Recombinant Listeria monocytogenes serotype 4b 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE)

What is the relationship between ispE and other characterized surface proteins in Listeria monocytogenes serotype 4b?

While ispE is not specifically mentioned in the search results as a surface protein, Listeria monocytogenes serotype 4b expresses several well-characterized surface proteins that are important for virulence and detection. IspC, an 86-kDa protein consisting of 774 amino acids, has been identified as a target of humoral immune response to listerial infection. IspC contains a C-terminal cell wall binding domain made up of seven GW modules of 81 to 82 amino acids each, containing a glycine-tryptophan dipeptide . Understanding the relationship between ispE and surface proteins like IspC could provide insights into bacterial metabolism and pathogenesis networks.

How does Listeria monocytogenes serotype 4b differ from other serotypes in terms of surface protein expression?

Listeria monocytogenes strains are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV) . Serotype 4b has several distinctive surface proteins, including IspC, which is highly conserved within this serotype. Monoclonal antibodies studies have demonstrated that five specific MAbs (M2774, M2775, M2780, M2790, and M2797) showed specificity for L. monocytogenes serotype 4b and only cross-reacted with serotype 4ab isolates . Another surface protein, LMOf2365_0148, which contains an LPXTG motif for covalent linkage to cell wall peptidoglycan, has been identified as a potential broad-range surface biomarker for L. monocytogenes across multiple lineages .

What are the general characteristics of Listeria monocytogenes that should be considered when studying ispE?

Listeria monocytogenes is a gram-positive bacillus characterized by beta-hemolysis, tumbling motility with flagella outside the cell, and "actin rocket" propulsion when intracellular. The bacterium can survive and multiply in near-freezing temperatures, which enables contamination of refrigerated food items . This cold tolerance should be considered when designing experiments involving ispE expression or activity. Additionally, L. monocytogenes is a facultative anaerobic, intracellular bacterium, so studies of ispE should consider both aerobic and anaerobic conditions as well as intracellular expression patterns .

What expression systems are most effective for recombinant Listeria monocytogenes proteins?

Based on previous studies with L. monocytogenes proteins, Escherichia coli remains the preferred expression system for recombinant proteins. For example, recombinant IspC was successfully expressed in E. coli, purified, and used to raise specific rabbit polyclonal antibodies for protein characterization . Similarly, other Listeria proteins such as phospholipase C (PC-PLC) have been successfully expressed in E. coli systems . When designing an expression strategy for ispE, researchers should consider:

What purification strategies should be employed for recombinant ispE?

A multi-step purification strategy is typically necessary to obtain pure, active recombinant ispE:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

• Intermediate purification using ion-exchange chromatography

• Polishing step using size-exclusion chromatography to remove aggregates

• Buffer optimization to maintain enzyme stability and activity

When designing purification protocols, researchers should consider that enzyme activity may be dependent on Zn²⁺ ions, as seen with other Listeria enzymes like PC-PLC . It's advisable to include activity assays at each purification step to ensure the enzyme remains functional throughout the process.

How can the expression and stability of recombinant ispE be optimized?

Optimizing expression and stability requires systematic testing of multiple conditions:

• Codon optimization for E. coli expression

• Testing different growth media (LB, TB, auto-induction media)

• Varying induction parameters (OD600 at induction, IPTG concentration, post-induction time)

• Screening buffers with different pH values, salt concentrations, and additives

• Including protease inhibitors during extraction and purification

• Testing stabilizing agents such as glycerol, reducing agents, and specific metal ions

For Listeria proteins, expression at lower temperatures (16-25°C) often improves solubility, as demonstrated in studies with other recombinant Listeria proteins .

What methods are appropriate for assessing the enzymatic activity of recombinant ispE?

The enzymatic activity of ispE can be assessed using several complementary approaches:

• Spectrophotometric assays coupling ADP production to NADH oxidation

• HPLC-based assays measuring substrate depletion and product formation

• Radiometric assays using ³²P-labeled ATP

• Mass spectrometry to identify and quantify reaction products

How can the structure-function relationship of ispE be investigated?

