Recombinant Listeria monocytogenes serotype 4b Fumarate hydratase class II (fumC)

What is Fumarate Hydratase Class II (fumC) in Listeria monocytogenes and how does it differ from other fumarase classes?

Fumarate hydratase (FH, fumarase) catalyzes the reversible conversion of fumarate into L-malate and plays essential roles in cellular metabolism. In prokaryotes like Listeria monocytogenes, there are two distinct classes of fumarases: Class I (iron-sulfur cluster-containing) and Class II (iron-independent). The fundamental difference is that Class I and Class II fumarases show no sequence similarity and have different evolutionary origins .

Class II fumarase (fumC) in L. monocytogenes has several distinctive features:

• Iron-independent activity, making it functional during iron limitation

• Greater stability under oxidative stress conditions

• Serves as a backup enzyme under conditions where Class I fumarases might be compromised

• Contains approximately 467 amino acid residues with a molecular weight of about 50 kDa

• Functions primarily in the tricarboxylic acid (TCA) cycle

Unlike in E. coli, where Class I fumarases (fumA and fumB) participate in DNA damage response and Class II (fumC) in respiration , the specific functional distribution in L. monocytogenes requires further investigation.

What serotypes of Listeria monocytogenes exist and why is serotype 4b significant for research?

Listeria monocytogenes can be classified into multiple serotypes based on somatic (O) and flagellar (H) antigens, including 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 . These serotypes cluster into two major genomic divisions:

• Division I: serotypes 1/2a, 1/2c, 3a, and 3c

• Division II: serotypes 1/2b, 4b, and 3b

Serotype 4b is particularly significant for research because:

• It exhibits the highest pathogenicity level among all serotypes

• Patients infected with serotype 4b have significantly higher mortality rates

• It is over-represented in outbreaks and sporadic cases of listeriosis

• Well-characterized strains like F2365 (a serotype 4b cheese isolate from the Jalisco cheese outbreak of 1985) serve as important reference strains

• Genome analysis revealed unique genomic features that may contribute to its increased virulence

This serotype has been implicated in several major outbreaks, making it a critical target for vaccine development, pathogenesis studies, and diagnostic method development.

How does Listeria monocytogenes cause disease and what role might fumC play in pathogenesis?

Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis, a disease with a mortality rate of 20-30% . The pathogenesis involves several steps:

• Entry: L. monocytogenes enters the host through contaminated food. It uses D-galactose residues on its surface to attach to D-galactose receptors on host cell walls, enabling translocation across the intestinal epithelium .

• Invasion and spread: The bacterium can invade and multiply within various host cells, including epithelial cells, macrophages, and dendritic cells. It can cross the blood-brain barrier and the placental barrier in pregnant women .

• Immune response: Infection triggers recruitment of inflammatory neutrophils and macrophages that produce CXCL9, promoting the infiltration of CXCR3-expressing T cells. In pregnancy, this can lead to fetal wastage .

While the specific role of fumC in L. monocytogenes pathogenesis remains under investigation, several potential functions can be hypothesized based on research in other organisms:

• Metabolic adaptation: fumC may be critical during iron limitation within host cells

• Oxidative stress resistance: As shown in E. coli, fumC functions as a backup enzyme under oxidative stress conditions

• Potential virulence factor: In some pathogens, metabolic enzymes can have moonlighting functions that contribute to virulence

The TCA cycle, in which fumC participates, is essential for bacterial adaptation to different host environments and nutrient conditions during infection.

What are the optimal conditions for expressing recombinant L. monocytogenes fumC in heterologous systems?

