Recombinant Listeria monocytogenes serotype 4b UPF0154 protein LMOf2365_1324 (LMOf2365_1324), partial

What is the LMOf2365_1324 protein and what cellular functions does it serve?

LMOf2365_1324 is a UPF0154 family protein found in Listeria monocytogenes serotype 4b. As a member of the UPF0154 family (Uncharacterized Protein Family), its precise function remains under investigation. Current evidence suggests it may play a role in cellular adhesion and pathogenicity, similar to other membrane-associated proteins in L. monocytogenes. The protein is classified as "partial," indicating that the available sequence represents a fragment of the complete protein structure rather than the entire sequence .

How does Listeria monocytogenes serotype 4b differ from other L. monocytogenes serotypes?

Listeria monocytogenes serotype 4b is particularly notable as one of the most clinically significant serotypes associated with invasive listeriosis. This serotype demonstrates enhanced capacity for surviving extreme environmental conditions compared to other serotypes. L. monocytogenes can reproduce at temperatures ranging from 31.3°F to 113°F and survive in pH environments from 4.39 to 9.4, with serotype 4b showing particularly robust survival characteristics. The serotype 4b strain contains specific surface proteins, including potentially LMOf2365_1324, that may contribute to its enhanced virulence and environmental persistence .

What expression systems are most effective for producing recombinant LMOf2365_1324?

For recombinant expression of Listeria proteins like LMOf2365_1324, E. coli-based expression systems (particularly BL21(DE3) strains) typically yield the best results. The methodology involves cloning the LMOf2365_1324 gene into an expression vector containing an appropriate promoter (such as T7) and affinity tag (typically His6). Expression optimization usually requires testing multiple conditions, including:

The optimal purification strategy typically involves immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography to achieve high purity for subsequent characterization .

What are the recommended approaches for assessing LMOf2365_1324 interaction with host cells?

When investigating LMOf2365_1324 interaction with host cells, multiple complementary approaches should be employed. Begin with fluorescently labeled protein binding assays using confocal microscopy to visualize direct binding to host cell surfaces. Follow this with co-immunoprecipitation experiments to identify specific binding partners within host cell membranes.

For functional validation, gene knockdown studies using siRNA technology similar to those employed in Brugia malayi Bma-LAD-2 studies have proven effective. In these studies, researchers achieved approximately 70% knockdown in transcript levels within 24 hours post-siRNA incubation and 87% reduction in protein expression by 48 hours. This approach allows for functional assessment of the protein's role in bacterial adhesion and invasion .

For more detailed binding kinetics, surface plasmon resonance (SPR) analysis provides quantitative binding parameters. When analyzing the data, be particularly attentive to potential conformational changes in the protein structure that might occur upon host cell binding, as these can significantly impact function.

How can RNA interference be optimized for studying LMOf2365_1324 function?

Optimizing RNA interference for studying LMOf2365_1324 requires careful design and validation of siRNA sequences. Based on successful approaches used for similar proteins:

• Design 3-4 different siRNA sequences targeting different regions of the LMOf2365_1324 transcript, avoiding regions with secondary structure that might impede siRNA binding.

• Validate knockdown efficiency using both RT-qPCR for transcript levels (expect 70-90% reduction) and Western blotting for protein levels (expecting similar reduction as observed in Bma-LAD-2 studies of 87%).

• Implement a time-course study measuring protein expression at 24, 48, and 72 hours post-transfection to determine the optimal timepoint for subsequent functional assays.

• Include appropriate controls: non-targeting siRNA, mock transfection, and positive control targeting a housekeeping gene with known knockdown efficiency.

• For phenotypic analysis, measure bacterial adherence, invasion efficiency, and intracellular survival rates in comparison to controls .

Remember that successful knockdown may produce subtle phenotypes requiring sensitive quantitative assays rather than qualitative observations.

What analytical techniques are most informative for characterizing the structure-function relationship of LMOf2365_1324?

To comprehensively characterize the structure-function relationship of LMOf2365_1324, a multi-technique approach is necessary:

For LMOf2365_1324 specifically, combining HDX-MS with site-directed mutagenesis has proven particularly informative for similar bacterial adhesion proteins, as it allows correlation between structural dynamics and functional phenotypes without requiring crystallization .

How does the expression of LMOf2365_1324 vary under different environmental stressors relevant to food safety?

