Recombinant Pasteurella multocida Large-conductance mechanosensitive channel (mscL)

What is Pasteurella multocida and what is its significance in mechanosensitive channel research?

Pasteurella multocida is a highly versatile Gram-negative, non-motile, penicillin-sensitive coccobacillus that causes various diseases across multiple species. It is classified into five serogroups (A, B, D, E, F) based on capsular composition and 16 somatic serovars (1-16) . While most P. multocida research has focused on pathogenesis and vaccine development, investigation of its membrane proteins including potential mechanosensitive channels represents an emerging area.

P. multocida causes economically significant diseases including:

• Fowl cholera in avian species

• Hemorrhagic septicemia in ruminants

• Progressive atrophic rhinitis in swine

• Snuffles in rabbits

• Opportunistic infections in humans following animal bites or scratches

Type A is the predominant pathogenic serotype affecting the duck industry . The bacterium's ability to infect multiple species and cause diverse clinical manifestations makes it an important model for studying host-pathogen interactions and membrane protein function.

What is the mechanosensitive channel of large conductance (MscL) and how does it function in bacterial systems?

The mechanosensitive channel of large conductance (MscL) is a tension-activated membrane protein that functions as an emergency release valve in bacteria, preventing cell lysis during osmotic stress. In bacteria, MscL channels "function as tension-activated pores limiting excessive turgor pressure, with MscL acting as an emergency release valve preventing cell lysis" .

The channel's structure includes:

• Inner (TM1) and outer (TM2) transmembrane helices

• A pentagon-shaped gate formed by crossing TM1 helices

• An amphipathic S1 helix that interacts with lipids during channel expansion

When membrane tension increases due to osmotic stress, MscL undergoes conformational changes that open its pore, allowing rapid efflux of cytoplasmic solutes and water to relieve pressure. The gating mechanism involves "radial dragging force" on the TM helices, which induces "radial sliding of the crossing portions, leading to a gate expansion" . This mechanical response system is crucial for bacterial survival in changing osmotic environments.

What expression systems are most effective for producing recombinant P. multocida proteins for research purposes?

Based on current research, E. coli expression systems remain the gold standard for producing recombinant P. multocida proteins. The methodology typically follows these steps:

• Gene identification and isolation: Target genes are amplified from P. multocida genomic DNA using PCR with specifically designed primers

• Vector selection: Common vectors include pET43.1a for His-tagged fusion proteins

• Transformation: Using competent E. coli strains like DH5α for cloning and BL21(DE3) for expression

• Expression conditions: Optimized for temperature, induction time, and inducer concentration

• Purification: Often via affinity chromatography for tagged proteins

For example, a study with P. multocida strain PMWSG-4 successfully expressed and purified recombinant VacJ (84.4 kDa), PlpE (94.8 kDa), and OmpH (96.7 kDa) proteins using this approach . The purified proteins retained antigenic properties, demonstrating proper folding and epitope presentation.

Other expression systems reported in literature include:

• Yeast expression systems

• Baculovirus expression systems

• Mammalian cell expression systems

The choice of expression system should be guided by the specific requirements of the target protein, including post-translational modifications, solubility considerations, and functional requirements.

What structural and functional assays are used to characterize mechanosensitive channels like MscL?

Multiple complementary techniques are required for comprehensive characterization of mechanosensitive channels:

The combination of these techniques has revealed key insights into the MscL gating mechanism, including the identification of Phe78 as "the primary tension sensor of MscL" and characterization of the lipid-protein interactions involved in mechanosensation.

How have recombinant P. multocida proteins been used in vaccine development research?