Based on research practices with similar proteins, optimal expression conditions for recombinant L. monocytogenes fumC include:

Expression Systems:

• E. coli: Most commonly used for initial characterization due to high yield and simplicity

• Yeast: Useful when post-translational modifications might be important

• Baculovirus: For higher eukaryotic expression with proper folding

• Mammalian cells: When authentic folding and modifications are critical

Expression Protocol for E. coli System:

• Vector selection: pET system vectors with T7 promoter are recommended for high-level expression

• Host strain: BL21(DE3) or T7 Express strains perform well for recombinant L. monocytogenes proteins

• Induction conditions: 0.5-1 mM IPTG at OD600 of 0.6-0.8

• Temperature: Lower temperatures (16-18°C) after induction may improve solubility

• Expression time: 4-16 hours depending on temperature

Purification Strategy:

• Cell lysis in buffer containing 50 mM Tris pH 8.5, 150 mM NaCl

• Nickel affinity chromatography for His-tagged proteins

• Optional addition of 1 mM DTT to all buffers to maintain protein stability

• Perform all steps at 4°C, preferably in anaerobic conditions for optimal activity

The purified enzyme can be assayed for activity by measuring the production or consumption of fumarate spectrophotometrically at 250 nm (ε₂₅₀ₙₘ = 1,450 M⁻¹cm⁻¹) or 300 nm (ε₃₀₀ₙₘ = 36.6 M⁻¹cm⁻¹) .

What methods are most effective for detecting L. monocytogenes serotype 4b in environmental or food samples?

Detecting L. monocytogenes serotype 4b in environmental or food samples requires sensitive and specific methods. Recent advancements have improved both detection time and accuracy:

Conventional Culture Methods:

• Enrichment: Traditional methods use two-stage enrichment in selective broths

• Selective plating: On media like Oxford, PALCAM, or RAPID'L.mono agar

• Biochemical confirmation: Tests for esculin hydrolysis, phosphatidyl-inositol phospholipase C activity, and xylose fermentation

• Serotyping: Using specific antisera against O and H antigens

Advanced Detection Methods:

• Streamlined workflow approach (recent innovation, 2025):

  • Abbreviated 5h culture enrichment in PALCAM liquid medium

  • Physical separation using filtration and centrifugation

  • Specific capture with bacteriophage endolysin-derived cell wall-binding domain

  • Molecular detection using MicroSEQ L. monocytogenes RTi-PCR detection kit

  • Can detect as few as 2 CFU in a 25g sample within an 8-hour workday

• FTIR spectroscopy technique:

  • Generates biochemical fingerprints for differentiation of strains

  • Successfully distinguishes between epidemic clones (EC III and IV)

  • 100% success rate in differentiation analysis

  • Low cost, high throughput technology

• Quasimetagenomic sequencing:

  • Combines enrichment with metagenomic sequencing

  • Allows early detection and population dynamics analysis

  • Can track strain-specific sequence types (STs) during enrichment

Comparative performance of detection methods on environmental samples:

How can researchers purify and characterize the enzymatic activity of L. monocytogenes fumC?

Purification and characterization of L. monocytogenes fumC enzymatic activity requires careful consideration of the protein's biochemical properties:

Purification Protocol:

• Expression: Express recombinant protein with affinity tag (His-tag recommended)

• Lysis: Use buffer containing 50 mM Tris (pH 8-9), 150 mM NaCl

• Chromatography:

  • Primary: Nickel affinity chromatography

  • Secondary: Size exclusion chromatography to obtain homogeneous tetrameric protein

• Storage: Store in buffer containing DTT (1 mM) to prevent oxidation

Enzymatic Activity Characterization:

• Spectrophotometric assay:

  • Forward reaction (malate → fumarate): Monitor increase in absorbance at 250 nm

  • Reverse reaction (fumarate → malate): Monitor decrease in absorbance at 250 nm

  • Temperature: 25°C (room temperature)

  • Buffer: 50 mM Tris, pH 9, 150 mM NaCl

• Kinetic parameters determination:

  • Vary substrate concentration (0.1-10 mM range)

  • Analyze using Michaelis-Menten kinetics

  • Account for the reversible nature of the reaction in calculations

• Inhibition studies:

  • Test with 2-thiomalate, a known selective inhibitor of Class I FHs

  • Determine IC₅₀ values using the dose-response equation:
    Inhibition (%) = Imin + (Imax - Imin) / (1 + 10^((log[IC₅₀]-log[I])*h))

  • Where Imin is minimum inhibition, Imax is maximum inhibition, h is the Hill coefficient

Class II fumarases typically show tetrameric quaternary structure with a molecular weight of approximately 200 kDa. The catalytic efficiency (kcat/Km) should be determined for both the forward and reverse reactions to fully characterize the enzyme's function.

