Recombinant Listeria monocytogenes serotype 4b Glutathione biosynthesis bifunctional protein gshAB (gshAB), partial

What is Listeria monocytogenes serotype 4b and why is it significant in research?

Listeria monocytogenes serotype 4b is one of the most clinically relevant serotypes, responsible for the majority of human listeriosis cases. Over 90% of human listeriosis cases are caused by L. monocytogenes serotypes 1/2a, 1/2b, and 4b, with serotype 4b making up the bulk of these infections . This serotype is particularly concerning as it is associated with severe forms of listeriosis, including sepsis and meningitis.

Serotype 4b strains have distinct genomic profiles compared to other serotypes. Research has identified 4b variant strains that show the presence of a 1/2a-3a specific amplicon in addition to standard 4b-4d-4e specific amplicons, suggesting genetic exchange between different serotypes . These variant strains have been linked to outbreaks, representing distinct genomic groups that differ from known reported 4b outbreak strains from major epidemic clones.

The ability to create recombinant L. monocytogenes strains makes this organism valuable for research into bacterial pathogenesis and as a potential vaccine vehicle. Its unique life cycle—where it enters host cells, escapes from endocytic vesicles, multiplies within the cytoplasm, and spreads directly from cell to cell—provides a platform for delivering antigens directly into the cytosol of host cells for MHC class I presentation .

What is the glutathione biosynthesis bifunctional protein GshAB and how is it different from GshF in Listeria?

Glutathione (GSH) is an essential component of cellular defense against oxidative stress in most aerobic organisms. In L. monocytogenes, glutathione is synthesized by a novel multidomain protein called GshF (Lmo2770), which catalyzes the complete synthesis of glutathione in a single enzyme .

The GshF protein contains two primary domains:

• An N-terminal γ-glutamylcysteine ligase domain (amino acids 1-441) exhibiting moderate sequence identity with E. coli GshA protein

• A C-terminal portion (amino acids 450-776) containing an ATP-grasp domain

This fusion protein is distinct from the typical two-enzyme pathway found in most organisms (like E. coli), where glutathione synthesis involves the consecutive action of two physically separate enzymes: γ-glutamylcysteine ligase (GshA) and glutathione synthetase (GshB) .

Interestingly, while L. monocytogenes uses GshF, certain other pathogenic bacteria like Streptococcus agalactiae use a bifunctional enzyme encoded by gshAB that serves a similar function . This suggests evolutionary convergence in pathogenic bacteria toward efficient glutathione synthesis systems.

The presence of GshF-like fusion proteins is particularly noteworthy because 95% of bacteria possessing these proteins are mammalian pathogens, suggesting a possible adaptation for virulence .

What are the established techniques for creating recombinant L. monocytogenes strains expressing specific proteins?

Creating recombinant L. monocytogenes strains requires specific genetic engineering techniques. Based on the search results, established methodologies include:

• Site-specific integration of expression cassettes: This approach involves stable integration of expression cassettes into the L. monocytogenes genome, allowing for consistent expression of foreign antigens .

• Homologous recombination: This technique can be used to integrate target genes into the L. monocytogenes genome or to create deletion mutants. The process typically involves:

  • Amplifying target fragments using PCR

  • Enzyme digestion and ligation into shuttle vectors

  • Electrotransformation into L. monocytogenes

  • Selection using antibiotic screening

  • Verification through genome PCR and sequencing

• Gene disruption for functional studies: An internal fragment of the target gene is amplified by PCR, cloned into a temperature-sensitive shuttle vector, introduced by electroporation, and integrated via homologous recombination. This approach was used to create GshF466 disruptant mutant strain to study the function of glutathione synthesis in L. monocytogenes .

• Expression of heterologous antigens: L. monocytogenes can be engineered to express and secrete foreign antigens. For example, researchers have created recombinant L. monocytogenes expressing the H-2Kb gB 498-505 peptide from HSV-1 to trigger robust CD8 T cell responses .

When designing recombinant L. monocytogenes strains, researchers must consider the protein secretion signals, promoter strength, and integration site to ensure proper expression and function of the heterologous protein.

How can we assess the efficiency of glutathione synthesis in recombinant L. monocytogenes strains?

