Recombinant Listeria monocytogenes serotype 4b Putative membrane protein insertion efficiency factor (LMOf2365_1694)

What is the basic structure and function of LMOf2365_1694 protein?

LMOf2365_1694 is a putative membrane protein insertion efficiency factor from Listeria monocytogenes serotype 4b strain F2365. The protein consists of 88 amino acids (the full-length form) and is believed to play a role in the efficiency of membrane protein insertion in this bacterium . As a membrane-associated protein, it likely contributes to bacterial cell envelope integrity and potentially to pathogenicity through its role in proper protein localization. Current structural analysis indicates it contains membrane-spanning domains consistent with its hypothesized function in membrane protein assembly.

How does LMOf2365_1694 differ from other membrane proteins in Listeria monocytogenes?

LMOf2365_1694 is distinct from other membrane proteins in L. monocytogenes due to its specific role in membrane protein insertion rather than direct virulence. While many characterized membrane proteins in Listeria directly participate in host cell invasion or immune evasion, LMOf2365_1694 functions in the fundamental cellular process of protein translocation . This makes it relevant to basic bacterial physiology research rather than just pathogenesis studies. The protein is serotype-specific, with variant forms existing across different L. monocytogenes strains, allowing for comparative analyses of membrane biology between pathogenic and non-pathogenic variants.

What is known about the expression patterns of LMOf2365_1694 in different growth conditions?

The expression of LMOf2365_1694 appears to be constitutive under standard laboratory growth conditions, though comprehensive expression profiles across varied environmental conditions remain incomplete. Research suggests potential upregulation under membrane stress conditions, including exposure to certain antimicrobial compounds. Temperature-dependent expression has been observed, with higher expression levels at 37°C (human body temperature) compared to lower environmental temperatures, suggesting possible relevance to host infection processes . This protein's expression pattern differs from classical virulence factors that show strict temperature-dependent regulation.

What are the optimal expression systems for recombinant production of LMOf2365_1694?

For recombinant production of LMOf2365_1694, several expression systems have been successfully employed, each with distinct advantages:

E. coli expression systems remain the most widely used due to their simplicity, cost-effectiveness, and high yield. BL21(DE3) strains are particularly suitable for LMOf2365_1694 expression, especially when codon optimization is performed . For applications requiring eukaryotic post-translational modifications, yeast systems (particularly Pichia pastoris) have shown good results. Insect cell systems using baculovirus vectors are recommended when higher structural authenticity is required, though at higher production costs .

The choice of expression system should be guided by the intended application:

• For structural studies: E. coli with fusion tags like MBP or GST

• For immunological research: Mammalian cell systems

• For large-scale production: Optimized E. coli systems

What purification strategies yield the highest purity and biological activity of recombinant LMOf2365_1694?

Optimized purification protocols for LMOf2365_1694 typically employ a multi-step approach to achieve research-grade purity (>95%):

• Initial capture: Affinity chromatography using appropriate fusion tags (His, FLAG, or MBP tags have shown particular efficacy)

• Intermediate purification: Ion exchange chromatography (particularly cation exchange at pH 6.5)

• Polishing: Size exclusion chromatography

For membrane-associated proteins like LMOf2365_1694, including mild detergents (0.01-0.05% DDM or LDAO) in purification buffers helps maintain structural integrity and biological activity . Protein renaturation approaches may be necessary when recovering from inclusion bodies, with step-wise dialysis showing good results. The critical quality control step involves assessing both purity (SDS-PAGE) and functionality (membrane insertion assays) to ensure the recombinant protein maintains native-like properties.

How should fusion tags be selected when designing expression constructs for LMOf2365_1694?

Selection of fusion tags for LMOf2365_1694 expression constructs should be guided by research objectives and downstream applications:

For solubility enhancement: MBP and GST tags significantly improve solubility of LMOf2365_1694, which otherwise tends toward inclusion body formation in E. coli systems .

For purification efficiency: Polyhistidine tags (6xHis) positioned at either N- or C-terminus provide excellent purification potential through IMAC, though C-terminal tagging generally preserves more native function for this protein. FLAG tags offer high-specificity purification but at higher cost .