How can recombinant L. monocytogenes serotype 4b be utilized as a vaccine vector?

Recombinant L. monocytogenes has emerged as a promising vaccine vector platform due to its ability to stimulate robust cellular and humoral immune responses. Particularly, attenuated L. monocytogenes strains carrying specific mutations have shown significant potential:

Key advantages of L. monocytogenes as a vaccine vector:

• Stimulates both innate and adaptive immunity

• Generates robust CD8+ T cell responses

• Can deliver heterologous antigens to the host immune system

• Accessible through multiple administration routes

Optimal strain engineering approach:
Research has demonstrated that recombinant L. monocytogenes carrying ΔactA and a prfA* mutation (r-Listeria ΔactA prfA*) offers superior properties as a vaccine vector:

• Secretes >100-fold more immunogen than wild-type r-Listeria

• Elicits greater cellular and humoral immune responses

Vaccination routes and immune responses:

The intranasal vaccination route is particularly noteworthy as it elicits "appreciable pulmonary IFN-γ+ cellular response" and "secretory immunogen-specific IgA titers that were similar to or higher in mucosal fluid than those induced by subcutaneous and intravenous immunizations" .

For designing L. monocytogenes-based vaccine vectors expressing fumC or other antigens, the ΔactA prfA* platform provides an optimal balance of safety and immunogenicity.

What are the key challenges in working with recombinant L. monocytogenes proteins and how can they be addressed?

Researchers working with recombinant L. monocytogenes proteins, including fumC, face several technical challenges:

1. Protein Solubility and Folding Issues:

• Challenge: L. monocytogenes proteins may form inclusion bodies when overexpressed

• Solution:

  • Lower expression temperature (16-18°C)

  • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

  • Optimize induction conditions (lower IPTG concentration, 0.1-0.5 mM)

  • Include stabilizing agents in lysis buffer (10% glycerol, 1 mM DTT)

2. Enzyme Activity Preservation:

• Challenge: Loss of enzymatic activity during purification

• Solution:

  • Perform purification in anaerobic conditions for iron-sulfur containing proteins

  • Add reducing agents (DTT, β-mercaptoethanol) to prevent oxidation

  • Include 1 mM DTT in all buffers to maintain protein stability

  • Avoid freeze-thaw cycles by preparing single-use aliquots

3. Biosafety Considerations:

• Challenge: L. monocytogenes is a Biosafety Level 2 (BSL-2) pathogen

• Solution:

  • Work in BSL-2 facilities with appropriate containment

  • Use attenuated strains for research where possible

  • Implement proper decontamination procedures for all materials

  • Handle injections and mice in a biosafety cabinet

4. Enzyme Assay Complications:

• Challenge: The reversible nature of fumC reaction complicates kinetic analysis

• Solution:

  • Simultaneously analyze both directions of the reaction in a single kinetics assay

  • Use the IC50-to-Ki web server to estimate inhibitory constants

  • Account for racemic mixtures when using inhibitors like 2-thiomalate

5. Protein Crystallization Challenges:

• Challenge: Obtaining crystals suitable for structural studies

• Solution:

  • Screen multiple buffer conditions (HEPES has been shown to interact with human FH at the C-terminal domain)

  • Consider the tetrameric nature of the protein when designing crystallization strategies

  • Use tag-less protein after proteolytic removal of purification tags

Addressing these challenges requires careful planning and optimization of protocols based on the specific properties of L. monocytogenes fumC.

How can researchers optimize detection of L. monocytogenes in complex food matrices?

