Recombinant Listeria monocytogenes serovar 1/2a UPF0754 membrane protein lmo2224 (lmo2224)

What is UPF0754 membrane protein lmo2224 and why is it significant in Listeria monocytogenes research?

The UPF0754 membrane protein lmo2224 is a membrane-associated protein encoded by the lmo2224 gene in Listeria monocytogenes. It belongs to the UPF0754 protein family (Uncharacterized Protein Family 0754), indicating that while the protein has been identified, its full functions remain to be thoroughly characterized. The protein is significant in L. monocytogenes research due to its potential role in the organism's membrane structure and function, which may contribute to the pathogen's virulence characteristics and survival within host cells .

L. monocytogenes is a foodborne pathogen that causes listeriosis in humans, with severity depending on multiple factors including host characteristics and pathogen attributes. As a membrane protein, lmo2224 may play roles in cellular interactions, survival mechanisms, or virulence that are valuable to understand both for basic microbiology knowledge and potential therapeutic applications .

Which expression systems are most effective for producing recombinant lmo2224 protein?

For the recombinant expression of UPF0754 membrane protein lmo2224, multiple expression systems can be utilized, each with distinct advantages. E. coli and yeast expression systems provide the highest protein yields and shorter production times, making them practical for initial studies and situations where large quantities of protein are needed .

For applications requiring proper protein folding or maintenance of biological activity, insect cells with baculovirus expression systems or mammalian cell expression systems are preferable. These systems can provide many of the post-translational modifications necessary for correct protein folding or retention of the protein's functional activity .

The selection of expression system should be guided by the specific research questions being addressed:

How does L. monocytogenes serovar 1/2a differ from other serovars in terms of research applications?

L. monocytogenes serovar 1/2a is one of the three major serovars (along with 1/2b and 4b) responsible for approximately 90-95% of human listeriosis cases . This serovar belongs to PCR serogroup IIa (comprising serovars 1/2a and 3a) and has distinctive research applications compared to other serovars.

Serovar 1/2a strains are frequently isolated from food samples and food processing environments. They typically belong to genetic lineage II, which contains both virulent and less virulent strains. While serovar 4b (lineage I) strains are more commonly associated with severe clinical cases and outbreaks, serovar 1/2a strains show remarkable genetic diversity and adaptability to various environments .

For research applications, this means:

• Serovar 1/2a strains are often used in food safety studies due to their prevalence in food products

• They provide valuable models for studying genetic diversity within L. monocytogenes

• The comparative study of 1/2a vs. 4b strains helps understand virulence determinants

• Many laboratory strains (such as EGD-e) are serovar 1/2a, making them widely used reference organisms

Martinez et al. (2017) demonstrated that strains from different serotypes (1/2a, 4b, 1/2b) can show similar pathogenicity in some infection models, highlighting that virulence potential is both dose and strain-dependent rather than strictly serovar-dependent .

What methodologies are recommended for purification of recombinant lmo2224 protein while maintaining functional integrity?

Purification of recombinant UPF0754 membrane protein lmo2224 while maintaining its functional integrity requires careful consideration of the protein's membrane-associated nature. The following methodological approach is recommended:

• Expression system selection: Begin with the expression system that best balances yield with proper folding. For initial characterization studies, E. coli systems with membrane protein-specific vectors (containing signal sequences for proper membrane insertion) may be sufficient. For functional studies, insect or mammalian cell expression systems may be preferable despite lower yields .

• Cell lysis optimization: Use gentle lysis methods that preserve membrane integrity, such as enzymatic lysis with lysozyme for E. coli or non-ionic detergents for eukaryotic cells.

• Detergent selection: Since lmo2224 is a membrane protein, detergent selection is critical. Begin screening with mild non-ionic detergents (e.g., n-dodecyl β-D-maltoside, digitonin) that maintain protein structure and function.

• Affinity purification: Use affinity tags (His-tag, FLAG-tag) positioned to minimize interference with protein function. For membrane proteins, C-terminal tags often cause less disruption than N-terminal tags.