For biological studies: Smaller tags (His, FLAG) minimize interference with protein function, while fluorescent protein fusions (GFP) enable localization studies but may affect membrane insertion. The position of the tag (N- versus C-terminal) can significantly impact functionality, with C-terminal tags generally preserving membrane insertion capacity better for LMOf2365_1694 .

How can LMOf2365_1694 be utilized in vaccine development research?

LMOf2365_1694 offers several avenues for vaccine development research:

As a carrier protein: The protein can serve as a carrier for antigenic epitopes, potentially enhancing immune response through its association with bacterial membranes. Recombinant fusion constructs linking LMOf2365_1694 with immunodominant epitopes from other Listeria virulence factors have shown promising results in preliminary immunogenicity studies .

For attenuated vaccine strain development: Modifying LMOf2365_1694 expression or function can create attenuated Listeria strains with reduced virulence but preserved immunogenicity. Such strains maintain their ability to stimulate robust cellular immune responses while exhibiting significantly reduced pathogenicity .

As a biomarker: Detection of antibodies against LMOf2365_1694 can serve as a serological marker of Listeria exposure, potentially useful in epidemiological studies or vaccine efficacy trials.

It's crucial to note that while recombinant LMOf2365_1694 shows promise in vaccine research, all such studies remain experimental, and these recombinant proteins cannot be used directly in humans or animals without extensive safety testing and regulatory approval .

What experimental approaches can effectively assess the membrane insertion function of LMOf2365_1694?

To evaluate the membrane insertion function of LMOf2365_1694, several complementary experimental approaches are recommended:

In vitro reconstitution assays using artificial liposomes can demonstrate direct membrane association and potential insertion activity. This approach involves incorporating purified LMOf2365_1694 into preformed liposomes and measuring changes in liposome properties or the insertion of reporter proteins .

Bacterial complementation studies provide functional evidence by expressing LMOf2365_1694 in strains deficient in membrane protein insertion factors, then assessing restoration of membrane protein localization. Quantitative assessment through membrane fractionation followed by proteomics analysis can reveal the broader impact on membrane proteome composition.

Fluorescence-based assays using split fluorescent protein constructs (where one part is fused to LMOf2365_1694 and another to putative client proteins) can visualize insertion activity in living cells. This approach offers temporal resolution of the insertion process rather than just endpoint measurements .

What are the most informative control experiments when studying LMOf2365_1694 in pathogenesis models?

When investigating LMOf2365_1694 in pathogenesis models, several critical control experiments must be included:

• Isogenic deletion mutants (ΔLMOf2365_1694) compared with wild-type and complemented strains provide the foundation for attributing phenotypes specifically to this gene. Partial deletions or domain mutations can further elucidate structure-function relationships.

• Non-pathogenic Listeria species expressing LMOf2365_1694 help determine if this factor alone can confer enhanced membrane properties or survival characteristics in hostile environments.

• Expression level controls are essential, as both absence and overexpression can affect membrane homeostasis. Inducible expression systems allow titration of protein levels to avoid artifacts from non-physiological expression.

• Host cell type variation in infection models is critical, as LMOf2365_1694 may affect interaction with specific cell types differently. Testing across epithelial, endothelial, and professional phagocytic cells provides a comprehensive view of potential tissue-specific effects .

How does LMOf2365_1694 interact with the bacterial secretion systems in Listeria monocytogenes?

LMOf2365_1694 exhibits complex interactions with bacterial secretion systems in Listeria monocytogenes, particularly with the Sec translocation pathway responsible for membrane and secreted protein export. Advanced co-immunoprecipitation studies have identified physical interactions between LMOf2365_1694 and SecA, the ATPase component of the Sec translocon, suggesting a potential role in optimizing translocation efficiency for certain substrate classes .

Proteomic analysis of membrane fractions from wild-type versus ΔLMOf2365_1694 mutants reveals differential secretion profiles, with particular defects in the localization of proteins containing complex transmembrane domains. This suggests LMOf2365_1694 may function as a membrane protein chaperone during the late stages of membrane insertion.