Detection of L. monocytogenes in complex food matrices presents numerous challenges that researchers must overcome:

Key Challenges and Solutions:

1. Low bacterial concentration:

• Challenge: Initial contamination levels are often below detection limits

• Solution:

  • Implement abbreviated 5-hour enrichment protocols

  • Use physical separation methods (filtration, centrifugation) to concentrate bacteria

  • Apply bacteriophage endolysin-derived cell wall-binding domains for specific capture

2. Interference from food components:

• Challenge: Food particles can inhibit PCR and other detection methods

• Solution:

  • Use multi-stage filtration systems to remove food particles

  • Apply BagFilter Pull-up filter bags followed by centrifugation

  • Incorporate magnetic separation to capture L. monocytogenes cells specifically from preprocessed samples

3. Stressed or viable but non-culturable (VBNC) cells:

• Challenge: Processing and environmental stresses can induce VBNC state

• Solution:

  • Optimize enrichment media formulations

  • Employ molecular methods targeting RNA or membrane-integrity-dependent dyes

  • Consider dual-approach methods combining culture and molecular techniques

4. Strain variations affecting detection:

• Challenge: Genetic diversity among strains may lead to false negatives

• Solution:

  • Target conserved regions in molecular detection methods

  • Use quasimetagenomic sequencing to monitor population dynamics during enrichment

  • Implement multiple primer sets or broad-range primers for PCR-based detection

Comparative enrichment strategies:
When enriching multiple strains, population dynamics can impact detection:

• Without background microbiota: Some STs showed higher relative abundance during late enrichment

• With background microbiota: Population dynamics remained more consistent over time

Optimized workflow performance:
The recent streamlined workflow for L. monocytogenes detection demonstrates remarkable sensitivity:

• Can detect as few as 12.5 CFUs in pure cultures via RTi-PCR

• Consistently detects as few as 2 CFU in a 25g sample following 5h enrichment

• Completes detection within a standard 8-hour workday

What are important considerations for studying fumC in the context of L. monocytogenes pathogenesis?

Studying fumC in the context of L. monocytogenes pathogenesis requires careful consideration of several factors:

1. Selection of appropriate infection models:

• In vitro cell culture models:

  • Human epithelial cell lines (Caco-2, HeLa)

  • Macrophage cell lines (J774, RAW264.7)

  • Primary cells (PBMCs, bone marrow-derived macrophages)

• In vivo models:

  • C57BL/6J mice are commonly used due to their Th1-prone immune response

  • Consider sex differences (LD50 is 10^5 CFU for males and 1.5×10^5 CFU for females)

  • Important to standardize mice age, vendor, and strain (genetic differences exist between C57BL/6 substrains from different vendors)

2. Infection protocol optimization:

• Inoculum preparation:

  • Culture in BHI media until target OD600

  • Wash bacteria twice with PBS

  • Standardize CFU counts carefully

• Infection routes:

  • Intraperitoneal injection is common for systemic infection models

  • Oral gavage better mimics natural infection route

  • Consider intranasal route for studying respiratory immunity

3. Virulence assessment approaches:

• Bacterial burden determination:

  • Harvest organs (spleen, liver) at specific time points

  • Homogenize tissues and plate serial dilutions on selective media

  • Compare bacterial loads between wild-type and fumC mutant strains

• Immunological readouts:

  • Measure IFN-γ production by flow cytometry

  • Assess recruitment of immune cells to infection sites

  • For pregnancy models, monitor fetal wastage as an endpoint

4. Genetic manipulation strategies:

• Gene deletion approaches:

  • Create precise fumC deletion mutants

  • Consider complementation with wild-type fumC to confirm phenotypes

  • Use inducible expression systems for essential genes

• Point mutations:

  • Target specific residues based on structural data

  • Create catalytically inactive variants to distinguish enzymatic from structural roles

5. Special considerations for metabolic studies:

• The TCA cycle, including fumC, functions differently under various oxygen concentrations

• Consider how host microenvironments might affect fumC expression and activity

• Account for potential metabolic redundancy in L. monocytogenes

• Investigate potential moonlighting functions of fumC beyond its enzymatic role

The infectious dose, timing of treatment, and appropriate controls (including uninfected controls) are critical considerations for ensuring reproducible and interpretable results .