• Size exclusion chromatography: As a final purification step to ensure homogeneity and remove protein aggregates.

• Functional validation: Verify that the purified protein maintains its native conformation using circular dichroism spectroscopy, limited proteolysis, or functional binding assays specific to known interactions of lmo2224.

When working with membrane proteins like lmo2224, maintaining a detergent micelle environment throughout the purification process is essential to prevent protein aggregation and denaturation. The choice between detergent solubilization and alternative methods like amphipol exchange or nanodisc incorporation should be guided by the intended downstream applications.

How does the lmo2224 protein potentially contribute to L. monocytogenes virulence mechanisms?

While the specific role of lmo2224 in L. monocytogenes virulence is not explicitly detailed in the search results, we can infer potential contributions based on our understanding of membrane proteins in this pathogen and related virulence factors:

The pathogenicity of L. monocytogenes is primarily determined by its ability to invade host cells, escape from phagosomes, multiply intracellularly, and spread to adjacent cells. Membrane proteins often play crucial roles in these processes, particularly in adhesion, invasion, and interaction with host cell components .

L. monocytogenes contains several pathogenicity islands, with LIPI-1 being essential for virulence. All strains carry LIPI-1, which clusters several fundamental genes for pathogenicity, including hly (encoding listeriolysin O) and prfA (the main regulator of virulence genes) . Membrane proteins can interact with these virulence systems in various ways:

• Cell surface interactions: As a membrane protein, lmo2224 may contribute to bacterial adhesion to host cells or recognition of environmental signals.

• Nutrient acquisition: Membrane transporters are essential for bacterial survival within the nutrient-limited intracellular environment.

• Stress response: Membrane proteins often function in responding to environmental stresses encountered during infection.

• Evasion of host defenses: Some membrane proteins contribute to resistance against antimicrobial peptides or other host defense mechanisms.

Research methodologies to investigate lmo2224's role in virulence could include:

• Creation of lmo2224 deletion mutants and assessment of their virulence in cell culture and animal models

• Protein localization studies during different stages of infection

• Identification of host proteins that interact with lmo2224

• Transcriptional analysis to determine if lmo2224 expression is regulated by PrfA or other virulence regulators

What experimental models are most appropriate for evaluating the function of lmo2224 in pathogenesis?

Several experimental models can be employed to evaluate the function of lmo2224 in L. monocytogenes pathogenesis, each with specific advantages and limitations:

• In vitro cell culture models:

  • Human intestinal epithelial cells (e.g., Caco-2) to study adhesion and invasion

  • Macrophage cell lines (e.g., J774, RAW264.7) to investigate intracellular survival

  • Brain microvascular endothelial cells to examine blood-brain barrier crossing

  • Placental trophoblast cells to study maternal-fetal transmission

• Galleria mellonella (wax moth) larval model:
  This invertebrate model has been validated for studying L. monocytogenes virulence and can distinguish between virulent and attenuated strains from different clonal complexes (CCs) . G. mellonella offers several advantages:

  • Simplicity and cost-effectiveness

  • Ability to conduct experiments at 37°C (human body temperature)

  • Possession of both cellular and humoral immune responses

  • Ethical advantages over vertebrate models for initial screenings

• Mouse models:

  • Intravenous infection to assess systemic spread to liver and spleen

  • Oral infection to model the natural route of infection

  • Pregnant mouse models to study placental transmission

The experimental approach should include:

• Construction of isogenic mutants lacking lmo2224

• Complementation of mutants to confirm phenotype specificity

• Comparison with wild-type strains in various pathogenesis assays:

  • Adhesion and invasion efficiency

  • Intracellular multiplication rates

  • Cell-to-cell spread capabilities

  • In vivo organ colonization and bacterial burden

  • Host immune response assessment

When using G. mellonella, standardization of larvae diet is crucial, as it impacts hemolymph volume, hemocyte concentration, and immune response, which can affect experimental outcomes . Parameters to evaluate include survival rate, LD50 (median lethal dose), and cytotoxicity measurements.