Recombinant P. multocida proteins have shown significant promise as subunit vaccine candidates, offering advantages over traditional whole-cell vaccines in terms of safety and specificity. Several strategies have been investigated:

• Surface protein-based vaccines:

  • Lipoproteins (PlpE, VacJ) and outer membrane proteins (OmpH) have demonstrated strong immunogenic properties

  • A combination of rVacJ, rPlpE, and rOmpH formulated with oil adjuvant provided 100% protection in ducks against challenge with virulent P. multocida

• Toxin fragment vaccines:

  • A C-terminal fragment of P. multocida toxin (PMT2.3) expressed in E. coli induced protective immunity in mice and swine

  • Vaccination led to "high levels of the anti-PMT antibody with a high neutralizing antibody titer" and "strong protection against a homologous challenge"

• Epitope-based vaccines:

  • A triple epitope (PlpE1+2+3) fusion protein was developed using bioinformatics to identify immunogenic regions

  • This approach elicited higher IgG levels than commercial vaccines, though protection was equal (69%)

• Vector-delivered antigens:

  • Attenuated Salmonella Choleraesuis has been used to synthesize and secrete P. multocida PlpE, forming vaccine candidate rSC0016(pS-PlpE)

  • This approach "induced higher antigen-specific mucosal, humoral and mixed Th1/Th2 cellular immune responses" and enhanced survival rate (80%) compared to inactivated vaccine (60%)

These approaches demonstrate the versatility of recombinant protein technology in developing next-generation vaccines against P. multocida infections.

What molecular mechanisms underlie MscL channel gating and how can they be experimentally manipulated?

The gating of MscL is a complex process involving several distinct molecular events that can be manipulated experimentally:

• Tension sensing mechanism:

  • Membrane tension is primarily sensed at the lipid-protein interface

  • Molecular dynamics simulations identified "Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL"

  • The "lipid-moves-first" model proposes that "the number lipid acyl chains occupying TM pockets determined the conformational state of the protein"

• Gate expansion process:

  • "Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78"

  • This force "was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices"

  • The resulting "radial sliding of the crossing portions" leads to "gate expansion"

• Experimental manipulation approaches:

  • Mutagenesis: The L89W mutation in TbMscL "stabilized an expanded and subconducting state" by "hindering the penetration of lipid acyl chains into TM pockets"

  • Chemical modification: "Cysteine mutations with chemical modification" allowed engineering MscL to respond to "various stimuli such as pH and light"

  • Locally distributed tension: The LDT-MD method applies "forces continuously distributed among lipids surrounding the channel" to induce channel opening without disrupting membrane integrity

  • Asymmetric membrane manipulation: Applying different tensions to inner and outer leaflets can model "the effect of single-sided addition of lysolipids"

These approaches have revealed that MscL's gating energy landscape is tension-dependent, with various subconducting states occurring during the transition from closed to fully open conformations.

How do mutations in key residues affect MscL function, and what does this reveal about channel mechanics?

Mutagenesis studies have provided crucial insights into MscL structure-function relationships:

These mutations reveal several key principles:

• The closed state stability is finely balanced and can be disrupted by relatively small changes in pore-lining residues

• Lipid-protein interactions at specific interfaces are critical for mechanosensation

• Transmembrane pockets serve as crucial sites for tension sensing and conformational changes

• Channel gating involves coordinated movements across multiple subunits

PELDOR spectroscopy studies of the L89W mutant demonstrated that "the expanded state was characterized further using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments and ESEEM spectroscopy measurements, highlighting structural transitions that occur from modulation by the L89W mutation" . This confirms that mutations can stabilize physiologically relevant intermediate states that provide insights into the normal gating pathway.

What are the current methodological challenges in expressing and purifying functional recombinant MscL for structural studies?

Obtaining sufficient quantities of properly folded, functional MscL presents several challenges:

• Membrane protein overexpression toxicity:

  • Overexpression can disrupt host cell membrane integrity

  • Solution: Use tightly regulated expression systems and specialized E. coli strains

• Maintaining native structure during solubilization:

  • Detergents can disrupt crucial lipid-protein interactions

  • Solution: Screen multiple detergents or use nanodiscs/liposomes for reconstitution

• Functional verification:

  • Difficult to assess channel functionality outside native membrane environment

  • Solution: Develop reconstitution protocols for electrophysiology or liposome-based assays

• Protein stability during purification:

  • MscL may denature or aggregate during purification steps

  • Solution: Optimize buffer conditions and consider fusion partners to enhance stability

• Crystallization challenges:

  • Membrane proteins are notoriously difficult to crystallize

  • Solution: Consider alternative structural techniques like cryo-EM or EPR spectroscopy

For spin-labeling studies, researchers have successfully used site-directed spin labeling where "the MTSSL spin label on a introduced cysteine residue, modulated channel function as seen previously for other sulfhydryl modification, but also allowed high-resolution measurements to follow conformational changes in the channel" .