What are promising areas for future research on L. monocytogenes fumC and its applications?

Several promising areas for future research on L. monocytogenes fumC warrant investigation:

1. Structure-Function Relationships:

• Determine the crystal structure of L. monocytogenes fumC to identify unique structural features

• Investigate whether, like human FH, L. monocytogenes fumC has allosteric regulatory sites

• Examine the impact of small molecules (like HEPES identified in human FH studies) on fumC activity

• Compare structures across different L. monocytogenes strains to identify strain-specific adaptations

2. Role in Pathogenesis and Virulence:

• Determine if fumC participates in DNA damage response similar to Class I fumarases in E. coli

• Investigate potential moonlighting functions beyond its metabolic role

• Examine whether fumC expression changes during different stages of infection

• Explore potential interactions with host proteins or immune factors

3. Novel Therapeutic Targets:

• Develop selective inhibitors against L. monocytogenes fumC

• Explore whether 2-thiomalate, which inhibits Class I fumarases but not human Class II FH, has effects on L. monocytogenes metabolism

• Screen for other class-specific inhibitors with minimal host toxicity

• Investigate fumC as a potential drug target for listeriosis treatment

4. Vaccine Development:

• Further optimize recombinant L. monocytogenes ΔactA prfA* as a vaccine vector

• Explore the potential of fumC as an antigen for subunit vaccines

• Investigate combination approaches using fumC with other L. monocytogenes antigens

• Develop intranasal vaccination strategies to leverage the robust mucosal IgA response observed with recombinant L. monocytogenes

5. Diagnostic Applications:

• Develop fumC-targeted detection methods for improved specificity

• Explore fumC sequence variations as potential markers for virulent strains

• Integrate fumC-based detection into multi-target approaches for comprehensive L. monocytogenes surveillance

• Investigate whether fumC expression levels correlate with strain virulence

These research directions would contribute significantly to our understanding of L. monocytogenes metabolism, pathogenesis, and the development of novel interventions against this important foodborne pathogen.

How might integrated -omics approaches advance our understanding of L. monocytogenes metabolism and pathogenesis?

Integrated -omics approaches offer powerful tools to advance our understanding of L. monocytogenes metabolism and pathogenesis, particularly regarding fumC and related pathways:

1. Multi-omics Integration Strategies:

• Combine genomics, transcriptomics, proteomics, and metabolomics data to build comprehensive models

• Apply systems biology approaches to understand metabolic network dynamics during infection

• Use computational modeling to predict metabolic adaptations under different environmental conditions

• Integrate structural biology with other -omics data to understand protein function in context

2. Genomics Applications:

• Pan-genomic analysis has already revealed that L. monocytogenes strains consist of approximately 2,200 shared genes with a much larger accessory genome

• Continue comparative genomics between serotypes to identify virulence-associated genetic elements

• Apply phylogenomics to track evolutionary relationships between strains

• Use whole-genome sequencing for outbreak investigations and surveillance

3. Transcriptomics Approaches:

• RNA-Seq analysis during different growth phases and environmental conditions

• Dual RNA-Seq to simultaneously capture host and pathogen transcriptional responses

• Small RNA profiling to identify regulatory networks affecting metabolism

• Investigation of fumC expression regulation under various stress conditions

4. Proteomics Contributions:

• Quantitative proteomics to measure fumC and other TCA cycle enzyme abundance

• Secretome analysis to identify proteins released during infection

• Post-translational modification profiling to understand regulatory mechanisms

• Protein-protein interaction studies to identify fumC binding partners

5. Metabolomics Insights:

• Measure TCA cycle intermediates during infection

• Investigate metabolic flux through fumC using stable isotope labeling

• Examine how metabolite levels change in response to environmental stresses

• Study the role of fumarate and other metabolites as potential signaling molecules

Recent research has highlighted how metabolites like α-ketoglutarate and fumarate can function as signaling molecules affecting DNA damage repair in E. coli . Similar signaling roles may exist in L. monocytogenes, where metabolites could influence processes beyond central metabolism.