How can recombinant L. monocytogenes expressing modified lmo2224 be utilized in vaccine development strategies?

Recombinant L. monocytogenes expressing modified lmo2224 proteins represents a promising approach for vaccine development, leveraging the unique intracellular lifecycle of this bacterium. L. monocytogenes has several properties that make it an ideal vector for vaccine design:

• It can enter host cells, escape from endocytic vesicles, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular environment .

• Proteins secreted by L. monocytogenes efficiently enter the major histocompatibility complex (MHC) class I antigen processing pathway, stimulating CD8+ T cell responses .

• Genetic systems exist for stable site-specific integration of expression cassettes into the L. monocytogenes genome .

To utilize recombinant L. monocytogenes expressing modified lmo2224 in vaccine strategies, researchers could employ the following methodological approach:

Step 1: Engineering the expression construct

• Design expression cassettes with lmo2224 fused to immunogenic epitopes of interest

• Ensure proper secretion signals for efficient delivery to the MHC class I pathway

• Consider using the site-specific integration system demonstrated with lymphocytic choriomeningitis virus (LCMV) antigens

Step 2: Attenuation strategies for safety

• Use attenuated L. monocytogenes strains that maintain immunogenicity but reduce pathogenicity

• Consider deletion of virulence genes while preserving the ability to access the cytosol

• Balance attenuation with maintenance of sufficient persistence to induce robust immunity

Step 3: Validation of antigen presentation

• Confirm expression and secretion of the modified lmo2224 constructs

• Verify processing and presentation of desired epitopes using T cell assays

• Assess cross-presentation by dendritic cells

Step 4: Immunogenicity assessment

• Evaluate induction of antigen-specific CD8+ T cell responses

• Measure both primary and memory immune responses

• Confirm protective efficacy against challenge with relevant pathogens

This approach has been successfully demonstrated with LCMV antigens, where L. monocytogenes strains expressing either the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope conferred protection against challenge with virulent LCMV strains . The protection was abrogated by in vivo depletion of CD8+ T cells, confirming that protective immunity was mediated by this cell population .

When developing such vaccines, researchers should consider:

• Selecting appropriate serovar 1/2a strains with known virulence characteristics

• Engineering strains that provide optimal balance of safety and immunogenicity

• Addressing regulatory considerations for recombinant live bacterial vaccines

What are the methodological challenges in differentiating the functions of lmo2224 from other membrane proteins in L. monocytogenes, and how can these be addressed?

Differentiating the specific functions of lmo2224 from other membrane proteins in L. monocytogenes presents several methodological challenges that require sophisticated experimental approaches:

Challenge 1: Functional redundancy
Membrane proteins often have overlapping functions, making phenotypic effects of single gene deletions difficult to detect. This is particularly relevant in bacteria like L. monocytogenes, which possess multiple virulence factors and adaptation mechanisms.

Methodological solution:

• Generate combinatorial deletion mutants of lmo2224 with functionally related membrane proteins

• Employ synthetic lethality screens to identify genetic interactions

• Use CRISPRi for partial and tunable repression of multiple genes simultaneously

Challenge 2: Context-dependent function
The function of lmo2224 may vary depending on environmental conditions, growth phase, or host cell type.

Methodological solution:

• Conduct phenotypic assays under diverse conditions (temperature, pH, nutrient availability)

• Use time-course experiments to capture temporal dynamics of protein function

• Employ tissue-specific or cell-type-specific infection models

Challenge 3: Technical difficulties in membrane protein characterization
Membrane proteins are notoriously challenging to work with due to their hydrophobicity and requirement for lipid environments.

