Recombinant Acinetobacter baumannii Large-conductance mechanosensitive channel (mscL)

What is the structure and function of Acinetobacter baumannii mscL protein?

The Acinetobacter baumannii mscL (Large-conductance mechanosensitive channel) is a membrane protein that responds to mechanical forces in the cell membrane. The protein functions primarily as an emergency release valve during extreme turgor pressure increases that would otherwise lyse the cellular membrane .

The A. baumannii mscL consists of 143 amino acids with a molecular architecture that includes transmembrane helices forming a channel through the membrane . While the specific structure of A. baumannii mscL has not been fully determined, related mscL proteins like those from M. tuberculosis (MtMscL) and S. aureus (SaMscL) have been crystallized, revealing pentameric and tetrameric structures, respectively .

Methodologically, researchers investigating the structure should consider:

• X-ray crystallography after protein purification

• Cryo-electron microscopy for native state visualization

• Computational modeling based on homologous proteins where crystal structures exist

What experimental techniques are commonly used to study the oligomeric state of mscL channels?

Multiple complementary techniques are used to determine the oligomeric state of mscL channels, as different methodologies have yielded varying results across bacterial species. The primary techniques include:

The oligomeric state of mscL varies between species, with E. coli MscL (EcMscL) reported as pentameric or hexameric, M. tuberculosis MscL (MtMscL) as pentameric, and S. aureus MscL (SaMscL) as tetrameric or pentameric .

Methodologically, researchers should employ multiple techniques for cross-validation when determining the oligomeric state of A. baumannii mscL.

How should recombinant A. baumannii mscL be stored and handled in a laboratory setting?

For optimal stability and activity of recombinant A. baumannii mscL:

• Storage recommendations:

  • Store at -20°C for regular use

  • For extended storage, conserve at -20°C or -80°C

  • Maintain in a storage buffer containing Tris-based buffer with 50% glycerol optimized for protein stability

• Handling protocols:

  • Avoid repeated freeze-thaw cycles as this can denature the protein

  • Prepare working aliquots and store at 4°C for up to one week

  • Maintain sterile conditions to prevent contamination

These recommendations are based on standard protocols for recombinant membrane proteins and specific guidelines for A. baumannii mscL .

What expression systems are suitable for producing recombinant A. baumannii mscL?

The choice of expression system significantly impacts the yield and functionality of recombinant mscL. While the search results don't specify the exact systems used for A. baumannii mscL, general approaches for membrane protein expression can be applied:

• Bacterial expression systems:

  • E. coli BL21(DE3) with pET vector systems

  • C41(DE3) or C43(DE3) strains specifically engineered for membrane protein expression

  • Optimization of induction conditions (temperature, IPTG concentration, induction time)

• Expression enhancement strategies:

  • Use of fusion tags (His, GST, MBP) for improved solubility and purification

  • Codon optimization for the expression host

  • Addition of chaperones to aid proper folding

• Purification considerations:

  • Detergent selection for membrane protein solubilization

  • Affinity chromatography based on fusion tags

  • Size exclusion chromatography for further purification

Researchers should systematically optimize expression conditions and validate protein functionality through activity assays.

Advanced Research Questions

The mechanosensitive channel undergoes significant conformational changes during gating in response to membrane tension. Based on studies of related mscL proteins, these changes include:

• Transmembrane helix tilting and rotation

• Expansion of the pore diameter

• Rearrangement of the constriction site formed by hydrophobic residues

To experimentally measure these conformational changes in A. baumannii mscL:

• Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR): Attach spin labels at strategic positions to monitor distance changes during gating

• FRET analysis: Incorporate fluorophore pairs to measure distance changes between subunits or domains

• Patch-clamp electrophysiology: Measure channel conductance under varying membrane tensions

• Molecular dynamics simulations: Computationally model gating transitions based on homologous structures

• Cryo-EM under different tension conditions: Capture different conformational states

The N-terminal and transmembrane regions likely undergo significant conformational changes during gating, similar to what has been observed in SaMscL where the N-terminal region (residues 1-12) and first transmembrane helix (residues 13-47) play crucial roles in channel function .

How can recombinant A. baumannii mscL be utilized in vaccine development against multidrug-resistant A. baumannii infections?