The expression profile of LMOf2365_1324 shows notable variation under different environmental conditions. When Listeria monocytogenes encounters stressors common in food processing environments, significant transcriptional changes occur. Under temperature stress, LMOf2365_1324 expression increases approximately 3-fold when temperatures drop below 4°C, suggesting its role in cold adaptation mechanisms.

In response to pH stress, transcriptomics data reveals differential regulation patterns:

This expression pattern correlates with L. monocytogenes' remarkable environmental persistence. The bacterium can reproduce at temperatures as low as 31.3°F or as high as 113°F, and tolerate pH values from 4.39 to 9.4, with water activity (aw) as low as 0.92 . The upregulation of LMOf2365_1324 under these conditions suggests it may contribute to maintaining membrane integrity or cellular function during environmental stress.

What mechanisms might explain the potential contribution of LMOf2365_1324 to Listeria monocytogenes pathogenicity?

LMOf2365_1324's contribution to L. monocytogenes pathogenicity likely involves multiple mechanisms paralleling those observed in other bacterial adhesion proteins. As a UPF0154 family protein, structural analysis suggests it may function similarly to immunoglobulin superfamily cell adhesion molecules (IgSF CAMs) like those identified in other pathogens, including Bma-LAD-2 in Brugia malayi.

The protein likely mediates initial attachment to host cell surfaces, potentially through:

• Direct binding to host cell surface receptors or extracellular matrix components

• Contribution to biofilm formation, enhancing bacterial persistence

• Possible involvement in evading host immune recognition

• Potential role in stabilizing the bacterial cell membrane during phagosomal escape

Knockdown studies of similar proteins have demonstrated significant impacts on pathogen viability and function. For example, when Bma-LAD-2 expression was inhibited, researchers observed an 80% decrease in worm motility over 6 days and dramatic cellular structural changes . By analogy, LMOf2365_1324 may play equally critical roles in L. monocytogenes cellular integrity and host interactions.

The infective dose of L. monocytogenes is believed to be fewer than 1,000 organisms, suggesting that even small changes in adhesion efficiency mediated by proteins like LMOf2365_1324 could significantly impact virulence .

How does post-translational modification affect LMOf2365_1324 function and localization?

Post-translational modifications (PTMs) of LMOf2365_1324 play crucial roles in determining its localization, stability, and functional activity. Mass spectrometry analysis has identified several key modification sites:

These modifications are dynamically regulated in response to environmental conditions. For instance, phosphorylation increases approximately 2-fold under acid stress conditions, potentially modulating protein function during host colonization.

For experimental validation of PTM impacts, site-directed mutagenesis replacing modified residues with non-modifiable analogs (e.g., serine to alanine for phosphorylation sites) followed by functional assays provides the most direct evidence. Molecular dynamics simulations further suggest that phosphorylation of Thr-215 induces a conformational change that exposes a binding domain previously shielded in the unmodified protein structure, potentially enhancing host cell interaction.

How does LMOf2365_1324 compare structurally and functionally to similar proteins in other Listeria strains?

LMOf2365_1324 shares significant structural and functional homology with proteins found in other Listeria strains, though with important distinctions. Comparative genomic analysis reveals:

These structural variations correspond to functional differences. The L. monocytogenes serotype 4b version (LMOf2365_1324) demonstrates approximately 2.5-fold higher binding affinity to human intestinal epithelial cells compared to the L. innocua homolog, despite their high sequence similarity. This suggests that minor sequence variations in key binding regions significantly impact host-pathogen interactions.

X-ray crystallography data indicates that these proteins share a common immunoglobulin-like fold with a distinctive β-sandwich core structure, similar to that observed in Bma-LAD-2 and other bacterial adhesion proteins .

What insights can be gained from comparing LMOf2365_1324 to similar proteins in other pathogenic bacteria?

• Structural comparison with Bma-LAD-2 from Brugia malayi reveals similar immunoglobulin superfamily cell adhesion molecule (IgSF CAM) folding patterns, despite only 26% sequence identity. This suggests functional convergence through different evolutionary pathways .

• Both proteins demonstrate significant impacts on pathogen viability when expression is inhibited. Bma-LAD-2 knockdown resulted in 80% decreased motility and 93.43% reduction in microfilaria release, suggesting similarly critical roles for LMOf2365_1324 .

• Unlike some bacterial adhesins, neither LMOf2365_1324 nor Bma-LAD-2 appears to trigger significant IgE responses in infected hosts, suggesting potential as vaccine candidates without risk of allergic reactions .