Methodological solution:

• Use protein-protein interaction methods optimized for membrane proteins (e.g., MYTH - Membrane Yeast Two-Hybrid)

• Employ proximity labeling approaches (BioID, APEX) to identify interaction partners in their native environment

• Apply advanced structural biology techniques (cryo-EM, NMR) for membrane proteins

Challenge 4: Distinguishing direct from indirect effects
Phenotypic changes in lmo2224 mutants may result from indirect effects on membrane integrity or other cellular processes.

Methodological solution:

• Conduct comprehensive membrane integrity assays

• Use metabolomic and proteomic profiling to identify pathway perturbations

• Perform carefully controlled complementation studies with point mutants affecting specific functional domains

Challenge 5: Limited knowledge of protein function
As a member of the UPF0754 family, lmo2224's function may not be easily predicted from sequence information alone.

Methodological solution:

• Employ comparative genomics across Listeria species and strains

• Use advanced bioinformatic tools for secondary and tertiary structure prediction

• Generate chimeric proteins with domains from better-characterized homologs

An integrated research strategy combining these approaches would involve:

• Initial bioinformatic characterization and structure prediction

• Generation of a panel of targeted mutations and expression constructs

• Systematic phenotypic assessment under varying conditions

• Identification of interaction partners in different cellular contexts

• Validation of function through complementation and heterologous expression

How does the expression and function of lmo2224 vary across different L. monocytogenes clonal complexes (CCs), and what are the implications for strain virulence assessment?

The expression and function of lmo2224 likely varies across different L. monocytogenes clonal complexes (CCs), potentially contributing to differences in virulence potential. While the search results don't provide specific information about lmo2224 variation across CCs, we can construct a methodological framework for investigating this question based on what is known about CC variation in L. monocytogenes.

L. monocytogenes comprises distinct genetic lineages and clonal complexes with varying virulence potential. Hypervirulent CCs (notably including CC1, CC2, CC4, and CC6) are predominantly associated with clinical cases and severe forms of listeriosis, while hypovirulent CCs (including CC9) are more commonly isolated from food and food processing environments .

Methodological approach to investigate lmo2224 variation across CCs:

• Comparative genomic analysis:

  • Sequence lmo2224 from representative strains across multiple CCs

  • Identify polymorphisms and structural variations

  • Analyze promoter regions for regulatory differences

  • Assess copy number variations

• Transcriptional analysis:

  • Compare lmo2224 expression levels across CCs using RT-qPCR

  • Perform RNA-seq to understand expression in context of the global transcriptome

  • Investigate condition-dependent expression (e.g., during infection vs. environmental growth)

  • Determine if expression is regulated by PrfA or other virulence regulators

• Functional characterization:

  • Create chimeric strains by swapping lmo2224 alleles between CCs

  • Evaluate impact on virulence using Galleria mellonella model, which has been validated for distinguishing virulence potential across CCs

  • Assess cytotoxicity and LD50 values as done for other virulence comparisons

• Protein structure-function analysis:

  • Express and purify lmo2224 variants from different CCs

  • Compare protein stability, localization, and interaction partners

  • Identify functional domains affected by CC-specific variations

Implications for strain virulence assessment:

Understanding lmo2224 variation across CCs has several important implications:

• Biomarker potential: If lmo2224 variants correlate with virulence potential, they could serve as biomarkers for rapid identification of hypervirulent strains, addressing the Food and Agriculture Organization's call for virulence biomarkers .

• Risk assessment refinement: Current risk assessments often treat all L. monocytogenes equally, despite clear differences in virulence potential. lmo2224 variation could help develop more nuanced risk models.

• Evolutionary insights: Patterns of lmo2224 conservation or variation across CCs may reveal evolutionary pressures and adaptation strategies.

• Therapeutic targeting: CC-specific variations in lmo2224 could inform the development of targeted antimicrobial strategies.

The proposed methodological framework would build upon existing knowledge of CC variation, such as the distribution of pathogenicity islands like LIPI-3 (present in CC1, CC2, and CC6) and LIPI-4 (associated with CC4 and CC87), which serve as potential markers of hypervirulence .