Assessment of glutathione synthesis efficiency in recombinant L. monocytogenes strains involves several complementary approaches:

• Quantification of intracellular glutathione levels: Direct measurement of glutathione content in wild-type versus mutant strains. This approach has been used in studies of Streptococcus suis, where deletion of gshAB led to lower intracellular glutathione content compared to wild-type strains .

• Oxidative stress resistance assays: Since glutathione plays a crucial role in protecting against oxidative stress, comparing the growth of wild-type and mutant strains under oxidative stress conditions (e.g., exposure to diamide, copper ions, or hydrogen peroxide) can indirectly assess glutathione synthesis efficiency .

• Growth kinetics analysis: Monitoring growth rates of wild-type versus gshAB mutant strains in defined media can reveal the importance of glutathione synthesis for bacterial growth. In the absence of stress, comparable growth rates may be observed, while differences become apparent under stress conditions .

• Virulence assessment in infection models: Testing the ability of wild-type versus gshAB mutant strains to survive and proliferate in macrophages or animal models can reveal the role of glutathione synthesis in pathogenesis .

• Complementation studies: Restoring the deleted gshAB gene in a knockout strain should restore glutathione synthesis and related phenotypes, confirming the specific role of the protein in observed phenotypes.

For example, a study comparing wild-type and gshAB knock-in strains of Streptococcus agalactiae demonstrated that deletion mutants were more sensitive to killing and growth inhibition by reactive oxygen species, supporting the role of glutathione synthesis in oxidative stress resistance .

What is the current understanding of the role of glutathione in L. monocytogenes virulence and stress response?

Current research indicates that glutathione plays multiple crucial roles in L. monocytogenes virulence and stress response:

• Virulence regulation: Glutathione modifies virulence factors in L. monocytogenes, particularly the master virulence regulator PrfA and the pore-forming toxin listeriolysin O (LLO) . GSH acts as an allosteric activator of PrfA, which controls the expression of key virulence genes.

• Oxidative stress tolerance: Glutathione is essential for L. monocytogenes to cope with oxidative stress. The deletion of gshF, which synthesizes glutathione, results in reduced bacterial growth when exposed to oxidative stressors such as diamide and copper ions .

• Intracellular survival and proliferation: Glutathione synthesis contributes to the efficiency of invasion and proliferation in macrophages and mouse organs. This suggests that glutathione plays a role in surviving the oxidative burst produced by phagocytic cells .

• Global transcriptional regulation: GshF influences global transcriptional profiles, particularly those related to carbohydrate and amino acid metabolism, such as the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) genes under oxidative stress conditions .

• Signaling role: Locally high GSH concentrations in the host cytosol may act as a signal for bacterial entry into the intracellular niche . This suggests that L. monocytogenes may sense GSH levels to adapt its metabolism and virulence gene expression when entering host cells.

It's important to note that unlike many bacteria that can synthesize cysteine, L. monocytogenes is auxotrophic for cysteine and must import exogenous cysteine or utilize glutathione for growth and virulence. This partial cysteine auxotrophy may have a pathoadaptive role, where locally high GSH/GSSG or thiosulfate concentrations signal arrival to distinct host niches .

How efficient is the processing of recombinant proteins expressed by L. monocytogenes compared to other expression systems?

Research demonstrates that recombinant proteins expressed by L. monocytogenes are processed remarkably efficiently for MHC class I presentation compared to proteins expressed by other systems. A comparative study revealed:

• High efficiency processing pathway: Proteins secreted from Listeria that are processed for MHC class I presentation follow an extremely efficient pathway that is only accessed by a subset of endogenously synthesized proteins .

• Independence from protein half-life: Unlike endogenously synthesized viral proteins whose pMHC production rate depends on protein half-life, pMHC production from secreted Listeria proteins occurs at the same rate regardless of the cellular half-life of the protein .

• Quantitative comparison: The efficiency of pMHC generation from recombinant Listeria proteins was at least 19-fold more efficient than that for the same recombinant proteins expressed by vaccinia virus .