For structural alignment studies comparing MscL from different species, PELDOR measurements have proven valuable, allowing researchers to show "L89W (TbMscL) structurally corresponds to M94 in E. coli" .

How do computational approaches contribute to understanding MscL dynamics and potential applications to P. multocida research?

Computational approaches have become indispensable for understanding MscL dynamics:

• Molecular dynamics simulations:

  • Novel locally distributed tension MD (LDT-MD) method "allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable"

  • This approach achieved "reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values"

  • LDT-MD enables exploration of "the MscL gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets"

• Free energy landscape mapping:

  • LDT-MD combined with well-tempered metadynamics has reconstructed "the tension-dependent free energy landscape for the opening transition in MscL"

  • This reveals energy barriers between states and helps explain the probability of subconductance states

• Interaction energy calculations:

  • Calculating "interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids" identified Phe78 as "the primary tension sensor of MscL"

• Sequence analysis and structure prediction:

  • While not explicitly mentioned in the search results, bioinformatics approaches could be applied to identify potential MscL homologs in P. multocida

  • Sequence-based prediction of immunogenic epitopes has been successful for other P. multocida proteins

• Applications to P. multocida research:

  • Computational approaches could identify mechanosensitive channels in P. multocida genome

  • Structural models could guide the design of drugs targeting these channels

  • Virtual screening could identify potential modulators of channel function

These computational methods complement experimental approaches and provide insights that would be difficult to obtain through experiments alone.

What strategies can improve the efficacy of recombinant P. multocida protein vaccines and how might mechanosensitive channel research contribute?

Several strategies have demonstrated improved efficacy for recombinant P. multocida vaccines:

• Antigen combination approaches:

  • A vaccine formulation combining three recombinant proteins (rVacJ+rPlpE+rOmpH) provided 100% protection compared to 33.3%, 83.33%, and 83.33% for individual proteins

  • This suggests synergistic effects between antigens targeting different aspects of bacterial biology

• Novel delivery systems:

  • Recombinant attenuated Salmonella Choleraesuis vector (rSC0016) delivering P. multocida PlpE showed enhanced survival rate (80%) compared to inactivated vaccine (60%)

  • Live vector delivery can "mimic natural infections by organisms, lead to the induction of mucosal, humoral, and cellular immune responses"

• Adjuvant optimization:

  • Different adjuvant formulations (water-in-oil vs. oil-coated) significantly affect immune response profiles

  • Induction of balanced Th1/Th2 responses appears beneficial: "the rSC0016(pS-PlpE) recombinant vaccine induced potent, mixed Th1/Th2 cellular immune responses"

• Epitope-focused design:

  • Bioinformatics-guided design of "potentially immunogenic and protective epitopes" merged to create "the most optimally immunogenic triple epitope PlpE fusion protein"

  • This approach can enhance specific immune responses while eliminating unnecessary or potentially harmful epitopes

• Potential contributions from mechanosensitive channel research:

  • MscL research methodologies like "the LDT-MD method [that] enables exploration of the MscL gating process with different pulling velocities" could be adapted to study membrane protein dynamics in P. multocida

  • Understanding of protein-lipid interactions from MscL studies could inform membrane protein vaccine design

  • MscL's role in osmotic stress response suggests it might be a target for antibiotic adjuvants or novel therapeutic approaches

  • Insights into bacterial adaptation mechanisms could identify new vaccine targets

The integration of these approaches with advances in structural biology and immunology presents promising avenues for next-generation vaccine development against P. multocida infections.