are the recommended protocols for purification of recombinant E. ictaluri mscS?

Purification of recombinant E. ictaluri mscS requires a tailored approach due to its membrane protein characteristics. A detailed purification methodology includes:

• Cell lysis using sonication or pressure-based methods in a buffer containing detergents suitable for membrane protein extraction

• Affinity chromatography utilizing the N-terminal His tag, typically with Ni-NTA resin

• Size exclusion chromatography to separate the properly folded protein from aggregates and contaminants

The final purified protein typically achieves >90% purity as determined by SDS-PAGE . After purification, the protein is often lyophilized for storage stability, and reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added as a cryoprotectant for long-term storage at -20°C/-80°C .

How can researchers investigate the conformational changes of E. ictaluri mscS during channel gating?

Investigating conformational changes of E. ictaluri mscS during gating requires specialized techniques that capture the dynamic nature of channel opening and closing. Methodological approaches include:

• Site-directed spin labeling coupled with electron paramagnetic resonance (EPR) spectroscopy:

  • This allows monitoring of distance changes between specific residues during channel gating

  • Key residues in transmembrane domains should be selected based on structural predictions

• Disulfide crosslinking experiments:

  • Introduction of cysteine residues at strategic positions to trap different conformational states

  • Similar studies with E. coli MscS have shown that disulfide bridges between Cys 20 and Cys 36 of adjacent M1 helices can trap the channel in an open conformation

• Fluorescence resonance energy transfer (FRET):

  • Labeling of specific residues with fluorophore pairs

  • Allows real-time monitoring of conformational changes during channel activation

These approaches can be combined with electrophysiological measurements to correlate structural changes with channel function.

What experimental methods are most reliable for assessing E. ictaluri mscS channel activity?

Several complementary approaches are recommended for comprehensive functional characterization of E. ictaluri mscS:

• Patch-clamp electrophysiology:

  • Giant spheroplast preparation or reconstitution in liposomes for patch-clamp recording

  • Allows direct measurement of channel conductance (expected to be in the range of 2.5 nanosiemens based on homologous channels)

  • Enables quantification of mechanosensitivity by applying defined suction pressures

• Osmotic shock survival assays:

  • Bacterial cells expressing recombinant mscS are subjected to sudden osmotic downshift

  • Survival rates directly correlate with functional channel activity

  • Allows assessment of physiological relevance in cellular contexts

• Fluorescence-based flux assays:

  • Reconstitution of purified channels in liposomes loaded with fluorescent dyes

  • Measurement of dye efflux upon mechanical stimulation provides a readout of channel activity

  • Allows high-throughput screening of channel modulators

A combination of these methods provides a comprehensive understanding of channel function, from single-molecule biophysics to physiological relevance.

How does E. ictaluri mscS compare structurally and functionally to E. coli mscS?

Structural and functional comparison between E. ictaluri and E. coli mscS reveals important similarities and distinctions:

This comparative analysis highlights the need for specific investigations into potential non-canonical functions of E. ictaluri mscS, particularly regarding interactions with cell division machinery and roles in stress response.

What are the key molecular determinants of mechanosensitivity in E. ictaluri mscS?

Based on studies of homologous mechanosensitive channels, several molecular features likely determine the mechanosensitivity of E. ictaluri mscS:

• Lipid-protein interactions:
  Research on related mscS channels indicates that specific protein-lipid interactions are crucial for mechanosensitivity. The region just distal to the cytoplasmic end of the second transmembrane helix appears particularly important, potentially acting as an anchor for transmembrane domain tilting during gating .

• Transmembrane domain architecture:
  The arrangement of transmembrane helices creates the tension-sensing apparatus. In E. coli MscS, M1 transmembrane α-helices undergo an iris-like expansion and flattening when perturbed by membrane tension . Similar mechanisms likely exist in E. ictaluri mscS.

• Subunit organization:
  Functional mechanosensitive channels typically form homo-oligomeric complexes. E. coli MscS forms a homo-hexamer , and E. ictaluri mscS likely adopts a similar quaternary structure that enables coordinated response to membrane deformation.

To experimentally determine these mechanosensitivity determinants, researchers should consider mutagenesis studies targeting:

• Residues at lipid-protein interfaces

• Amino acids involved in inter-subunit interactions

• Regions undergoing conformational changes during gating

How can E. ictaluri mscS be utilized to study bacterial pathogenesis mechanisms?

E. ictaluri mscS presents a valuable model for investigating pathogenesis mechanisms in several ways:

• Role in stress adaptation during infection:

  • E. ictaluri causes bacillary necrosis (BNP) in striped catfish (Pangasianodon hypophthalmus)

  • The mechanosensitive channel may be essential for bacterial adaptation to osmotic conditions in host tissues

  • Experimental approaches could include generating mscS deletion mutants and assessing virulence in infection models

• Connection to antimicrobial resistance:

  • E. ictaluri strains often carry multiple antimicrobial resistance genes, including tetA (63% of isolates) and floR (77% of isolates)

  • Research should investigate potential links between mechanosensitive channel function and antibiotic resistance mechanisms

  • The interaction of mscS with FtsZ in other bacteria suggests roles in cell division and cell wall integrity that may impact antibiotic susceptibility

• Vaccine development considerations:

  • E. ictaluri proteins have been explored as vaccine candidates against fish pathogens

  • The extracellular portions of mscS could potentially serve as antigenic determinants

  • Research could focus on whether antibodies against mscS neutralize bacterial fitness or virulence

What role might E. ictaluri mscS play in bacterial responses to antibiotics?