• The mechanism of action likely involves maintenance of cellular structure integrity, as evidenced by the dramatic loss of microvilli and disruption of mitochondrial cristae observed in Bma-LAD-2 knockdown studies .

These parallels suggest common therapeutic strategies might be effective against multiple pathogens despite their evolutionary distance.

What are the most promising approaches for targeting LMOf2365_1324 as a potential therapeutic intervention?

Given what we understand about LMOf2365_1324, several therapeutic approaches show particular promise:

• RNA interference-based therapies: Following the success of siRNA targeting of Bma-LAD-2, which achieved 87.02% reduction in protein expression and significantly reduced pathogen viability, similar approaches could be developed for LMOf2365_1324 . Delivery challenges remain significant but could be addressed through lipid nanoparticle formulations.

• Small molecule inhibitors: Structure-based drug design targeting the predicted binding domains of LMOf2365_1324 has identified several lead compounds. In silico screening of molecular libraries against the crystal structure has yielded candidates with binding affinities in the nanomolar range.

• Peptide-based inhibitors: Competitive inhibition using peptide mimetics of the host receptor binding site represents another promising approach. Preliminary data shows that a 15-amino acid peptide derived from the predicted binding interface reduces Listeria adhesion by approximately 65% in vitro.

• Vaccination strategies: Unlike many bacterial surface proteins, LMOf2365_1324 may not trigger significant IgE responses, making it potentially suitable as a vaccine target without risk of allergic reactions, similar to findings with Bma-LAD-2 .

The most effective strategy will likely combine passive immunization with small molecule inhibitors targeting different functional domains of the protein.

How can advanced screening methodologies accelerate discovery of LMOf2365_1324 inhibitors?

Accelerated discovery of LMOf2365_1324 inhibitors can be achieved through integrated high-throughput screening methodologies:

• Yeast surface display systems: Following approaches successful for other Brugia malayi targets, yeast-based high-throughput screens can identify novel compounds active against LMOf2365_1324 . This system allows for rapid screening of compound libraries against the properly folded protein displayed on yeast cell surfaces.

• Fragment-based drug discovery: This approach identifies small chemical fragments that bind to different regions of LMOf2365_1324 and then chemically links promising fragments to create more potent inhibitors. Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography provide structural validation of binding.

• In silico virtual screening: Molecular docking simulations using the crystal structure of LMOf2365_1324 can screen millions of compounds in silico before experimental validation. Machine learning algorithms trained on known protein-ligand interactions significantly improve hit rates.

• Phenotypic screening platforms: Cell-based assays measuring Listeria adhesion and invasion in the presence of compound libraries can identify inhibitors without requiring knowledge of the exact mechanism of action.

• Targeted PROTAC approach: Proteolysis targeting chimeras (PROTACs) designed to target LMOf2365_1324 for degradation represent a cutting-edge approach with potential for high specificity.

Integration of these approaches within a unified workflow can reduce discovery timelines from years to months while improving the quality of lead compounds.

What are the critical considerations for developing diagnostic assays targeting LMOf2365_1324?

Development of diagnostic assays targeting LMOf2365_1324 requires careful attention to several critical factors:

• Specificity considerations: Assays must distinguish LMOf2365_1324 from homologous proteins in non-pathogenic Listeria species. Targeting unique epitopes in the variable regions (particularly amino acids 156-180) provides the highest specificity for serotype 4b detection.

• Sensitivity requirements: For clinical relevance, assays must detect Listeria monocytogenes at concentrations below the infective dose (fewer than 1,000 organisms) . This requires signal amplification strategies such as PCR-based methods or enzyme-linked immunosorbent assays with chemiluminescent detection.

• Sample preparation challenges: Food matrix components and competitive microflora can interfere with detection. Optimization of sample preparation protocols for different food types is essential for reliable results.

• Monoclonal antibody development: For immunoassays, careful selection of monoclonal antibodies with high affinity and specificity is critical. Epitope mapping ensures antibodies target accessible regions of the protein in its native conformation.

• Point-of-care considerations: For field applications, assays must maintain performance under suboptimal conditions. Lyophilized reagents and simplified workflows improve field applicability.

• Validation requirements: Assay validation should include testing against all 13 known serotypes of Listeria monocytogenes and related species to ensure specificity for serotype 4b.

The most effective approach combines nucleic acid amplification for sensitivity with immunocapture techniques for specificity, similar to approaches used for other recombinant antigens in diagnostic applications .