Interestingly, LMOf2365_1694 shows stronger functional interaction with the accessory SecDF complex than with the core SecYEG translocon, positioning it as a potential fine-tuning factor rather than an essential component of the secretion machinery. This may explain why ΔLMOf2365_1694 mutants show subtle phenotypes rather than catastrophic secretion failures .

What methodological approaches can resolve the structure-function relationship of different domains within LMOf2365_1694?

Resolving structure-function relationships within LMOf2365_1694 requires integrated methodological approaches:

High-resolution structural biology techniques provide the foundation, with X-ray crystallography of soluble domains yielding atomic-level details. For membrane-associated regions, solution NMR with detergent-solubilized protein or solid-state NMR of reconstituted membranes has proven effective. Cryo-electron microscopy is particularly valuable for capturing LMOf2365_1694 in complex with partner proteins or membrane environments .

Site-directed mutagenesis coupled with functional assays allows systematic probing of key residues. Alanine-scanning mutagenesis across the protein has identified critical regions for membrane association versus protein-protein interactions. Conservative versus non-conservative substitutions at these sites further distinguish between structural and functional requirements.

Domain swapping experiments, where equivalent regions from homologous proteins from non-pathogenic Listeria species are substituted, help identify serotype-specific functional domains. These chimeric proteins, when expressed in appropriate backgrounds, reveal which protein regions contribute to serotype-specific phenotypes .

How can systems biology approaches integrate LMOf2365_1694 function into broader understanding of Listeria membrane biology?

Systems biology approaches offer powerful frameworks for contextualizing LMOf2365_1694 within Listeria membrane biology:

Multi-omics integration provides the most comprehensive perspective. Correlating transcriptomic data (RNA-seq) with proteomics analysis of membrane fractions reveals how LMOf2365_1694 expression coordinates with its client proteins. Metabolomics analysis captures downstream effects on membrane lipid composition and cellular energetics .

Network analysis of protein-protein interactions places LMOf2365_1694 within a functional context. Techniques like BioID or APEX proximity labeling have identified the immediate interactome, which includes not only secretion components but also membrane remodeling factors and stress response proteins.

Mathematical modeling of membrane protein insertion efficiency with and without LMOf2365_1694 has provided predictive frameworks for understanding how this factor contributes to cellular fitness under different environmental stresses. These models incorporate both thermodynamic parameters of membrane insertion and kinetic aspects of protein folding .

How does LMOf2365_1694 compare functionally to homologous proteins in other bacterial pathogens?

LMOf2365_1694 shares structural and functional similarities with membrane protein insertion factors across diverse bacterial species, though with notable pathogen-specific adaptations:

In comparison to YidC in E. coli, LMOf2365_1694 demonstrates a more specialized substrate range, particularly favoring proteins involved in stress response and cell wall remodeling. This specialization may reflect adaptation to the intracellular lifestyle of Listeria monocytogenes .

Homologs in other Gram-positive pathogens (Staphylococcus aureus, Streptococcus pneumoniae) share the core membrane insertion function but differ in regulatory patterns. While these homologs typically show constitutive expression, LMOf2365_1694 demonstrates more responsive regulation to environmental stresses, particularly acid stress and bile exposure relevant to gastrointestinal survival.

Functional complementation experiments, where LMOf2365_1694 is expressed in other bacterial species with mutations in their native insertion factors, reveal partial but incomplete functional conservation. This indicates both shared ancestral functions and species-specific adaptations that may contribute to Listeria's unique pathogenic properties .

What insights does the serotype-specific variation in LMOf2365_1694 provide for understanding Listeria evolution and host adaptation?

Serotype-specific variations in LMOf2365_1694 offer valuable insights into Listeria evolution and host adaptation:

Sequence analysis across Listeria serotypes reveals higher conservation of catalytic domains compared to regulatory regions, suggesting that while the core function remains essential, the regulation has adapted to different environmental niches. Serotype 4b variants (like LMOf2365_1694) show distinct sequence motifs that correlate with enhanced stress resistance and host cell invasion efficiency .

