Recombinant Yersinia pseudotuberculosis serotype O:3 NAD-dependent malic enzyme (maeA), partial

What is Yersinia pseudotuberculosis and how does it cause disease?

Yersinia pseudotuberculosis is an Enterobacteriaceae family member that primarily transmits through the fecal-oral route, causing infections in humans and animals. It invades through Peyer's patches and lymph vessels to infect mesenteric lymph nodes (MLNs), resulting in the clinical presentation of mesenteric lymphadenitis. A hallmark of Y. pseudotuberculosis infection is the formation of pyogranulomas in MLNs, which are composed of neutrophils, inflammatory monocytes, and lymphocytes surrounding extracellular microcolonies of the bacteria . The virulence plasmid pYV is essential for pathogenesis, encoding important virulence factors including the YadA adhesin and the Ysc-Yop type III secretion system (T3SS) that translocate multiple effector proteins into host cells .

What is the NAD-dependent malic enzyme (maeA) and what is its metabolic function?

NAD-dependent malic enzymes like MaeA catalyze the oxidative decarboxylation of malate to pyruvate and CO₂ while reducing NAD to NADH. This reaction plays a crucial role in bacterial carbon metabolism by:

• Generating pyruvate for various metabolic pathways

• Producing reducing power in the form of NADH

• Contributing to tricarboxylic acid (TCA) cycle intermediates

• Potentially enabling metabolic adaptation to different carbon sources

Recent research has revealed that MaeA from E. coli also possesses fumarase activity, catalyzing the conversion of fumarate to malate with a K₀.₅ value for fumarate of approximately 13 mM . This dual enzymatic activity suggests MaeA serves a more complex role in bacterial metabolism than previously understood, potentially providing metabolic flexibility under varying environmental conditions.

How does serotyping impact research on Y. pseudotuberculosis and what is significant about serotype O:3?

Serotyping in Y. pseudotuberculosis is based on variations in the O-antigen structure of lipopolysaccharide. Serotype O:3 represents one of several clinically significant serotypes, affecting epidemiological studies, infection models, and immune responses. When designing experiments with Y. pseudotuberculosis, researchers should consider:

• Serotype-specific variations in virulence determinants

• Potential differences in host immune recognition

• Regional prevalence patterns that might affect translational relevance

• Serotype-specific metabolic adaptations that could influence experimental outcomes

What are the optimal methods for constructing recombinant Y. pseudotuberculosis strains expressing modified maeA?

Based on successful approaches with related Yersinia species, the following methodology is recommended:

• Gene isolation and vector construction:

  • PCR amplification of maeA from Y. pseudotuberculosis genomic DNA

  • Insertion into an appropriate vector (commercial vectors like pTurboGFP-B have been successfully used with Yersinia species)

  • Introduction of desired modifications (point mutations, tags, or fusion proteins)

• Transformation protocol:

  • Electroporation is typically the most effective method for Yersinia species

  • Use of antibiotic selection markers (e.g., ampicillin resistance)

  • Verification of transformants by PCR and sequencing

• Expression verification:

  • Western blotting to confirm protein production

  • Enzymatic activity assays to verify functionality

  • Microscopy for tagged constructs to assess localization

The constructed recombinant strains should be extensively characterized to confirm they maintain the cultural-morphological and biochemical properties of the parent strain, particularly if virulence studies are planned .

What in vitro assays are most informative for studying maeA enzymatic activity?

The following assays provide comprehensive characterization of maeA enzymatic functions:

Table 1: Recommended Enzymatic Assays for maeA Characterization

When designing these assays, researchers should consider:

• Purification method effects on enzymatic activity

• Buffer composition impacts on catalytic efficiency

• Potential allosteric regulators of enzyme function

• Comparison with MaeB (NADP-dependent) to understand cofactor specificity differences

How can fluorescent protein tagging be optimized for studying maeA localization and function?

Based on successful approaches with Y. pestis fluorescent protein expression, researchers should consider:

• Fusion construct design:

  • C-terminal vs. N-terminal tagging (C-terminal generally preferable for secreted or membrane proteins)

  • Inclusion of flexible linker sequences to minimize functional interference

  • Selection of appropriate fluorescent protein (GFP variants have been successfully used in Yersinia species)

• Expression optimization:

  • Promoter selection based on experimental needs (constitutive vs. inducible)

  • Codon optimization for Y. pseudotuberculosis if necessary

  • Careful validation that fusion does not disrupt enzymatic activity

• Imaging considerations:

  • Fixed vs. live cell imaging approaches

  • Counterstaining strategies for host-pathogen interaction studies

  • Quantitative image analysis methods

• Applications:

  • Subcellular localization studies under different metabolic conditions

  • Host-pathogen interaction visualization

  • Protein dynamics using FRAP or similar techniques

The constructed strain should be verified to maintain virulence and survivability characteristics similar to the wild-type strain to ensure biological relevance of observations .

How might the dual enzymatic activity of maeA (malic enzyme and fumarase) affect Y. pseudotuberculosis pathogenesis?