This research conducted a detailed quantitative analysis, summarized in the following table:

The researchers calculated that at least 19 recombinant NP molecules must be synthesized in vaccinia virus-infected cells for every 1 recombinant NP molecule secreted by Listeria to generate equivalent levels of surface Kb-SIINFEKL complexes. This translates to an efficiency of approximately 1 surface pMHC complex for every 160 protein molecules processed from Listeria, compared to 1 in 3000 for vaccinia virus .

This high efficiency of antigen processing makes L. monocytogenes an excellent candidate for vaccine development, particularly for generating CD8+ T cell responses.

How can we design experiments to study the differential role of glutathione synthesis versus import in L. monocytogenes virulence?

Designing experiments to differentiate between the contributions of glutathione synthesis and import in L. monocytogenes virulence requires sophisticated approaches:

• Generation of specific mutant strains:

  • Create a gshF deletion mutant to eliminate glutathione synthesis

  • Generate a glutathione importer knockout by targeting the Ctp complex and OppDF ATPases

  • Develop a double knockout strain lacking both synthesis and import capabilities

  • Create complemented strains to confirm phenotypes

• In vitro characterization:

  • Compare growth kinetics of all strains in defined media with varying glutathione sources

  • Assess oxidative stress tolerance using challenges with H₂O₂, diamide, and copper ions

  • Measure intracellular glutathione levels using biochemical assays

  • Evaluate the expression of virulence genes using qRT-PCR or RNA-seq

• Transport assays:

  • Use radiolabeled glutathione to measure uptake rates in different strains

  • Compare affinity and specificity of transport using competition assays with various substrates

  • Determine kinetic parameters (Km, Vmax) for glutathione import

• Cell culture infection models:

  • Compare invasion and intracellular replication in macrophages and epithelial cells

  • Assess bacterial survival in the presence of oxidative stress inducing agents

  • Measure activation of host cell immune responses using cytokine profiling

• Animal infection studies:

  • Compare virulence of mutant strains in different mouse models

  • Analyze bacterial loads in various tissues (liver, spleen, brain)

  • Assess host immune responses to different strains

  • Conduct competition assays between mutant and wild-type strains in vivo

Recent research has identified that the Ctp complex is a high-affinity importer of GSH and GSSG, with the OppDF ATPases required for glutathione import in L. monocytogenes . The table below shows the homology between components of the E. coli GSH ABC importer and L. monocytogenes proteins:

This experimental framework would allow researchers to dissect the relative contributions of glutathione synthesis versus import to L. monocytogenes virulence and stress resistance in different environments.

What are the methodological challenges in evaluating the protective efficacy of recombinant L. monocytogenes strains as vaccine vectors?

Evaluating the protective efficacy of recombinant L. monocytogenes strains as vaccine vectors presents several methodological challenges that researchers must address:

• Strain attenuation while maintaining immunogenicity:

  • Challenge: Creating L. monocytogenes strains that are sufficiently attenuated for safety but retain immunogenicity

  • Approach: Use targeted deletions of virulence genes (e.g., actA) rather than random mutations to create stable, defined attenuation

  • Validation: Carefully assess in vivo growth kinetics and tissue distribution of attenuated strains compared to wild-type

• Antigen expression and delivery optimization:

  • Challenge: Ensuring stable expression and efficient delivery of heterologous antigens

  • Approach: Optimize promoters, signal sequences, and codon usage for target antigens

  • Validation: Quantify antigen expression levels and stability over multiple bacterial generations

• Immune response characterization:

  • Challenge: Comprehensively assessing multiple arms of the immune response

  • Approach: Analyze T cell responses using techniques like dimer staining to detect antigen-specific CD8 T cells, cytokine profiling (IFN-γ, IL-2, TNF-α), and cytotoxicity assays

  • Validation: In vivo depletion of specific cell populations (e.g., CD8+ T cells) to confirm their role in protection

• Protection assessment in appropriate models:

  • Challenge: Selecting appropriate challenge models that reflect the target pathogen's natural infection route

  • Approach: Develop challenge protocols with relevant pathogens at physiologically appropriate doses and routes

  • Validation: Include both immune-competent and immune-deficient animals to assess robustness of protection

  • Example: Studies have shown that recombinant L. monocytogenes expressing HSV antigens protected immune-competent mice but not IFN-γ-deficient or MyD88-deficient mice from HSV challenge