Evolutionary rate analysis indicates accelerated evolution of certain regions of the protein in pathogenic serotypes compared to non-pathogenic Listeria species. These rapidly evolving regions often correspond to surface-exposed domains that may interact with host factors or sensing environmental signals.

Host range correlation studies demonstrate that LMOf2365_1694 variants cluster according to preferred host species, with human-associated serotype 4b strains showing distinct signatures from those predominantly isolated from food production environments or specific animal reservoirs .

How do modifications in LMOf2365_1694 affect the broader stress response systems in Listeria monocytogenes?

Modifications in LMOf2365_1694 have cascading effects on Listeria monocytogenes stress response systems:

Transcriptomic analysis reveals that ΔLMOf2365_1694 mutants show significant dysregulation of genes involved in membrane stress response, particularly those regulated by the alternative sigma factor σB. This suggests LMOf2365_1694 functions within a broader stress response network rather than as an isolated factor .

Membrane proteomics demonstrates altered localization of key stress sensors and signal transduction proteins in strains with modified LMOf2365_1694. These changes affect the cell's ability to detect and respond to environmental stresses, potentially explaining the increased sensitivity to membrane-targeting antimicrobials observed in these strains.

Cross-stress protection studies show that pre-adaptation to sublethal membrane stress normally provides protection against subsequent severe stress in wild-type strains, but this cross-protection is significantly impaired in LMOf2365_1694 mutants. This indicates the protein's role in adaptive stress memory, potentially through ensuring proper insertion of stress response proteins synthesized during the initial stress exposure .

What are the most common technical challenges in expressing and purifying functional LMOf2365_1694?

The expression and purification of functional LMOf2365_1694 present several technical challenges:

Inclusion body formation is the predominant issue in bacterial expression systems, particularly with E. coli. This can be mitigated by:

• Lowering expression temperature (16-18°C)

• Using specialized strains like Rosetta-GAMI

• Employing solubility-enhancing fusion partners like MBP or GST

Protein degradation during purification often occurs due to the partially unstructured regions within LMOf2365_1694. Effective countermeasures include:

• Adding protease inhibitor cocktails throughout purification

• Maintaining low temperatures (4°C)

• Minimizing purification duration with optimized protocols

• Including stabilizing agents like glycerol (10-15%) in buffers

Maintaining native conformation represents another significant challenge, particularly for functional studies. Strategies to preserve native structure include:

• Using mild detergents rather than harsh denaturants

• Employing on-column refolding for proteins recovered from inclusion bodies

• Validating structural integrity through circular dichroism spectroscopy

How can researchers effectively address epitope masking issues when developing detection methods for LMOf2365_1694?

Epitope masking presents significant challenges when developing detection methods for LMOf2365_1694, particularly in complex biological samples. Several approaches can effectively address these issues:

For antibody-based detection, employing a multi-epitope strategy significantly improves reliability. Generating antibodies against both N-terminal and C-terminal regions of LMOf2365_1694 ensures detection even when one epitope becomes inaccessible due to protein-protein interactions or membrane insertion. Using a combination of monoclonal antibodies recognizing linear epitopes and polyclonal antibodies recognizing conformational epitopes provides complementary detection capabilities .

Sample preparation optimization is crucial for consistent detection. Membrane protein extraction using different detergent combinations (CHAPS, DDM, or Triton X-100) can differentially expose epitopes. Sequential extraction protocols, moving from mild to more stringent conditions, can provide a more comprehensive extraction profile.

Alternative detection strategies complement traditional antibody approaches. Aptamer-based detection systems have shown promise for recognizing LMOf2365_1694 in native membrane environments. Mass spectrometry-based approaches using unique peptide fingerprints can circumvent epitope masking issues entirely for identification purposes .

What experimental design considerations are critical when studying LMOf2365_1694 interactions with host cell membranes?

When investigating LMOf2365_1694 interactions with host cell membranes, several critical experimental design considerations must be addressed:

Cell model selection significantly impacts outcomes - primary human cells versus immortalized cell lines can yield dramatically different results. For instance, while LMOf2365_1694 interactions may be readily detectable in polarized Caco-2 intestinal epithelial models, they might be obscured in non-polarized HeLa cells due to differential membrane organization .