Structure-function studies of ispE could follow approaches similar to those used for other Listeria enzymes:

• X-ray crystallography to determine three-dimensional structure

• Site-directed mutagenesis of predicted catalytic residues

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Molecular dynamics simulations to study conformational changes during catalysis

• Truncation studies to identify minimal functional domains

Crystal structure determination has been valuable for understanding other Listeria enzymes like PC-PLC, which revealed distinctive structural details that translate into unique enzymatic properties . Similar approaches would likely provide valuable insights into ispE function.

What approaches can determine if ispE interacts with other proteins in Listeria monocytogenes?

Protein-protein interactions involving ispE can be investigated using:

• Pull-down assays with recombinant ispE as bait

• Bacterial two-hybrid systems

• Co-immunoprecipitation with anti-ispE antibodies

• Surface plasmon resonance (SPR) to measure binding kinetics

• Cross-linking followed by mass spectrometry (XL-MS)

The search results demonstrate the value of SPR for characterizing monoclonal antibody interactions with Listeria surface proteins, with several MAbs showing very low dissociation constants (4.5 × 10⁻⁹ to 1.2 × 10⁻⁸ M) when binding to IspC . Similar approaches could identify protein partners of ispE.

What strategies are effective for generating specific antibodies against ispE?

Based on successful approaches with other Listeria proteins, researchers could:

• Express and purify full-length recombinant ispE or immunogenic fragments

• Immunize rabbits to generate polyclonal antibodies

• Develop monoclonal antibodies through mouse immunization and hybridoma technology

• Screen antibodies for specificity against ispE from different Listeria serotypes

Previous studies have successfully generated 15 monoclonal antibodies against IspC, a surface-associated autolysin in L. monocytogenes serotype 4b . Five of these MAbs showed high specificity for serotype 4b. Similar approaches could yield specific anti-ispE antibodies.

How can antibodies be used to study ispE localization and expression patterns?

Antibodies against ispE can be employed in multiple techniques:

• Immunofluorescence microscopy to visualize protein distribution, as demonstrated with IspC which showed uneven distribution in clusters on the cell surface

• Immunogold electron microscopy for high-resolution localization studies

• Western blotting to monitor expression levels under different growth conditions

• Flow cytometry to quantify surface expression in bacterial populations

• Immunoprecipitation to isolate native protein complexes

Previous studies have shown that native IspC was detected in all growth phases at a relatively stable low level during in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase . Similar temporal expression studies could reveal important aspects of ispE regulation.

What considerations are important when using antibodies for quantitative analysis of ispE?

For quantitative applications, researchers should:

• Determine antibody specificity using recombinant ispE and Listeria lysates

• Establish standard curves with purified recombinant protein

• Validate detection limits and linear range of detection

• Include appropriate controls (knockout strains if available)

• Optimize antibody concentrations to avoid hook effects in immunoassays

When developing quantitative assays, researchers can learn from previous work with L. monocytogenes surface proteins, where ELISA was effectively used to test cross-reactivity of monoclonal antibodies against multiple isolates from different serotypes .

How might ispE contribute to Listeria monocytogenes pathogenesis?

While the specific role of ispE in pathogenesis is not addressed in the search results, insights can be drawn from other metabolic enzymes in bacterial pathogens:

• IspE likely contributes to isoprenoid biosynthesis, which is essential for bacterial membrane integrity

• Disruption of ispE might affect cell wall synthesis and stability

• Isoprenoid derivatives may play roles in virulence factor regulation

• The MEP pathway enzymes like ispE represent potential antibiotic targets due to their absence in humans

The search results highlight that other L. monocytogenes proteins like phospholipase C (PC-PLC) and listeriolysin O (LLO) are major virulence factors that enable the bacterium to spread in the host by destroying cell membranes . Understanding how ispE activity might support these primary virulence mechanisms would be valuable.

What infection models are appropriate for studying ispE's role in pathogenesis?

To investigate ispE's role in pathogenesis, researchers could employ:

• Tissue culture infection models using human cell lines (e.g., Caco-2, HeLa)

• Macrophage infection assays to assess intracellular survival

• Mouse models of listeriosis for in vivo relevance

• Galleria mellonella (wax moth) larvae for preliminary virulence screening

• Competitive index assays comparing wild-type and ispE mutant strains

These models should consider that L. monocytogenes serotype 4b is responsible for a high percentage of fatal cases of food-borne infection , suggesting potent virulence mechanisms that may depend on proper metabolic function, including ispE activity.