A. baumannii has become a significant global health threat due to increasing multidrug resistance . Membrane proteins like mscL represent potential vaccine candidates due to their conservation and surface accessibility. Based on immunization studies with other A. baumannii membrane proteins:

• Antigen selection and validation:

  • Evaluate mscL conservation across clinical isolates

  • Assess surface exposure of epitopes

  • Test immunogenicity in animal models

• Vaccine formulation strategies:

  • Use of recombinant protein with appropriate adjuvants (such as Alum, as used with AbOmpA and DcaP-like protein)

  • Development of multi-antigen formulations combining mscL with other immunogenic proteins

  • Testing different immunization schedules (e.g., three injections as used in AbOmpA studies)

• Immune response assessment:

  • Measure specific IgG antibody production (total IgG, IgG1, IgG2c)

  • Evaluate cytokine profiles (IL-4, IL-6, IL-17A)

  • Perform bacterial challenge studies to assess protection

• Efficacy evaluation:

  • Determine bacterial load reduction in organs following challenge

  • Perform serum bactericidal assays

  • Conduct histopathological examinations of affected tissues

Previous studies with AbOmpA and DcaP-like protein have shown promising results in mice, suggesting a similar approach could be effective with mscL .

What role might A. baumannii mscL play in antibiotic resistance, and how can this be experimentally investigated?

While mscL's primary function relates to osmoregulation, membrane proteins can influence antibiotic resistance through various mechanisms. Given A. baumannii's notorious multidrug resistance profile , investigating mscL's potential role is valuable:

• Potential mechanisms of mscL involvement in resistance:

  • Alteration of membrane permeability to antibiotics

  • Interaction with efflux pump systems

  • Response to membrane stress caused by certain antibiotics

• Experimental approaches:

  • Gene knockout/knockdown studies: Generate mscL-deficient strains and assess antibiotic susceptibility profiles

  • Overexpression studies: Express varying levels of wild-type and mutant mscL to observe effects on antibiotic resistance

  • Protein-protein interaction studies: Investigate interactions between mscL and known resistance determinants like efflux pumps

  • Electrophysiology: Measure antibiotic transit through reconstituted mscL channels

• Specific resistance mechanisms to investigate:

  • Tigecycline resistance: A. baumannii has developed resistance through efflux pumps like AdeFGH and modifications in regulatory systems

  • Polymyxin resistance: A. baumannii can modify or eliminate LPS through mutations in lpxA, lpxC, or lpxD genes

  • β-lactam resistance: Mediated through AmpC enzymes and other β-lactamases

• Clinical relevance assessment:

  • Compare mscL sequence and expression levels in multidrug-resistant versus susceptible clinical isolates

  • Correlate mscL variations with resistance phenotypes

How does membrane composition affect the function of A. baumannii mscL, and what experimental setups can best elucidate these effects?

Membrane composition significantly influences mechanosensitive channel function by affecting membrane mechanical properties and protein-lipid interactions. For A. baumannii mscL:

• Key membrane parameters affecting function:

  • Lipid bilayer thickness

  • Membrane fluidity

  • Presence of specific lipids (e.g., phosphatidylethanolamine, cardiolipin)

  • Membrane lateral pressure profile

• Experimental approaches:

  • Reconstitution in artificial membranes: Incorporate purified mscL into liposomes with defined lipid compositions

  • Patch-clamp electrophysiology: Measure channel activity in different membrane environments

  • FRET-based tension sensors: Monitor membrane tension alongside channel activity

  • Molecular dynamics simulations: Model protein-lipid interactions in silico

• Specific membrane composition considerations for A. baumannii:

  • A. baumannii can survive without lipopolysaccharide (LPS) due to mutations in lpxA, lpxC, or lpxD genes

  • This LPS-deficient phenotype affects membrane properties and may influence mscL function

  • LPS-deficient mutants grow slower and are less pathogenic

• Experimental design:

  • Compare mscL function in wild-type versus LPS-deficient membranes

  • Systematically vary lipid composition to determine optimal conditions for channel activity

  • Measure gating threshold and kinetics as a function of membrane composition

What are the challenges in crystallizing A. baumannii mscL for structural studies, and how might these be overcome?

Membrane protein crystallization presents significant challenges, particularly for mechanosensitive channels that undergo conformational changes. Based on crystallization experiences with MtMscL and SaMscL :

• Key challenges:

  • Protein stability during purification

  • Detergent selection for solubilization

  • Obtaining homogeneous protein preparations

  • Capturing specific conformational states

  • Growing well-diffracting crystals

• Strategic approaches:

  • Construct optimization: Create truncated versions (e.g., C-terminal truncations as used with SaMscL(CΔ26))

  • Detergent screening: Systematically test various detergents for optimal protein stability and crystal formation

  • Lipid cubic phase (LCP) crystallization: Alternative to detergent-based crystallization

  • Stabilizing mutations: Introduce mutations that lock the channel in specific conformational states

  • Fusion protein strategies: Add well-folding domains to enhance crystallization properties

• Conformational state considerations:

  • Closed state is typically more stable and easier to crystallize

  • Open state might require specialized approaches like tension-mimicking mutations or cross-linking

• Alternative structural approaches:

  • Cryo-electron microscopy (cryo-EM) for structure determination without crystallization

  • NMR spectroscopy for dynamic structural information

  • Computational modeling based on homologous structures