Emerging evidence suggests that mechanosensitive channels may have functions beyond osmotic regulation, particularly in bacterial responses to antibiotics:

• β-lactam antibiotic response:
  Studies with E. coli mscS have demonstrated that bacteria expressing mutants with reduced binding to cell division protein FtsZ show compromised growth on sublethal concentrations of β-lactam antibiotics . This suggests that the interaction between mscS and FtsZ could be important for bacterial cell responses to sustained stress in the presence of these antibiotics.

• Cell wall stress sensing:
  β-lactam antibiotics target cell wall synthesis, and the mechanical stress resulting from cell wall disruption might be sensed by mechanosensitive channels like mscS. This could trigger adaptive responses to maintain cell integrity.

• Potential impact on antibiotic efficacy:
  Understanding the role of mechanosensitive channels in antibiotic responses could inform strategies to enhance antibiotic efficacy. For instance, if mscS contributes to antibiotic tolerance, inhibiting its function might potentiate antibiotic activity.

For experimental investigations, researchers should consider:

• Susceptibility testing of E. ictaluri mscS mutants to various antibiotic classes

• Examining expression levels of mscS during antibiotic exposure

• Investigating potential interactions between mscS and cell division or cell wall synthesis machinery

What are common challenges in expressing and purifying functional E. ictaluri mscS?

Researchers working with recombinant E. ictaluri mscS often encounter several technical challenges:

• Protein aggregation:

  • As a membrane protein, mscS has hydrophobic regions that can cause aggregation during expression and purification

  • Solution: Optimize detergent selection and concentration; consider using mild detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

• Low expression yields:

  • Membrane proteins typically express at lower levels than soluble proteins

  • Solution: Test different E. coli expression strains (C41, C43) specifically designed for membrane protein expression; optimize induction conditions (temperature, IPTG concentration, induction time)

• Protein instability:

  • Purified mscS may lose activity during storage

  • Solution: Add 5-50% glycerol as a cryoprotectant and store in aliquots at -80°C; avoid repeated freeze-thaw cycles

• Functional validation:

  • Confirming that purified protein retains mechanosensitive properties can be challenging

  • Solution: Develop robust functional assays such as reconstitution into liposomes for patch-clamp studies or fluorescence-based activity assays

How can researchers assess the oligomeric state of recombinant E. ictaluri mscS?

Determining the oligomeric state of E. ictaluri mscS is crucial for structural and functional studies. Several complementary approaches are recommended:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS):

  • Provides accurate molecular weight determination in detergent solutions

  • Can distinguish between different oligomeric states based on molecular mass

• Blue native PAGE:

  • Allows visualization of native protein complexes

  • Can be combined with Western blotting for specific detection

• Analytical ultracentrifugation:

  • Provides detailed information about sedimentation properties

  • Can distinguish between different oligomeric species in solution

• Chemical crosslinking:

  • Use of crosslinkers with specific spacer lengths can capture oligomeric interactions

  • Analysis by SDS-PAGE reveals the presence of higher molecular weight species

  • Mass spectrometry analysis of crosslinked peptides can identify specific interaction interfaces

Based on studies of homologous proteins, E. ictaluri mscS likely forms a homo-hexameric complex, similar to E. coli mscS .

What are promising research avenues for understanding E. ictaluri mscS in pathogenesis?

Several emerging research directions warrant investigation to elucidate the role of E. ictaluri mscS in pathogenesis:

• Genomic epidemiology connections:
  Recent genomic studies have shown that E. ictaluri isolates from diseased striped catfish in the Mekong Delta belong to ST-26 and are genetically related, differing by a maximum of 90 single nucleotide polymorphisms . Research should investigate whether variations in the mscS gene correlate with virulence or host specificity.

• Investigating potential non-channel functions:
  Based on findings that E. coli mscS interacts with the cell division protein FtsZ , similar interactions should be explored in E. ictaluri to understand if mscS contributes to pathogenesis through effects on cell division or other cellular processes.

• Host-pathogen interaction studies:
  The role of mechanosensitive channels in sensing mechanical forces during host cell attachment or invasion remains poorly understood. Studies using cell culture models could reveal whether mscS contributes to the initial stages of infection.

• Vaccine development potential:
  Previous studies have explored E. ictaluri proteins as potential vaccine candidates . Research should assess whether antibodies against extracellular epitopes of mscS could neutralize bacterial function and provide protection in fish models.

How might structural studies of E. ictaluri mscS inform novel antimicrobial strategies?

Detailed structural analysis of E. ictaluri mscS could reveal targets for novel antimicrobial interventions:

• Channel-specific inhibitors:

  • High-resolution structural data could guide the design of small molecules that specifically block channel function

  • If mscS is essential for bacterial survival during infection, such inhibitors could have therapeutic potential

• Targeting protein-protein interactions:

  • If E. ictaluri mscS interacts with other proteins (like FtsZ) during pathogenesis, disrupting these interactions could be a novel antibiotic strategy

  • Structural data on interaction interfaces would be essential for designing such inhibitors

• Allosteric modulators:

  • Compounds that lock the channel in an open state could potentially cause constitutive solute leakage and compromise bacterial viability

  • Structure-based screening could identify binding pockets for such modulators

• Cross-species comparative approaches:

  • Structural comparisons between E. ictaluri mscS and mammalian mechanosensitive channels could reveal bacterial-specific features

  • Such differences could be exploited to develop selective antibacterial agents

These structural biology approaches should be complemented by functional studies to validate the physiological relevance of any identified targets.