The recently discovered dual functionality of NAD-dependent malic enzymes has important implications for Y. pseudotuberculosis pathogenesis:

• Metabolic flexibility during infection:

  • The ability to use both malate and fumarate provides metabolic options during transition through different host environments

  • This may enable adaptation to changing nutrient availability in intestinal lumen versus deeper tissues

• Redox balance maintenance:

  • Both enzymatic activities generate NADH, potentially contributing to redox homeostasis

  • NADH/NAD+ ratio affects numerous cellular processes and potentially virulence factor expression

• Integration with virulence regulation:

  • Metabolic enzymes may interact with virulence regulators similar to how MarA-like proteins in Y. pestis affect both metabolism and virulence

  • The metabolic state of the bacterium often influences virulence gene expression

• Host immune evasion:

  • Metabolic adaptability may enhance survival within phagocytes

  • Similar to how Y. pestis effectors inhibit innate immune defenses, metabolic flexibility might contribute to persistence

This dual functionality might be particularly important during the formation of pyogranulomas in MLNs, where the bacterium must adapt to the hostile environment while maintaining growth and virulence .

What experimental models best capture the role of maeA in Y. pseudotuberculosis infection?

Table 2: Experimental Models for Studying maeA Function in Y. pseudotuberculosis

When using these models, researchers should:

• Compare wild-type, maeA deletion mutant, and complemented strains

• Assess both early invasion and late persistence phases

• Consider how environmental conditions (oxygen, nutrients, pH) affect maeA contribution

• Integrate with systems biology approaches (transcriptomics, proteomics)

How do mutations in key catalytic residues of maeA affect enzyme function and bacterial fitness?

Structure-function analysis of maeA through site-directed mutagenesis can reveal:

• Catalytic mechanism insights:

  • Mutations in malate-binding residues versus fumarate-binding sites may differentially affect dual functionality

  • NAD-binding domain mutations can alter cofactor specificity (potentially converting NAD-dependence to NADP-dependence)

• Bacterial fitness impacts:

  • Growth rate differences in media with different carbon sources

  • Competitive index measurements in co-infection models

  • Metabolic profile alterations detectable through metabolomics

• Potential differential effects:

  • Mutations might differentially impact malic enzyme versus fumarase activity

  • Some mutations might enhance one function while diminishing another

  • Environmental conditions may alter the relative importance of different catalytic residues

How does maeA interact with other metabolic enzymes and virulence factors in Y. pseudotuberculosis?

Understanding the integration of maeA within the broader metabolic and virulence networks requires:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with metabolic enzymes and virulence regulators

  • Bacterial two-hybrid systems to identify interaction partners

  • Proximity labeling approaches to identify the maeA interactome

• Transcriptional regulation analysis:

  • Promoter studies to identify environmental signals affecting maeA expression

  • Potential cross-regulation with virulence genes (similar to MarA-like proteins in Y. pestis)

  • Response to host-derived signals during infection

• Metabolic network modeling:

  • Integration of maeA within central carbon metabolism models

  • Flux balance analysis to predict effects of maeA modulation

  • Identification of metabolic bottlenecks where maeA plays a crucial role

• Investigation of potential moonlighting functions:

  • Beyond its enzymatic activity, maeA might have secondary roles in protein complexes

  • Potential regulatory functions through protein-protein interactions

  • Possible involvement in bacterial stress responses

These interactions may have parallels to how MarA-like proteins in Y. pestis affect both antibiotic susceptibility and virulence, suggesting metabolic enzymes can have broader impacts on bacterial pathogenesis than their canonical functions would indicate .

Could maeA serve as a potential target for novel antimicrobial development?

Several factors make maeA potentially attractive as an antimicrobial target:

The efficacy of such approaches would likely depend on the degree to which Y. pseudotuberculosis relies on maeA during different stages of infection.

How can isotope labeling experiments enhance our understanding of maeA function during infection?

Isotope tracing methodologies provide powerful insights into maeA metabolic contributions:

• Ex vivo isotope tracing:

  • Infected cells or tissues cultured with ¹³C-labeled substrates

  • Mass spectrometry analysis of metabolite labeling patterns

  • Comparison between wild-type and maeA mutant strains

• In vivo metabolic labeling:

  • Administration of isotope-labeled substrates to infected animals

  • Tissue extraction and metabolite analysis

  • Spatial metabolomics to assess metabolic activity in different infection sites

• Key measurements:

  • Carbon flux through the malic enzyme reaction

  • Contribution to pyruvate and acetyl-CoA pools

  • Integration with TCA cycle activity

  • Potential channeling of metabolites to biosynthetic pathways

• Technical considerations:

  • Appropriate isotope selection (¹³C, ¹⁵N, or ²H depending on pathway)

  • Sampling timing to capture dynamic metabolic changes

  • Computational modeling to interpret complex labeling patterns

These approaches can reveal how Y. pseudotuberculosis metabolically adapts during infection and the specific role of maeA in this adaptation.