How can gene knockout or knockdown approaches be used to assess ispE function?

Genetic manipulation strategies to study ispE could include:

• CRISPR-Cas9 gene editing to create clean deletions

• Antisense RNA approaches if ispE is essential

• Inducible expression systems to control ispE levels

• Transposon mutagenesis for random insertions

• Complementation studies to confirm phenotypes are specifically due to ispE disruption

When analyzing such mutants, researchers should examine:

• Growth characteristics in different media and temperatures

• Cell morphology and division patterns

• Resistance to environmental stresses

• Virulence in appropriate infection models

• Metabolomic profiles to identify pathway disruptions

How can structural information about ispE inform antimicrobial development?

Crystal structure determination of ispE could guide antimicrobial development through:

• Identification of unique structural features not present in human kinases

• Structure-based virtual screening for potential inhibitors

• Fragment-based drug discovery approaches

• Analysis of the ATP-binding site for competitive inhibitor design

• Examination of substrate-binding regions for substrate-competitive inhibitors

The structural analysis approach has been valuable for other Listeria enzymes, as demonstrated with PC-PLC, where structural studies revealed distinctive features that translate into unique properties of the listerial phospholipase .

What high-throughput screening approaches could identify ispE inhibitors?

Effective screening strategies might include:

• Fluorescence-based enzymatic assays adaptable to 384-well plate format

• Virtual screening of compound libraries against the ispE structure

• Phenotypic screening using Listeria growth inhibition as a readout

• Thermal shift assays to identify compounds that bind to and stabilize ispE

• Surface plasmon resonance screening for direct binding interactions

How can systems biology approaches incorporate ispE into our understanding of Listeria monocytogenes metabolism?

Systems biology approaches could include:

• Metabolic flux analysis to quantify the contribution of the MEP pathway

• Integration of transcriptomic, proteomic, and metabolomic data

• Genome-scale metabolic modeling to predict the effects of ispE perturbation

• Network analysis to identify connections between ispE and virulence pathways

• Comparative genomics across Listeria serotypes to identify strain-specific features

These approaches should consider that Listeria monocytogenes expresses different proteins under different growth conditions. For example, IspC was shown to be upregulated in vivo during infection, despite being expressed at relatively stable low levels during in vitro culture .

How can recombinant ispE be utilized in developing diagnostics for Listeria monocytogenes serotype 4b?

Recombinant ispE could contribute to diagnostic development through:

• Generation of specific antibodies for immunoassay development

• Development of aptamers targeting ispE as alternative recognition elements

• Use as a positive control in PCR-based detection methods

• Creation of biosensor platforms based on enzyme activity detection

The search results highlight successful approaches with other Listeria proteins, particularly the promising use of monoclonal antibody M2775, which demonstrated high fidelity and affinity for the IspC protein and serotype 4b isolates, making it a strong candidate for diagnostic test development .

What advantages might ispE offer over existing biomarkers for Listeria detection?

Potential advantages of ispE as a biomarker could include:

• Expression across different growth conditions and environmental stresses

• Possible serotype-specific sequence variations useful for differentiation

• Enzymatic activity providing a functional readout for viable cells

• Potential presence in bacterial secretomes or outer membrane vesicles

For comparison, the search results describe LMOf2365_0148 as a useful novel surface biomarker for identifying, detecting, and isolating L. monocytogenes from food and environmental samples . IspE could potentially complement existing biomarkers for improved detection specificity and sensitivity.

What validation approaches ensure the reliability of ispE-based detection methods?

Rigorous validation should include:

• Testing against diverse L. monocytogenes isolates representing all serotypes

• Determining cross-reactivity with other Listeria species and related bacteria

• Establishing limits of detection in various food matrices and environmental samples

• Conducting interlaboratory validation studies

• Comparing performance against gold standard methods

The search results describe extensive characterization of monoclonal antibodies against IspC, including epitope localization experiments, cross-reactivity testing against multiple isolates from each L. monocytogenes serotype, and binding kinetics measurements using surface plasmon resonance . Similar comprehensive validation would be essential for ispE-based methods.

These FAQs provide a comprehensive framework for researchers working with recombinant Listeria monocytogenes serotype 4b 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE), addressing both fundamental questions and advanced research applications while emphasizing methodological approaches to address complex scientific challenges.