• Comparative studies with existing vaccine platforms:

  • Challenge: Providing meaningful comparisons to other vaccine strategies

  • Approach: Include control groups immunized with the same antigen delivered by different platforms

  • Validation: Standardize outcome measures across different vaccine platforms

Research has shown that recombinant Listeria monocytogenes expressing the H-2Kb gB 498-505 peptide from HSV-1 triggered a robust CD8 T cell response, with approximately 2% of peripheral CD8 T cells specific for this heterologous antigen at peak primary expansion. This immune response protected immune-competent mice from HSV-1-induced disease and showed marked reductions in recoverable virus .

How can whole genome sequencing approaches be applied to study the evolution and adaptation of glutathione metabolism systems in L. monocytogenes serotype 4b strains?

Whole genome sequencing (WGS) provides powerful tools for studying the evolution and adaptation of glutathione metabolism systems in L. monocytogenes serotype 4b strains. Here's a methodological framework for applying WGS approaches to this research question:

• Comparative genomics of diverse strain collections:

  • Sequence a diverse collection of L. monocytogenes serotype 4b strains from different geographical regions, time periods, and ecological niches

  • Include both clinical and environmental isolates to capture the full spectrum of genetic diversity

  • Apply standardized bioinformatic pipelines for genome assembly, annotation, and quality control

  • Establish a reference database of glutathione metabolism genes and related regulatory elements

• Phylogenetic analysis and evolutionary reconstruction:

  • Construct whole-genome phylogenies using maximum likelihood or Bayesian approaches

  • Map glutathione metabolism genes onto the phylogeny to trace their evolutionary history

  • Identify instances of potential horizontal gene transfer or recombination events

  • Calculate evolutionary rates for key components of the glutathione system

• Identification of facility-specific or niche-specific signatures:

  • Analyze allelic profiles across the whole genome to identify plant-specific molecular signatures

  • Examine the presence, absence, and sequence content of genomic hotspots that may contain glutathione-related genes

  • Compare closely related strains from different environments to identify adaptive changes

  • Assess the distribution of premature stop codons, prophage sequences, and other genetic elements that may influence glutathione metabolism

• Functional genomic analysis:

  • Identify single nucleotide polymorphisms (SNPs) and other genetic variations in the gshF gene and related pathways

  • Assess the impact of these variations on protein structure and function using predictive algorithms

  • Correlate genetic variations with phenotypic differences in glutathione synthesis, import, and utilization

  • Identify potential regulatory elements controlling glutathione metabolism

• Experimental validation of WGS findings:

  • Generate isogenic mutants based on WGS findings to confirm the functional significance of identified genetic variations

  • Perform transcriptomic and proteomic analyses to assess the impact of genetic variations on gene expression and protein function

  • Test mutants under conditions that mimic different ecological niches to assess adaptive significance

Research has demonstrated that WGS can reveal facility-specific signatures in L. monocytogenes strains. For example, comparative analysis of allelic variation across the whole genome has shown that allelic profiles can be specific to individual processing plants, even when closely-related strains from other sources are included in the analysis .

Additionally, WGS can identify novel strain variants, such as the 4b variant strains that show the presence of a 1/2a-3a specific amplicon in addition to standard 4b-4d-4e specific amplicons. These strains represent distinct genomic profiles compared to known 4b outbreak strains, with the acquisition of serotype 1/2a gene clusters appearing to be independent in origin, spanning large geographical and temporal spaces . Similar approaches could be applied to study the evolution and adaptation of glutathione metabolism systems in L. monocytogenes serotype 4b strains.

What is the current state of research on the interplay between glutathione metabolism and antibiotic resistance in L. monocytogenes?

The interplay between glutathione metabolism and antibiotic resistance in L. monocytogenes is an emerging area of research with several important connections being established:

• Co-localization of resistance determinants: Research has identified associations between glutathione-related genes and antimicrobial resistance genes in L. monocytogenes. For example, the cadmium resistance determinant cadA2 frequently accompanies the benzalkonium chloride (BC) resistance genes bcrABC in L. monocytogenes strains . This suggests potential genetic linkage between stress response systems and antimicrobial resistance.