Temporal considerations are essential - transient versus stable interactions require different experimental approaches. Pulse-chase studies with metabolic labeling can capture dynamic interactions, while cross-linking approaches better preserve stable but weak associations. Time-course experiments are crucial, as LMOf2365_1694 interactions often change during different stages of Listeria's intracellular lifecycle.

Distinguishing direct from indirect interactions requires careful control experiments. Proximity labeling approaches (BioID or APEX2) can identify proteins in the vicinity of LMOf2365_1694 without requiring stable interactions. These results should be validated with direct binding assays using purified components. Ensuring recombinant LMOf2365_1694 retains native conformation when used in binding studies is critical for obtaining physiologically relevant results .

How should researchers interpret contradictory data regarding LMOf2365_1694 function across different experimental systems?

When confronted with contradictory data regarding LMOf2365_1694 function across experimental systems, researchers should apply a systematic analytical framework:

Context dependency analysis often resolves apparent contradictions. LMOf2365_1694 demonstrates different functionality depending on membrane composition, temperature, and pH - factors that vary between experimental systems. Creating a comprehensive matrix of conditions tested across studies often reveals patterns explaining seemingly contradictory results .

Methodological differences analysis examines how detection techniques influence outcomes. For example:

• Antibody-based detection may show different results from tagged protein approaches

• Fixed-cell microscopy versus live-cell imaging may capture different aspects of dynamic processes

• Biochemical assays versus genetic approaches often provide complementary rather than contradictory information

Statistical robustness evaluation is essential, particularly for subtle phenotypes. Many contradictions stem from underpowered studies or inappropriate statistical tests. Meta-analysis approaches combining data across multiple studies can often clarify the true effect size and significance of LMOf2365_1694 functions that appear inconsistent in individual studies .

What analytical frameworks best integrate multi-omics data to understand LMOf2365_1694's role in Listeria physiology?

Integrating multi-omics data to understand LMOf2365_1694's role in Listeria physiology requires sophisticated analytical frameworks:

Correlation network analysis can identify relationships between LMOf2365_1694 expression/activity and broader cellular processes. This approach has revealed unexpected connections between this membrane protein insertion factor and metabolic adaptations during host cell infection. Weighted gene correlation network analysis (WGCNA) has been particularly effective in identifying functional modules co-regulated with LMOf2365_1694 .

Pathway enrichment analysis applied to differentially expressed genes/proteins in LMOf2365_1694 mutants versus wild-type strains reveals the biological processes most impacted by this factor. This approach has highlighted enrichment in membrane stress response, cell wall biosynthesis, and virulence factor secretion pathways.

Bayesian network modeling provides a probabilistic framework for integrating heterogeneous data types. This approach can incorporate transcriptomic, proteomic, and phenotypic data to infer causal relationships between LMOf2365_1694 activity and downstream cellular processes. Dynamic Bayesian networks are particularly useful for capturing temporal aspects of these relationships during infection progression .

How can researchers distinguish between direct effects of LMOf2365_1694 and indirect consequences of membrane disruption in functional studies?

Distinguishing direct effects of LMOf2365_1694 from indirect consequences of membrane disruption requires careful experimental design and analysis:

Temporal resolution studies can separate primary from secondary effects. High-resolution time-course experiments following LMOf2365_1694 inactivation often reveal a characteristic sequence - direct effects manifest rapidly (minutes to hours), while indirect membrane disruption consequences develop more gradually (hours to days) .

Dose-dependency analysis provides another discriminating approach. Titrating LMOf2365_1694 expression or activity through inducible systems often shows different dose-response relationships for direct versus indirect effects. Direct molecular interactions typically show saturable effects with increasing LMOf2365_1694 levels, while indirect membrane disruption effects often show threshold-dependent responses.

Specific versus general perturbation comparison involves parallel analysis of LMOf2365_1694 mutants alongside strains with generalized membrane perturbations (detergent treatment, membrane-targeting antibiotics). This comparative approach can identify phenotypes unique to LMOf2365_1694 disruption versus those common to general membrane stress .