• Horizontal gene transfer potential: Studies have demonstrated the potential for transfer of resistance genes between Listeria species. For instance, bcrABC can be transferred via conjugation from L. welshimeri to L. monocytogenes upon co-selection for cadmium resistance . This mechanism could potentially apply to glutathione-related genes as well.

• Oxidative stress and antibiotic tolerance: Glutathione plays a crucial role in protecting against oxidative stress, which is a common secondary effect of many antibiotics. Enhanced glutathione metabolism may therefore indirectly contribute to antibiotic tolerance by mitigating oxidative damage.

• Strain-specific variations in stress resistance: Different L. monocytogenes strains show variable tolerance to stressors like benzalkonium chloride. Some outbreak and sporadic clinical case-associated strains demonstrate BC tolerance, which might contribute to their survival in food processing environments . These strain-specific differences could be related to variations in glutathione metabolism.

• Methodological approaches to study this interplay:

  • Generate knockout strains targeting glutathione metabolism genes and assess their susceptibility to various antibiotics

  • Perform transcriptomic analysis of L. monocytogenes exposed to antibiotics to identify changes in glutathione-related gene expression

  • Use fluorescent probes to measure intracellular redox status and glutathione levels during antibiotic exposure

  • Conduct evolution experiments under antibiotic pressure and analyze genomic changes related to glutathione metabolism

Future research directions should focus on establishing a more direct causal relationship between glutathione metabolism and antibiotic resistance, potentially revealing new targets for combination therapies that could enhance antibiotic efficacy against L. monocytogenes.

How can advanced structural biology techniques be applied to study the bifunctional nature of the GshF protein in L. monocytogenes?

Advanced structural biology techniques offer powerful approaches to elucidate the bifunctional nature of the GshF protein in L. monocytogenes, providing insights into its mechanism, regulation, and potential as a therapeutic target:

• X-ray crystallography and cryo-electron microscopy (cryo-EM):

  • Determine the high-resolution structure of the complete GshF protein

  • Capture different conformational states during the catalytic cycle

  • Visualize substrate binding and coordination within each domain

  • Identify the structural basis for the fusion of two enzymatic activities

  • Methodology: Express recombinant GshF with affinity tags, purify to homogeneity, set up crystallization trials or prepare cryo-EM grids, collect diffraction data or EM images, and solve the structure

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map protein dynamics and conformational changes during catalysis

  • Identify regions involved in substrate binding and product release

  • Detect allosteric communication between the two catalytic domains

  • Methodology: Expose purified GshF to D2O under various conditions (with/without substrates), quench the reaction at different time points, digest with proteases, and analyze peptide fragments by mass spectrometry

• Single-molecule Förster Resonance Energy Transfer (smFRET):

  • Monitor real-time conformational changes during catalysis

  • Determine the coordination between the two enzymatic activities

  • Investigate potential substrate channeling between domains

  • Methodology: Introduce fluorescent labels at specific sites in the protein, immobilize on a surface, and monitor fluorescence signals from individual molecules during catalysis

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Characterize protein dynamics at the amino acid level

  • Monitor changes in chemical environment during catalysis

  • Study interactions with substrates and potential inhibitors

  • Methodology: Express isotopically labeled GshF, collect multidimensional NMR spectra, and analyze chemical shift perturbations under different conditions

• Computational approaches:

  • Perform molecular dynamics simulations to predict domain movements

  • Use quantum mechanics/molecular mechanics (QM/MM) methods to model the catalytic mechanism

  • Conduct virtual screening to identify potential inhibitors targeting the interdomain region

  • Methodology: Build structural models based on experimental data, set up simulation parameters, run calculations on high-performance computing resources, and analyze trajectories

The bifunctional nature of GshF is particularly interesting as it consists of a γ-glutamylcysteine ligase domain (amino acids 1-441) and an ATP-grasp domain (amino acids 450-776), connected by a ~160-amino-acid segment with high sequence conservation among related proteins . Understanding how these domains coordinate their activities could provide insights into the evolution of this fusion protein, which is found primarily in mammalian pathogens (95% of bacteria possessing GshF-like proteins are mammalian pathogens) .