Recombinant Yersinia pseudotuberculosis serotype O:3 Glycine dehydrogenase [decarboxylating] (gcvP), partial

What are the key characteristics of Y. pseudotuberculosis serotype O:3 compared to other serotypes?

Y. pseudotuberculosis serotype O:3 is a clinically significant strain that has been implicated in numerous human infections. This serotype is distinguished by its O-antigen structure, which defines its serological classification. Serotype O:3 has been associated with widespread foodborne outbreaks, particularly in Northern Europe. In Finland, for example, an extensive outbreak investigation revealed that serotype O:3 was responsible for 47 confirmed cases of infection with patients ranging from 2-77 years of age (median 19 years) .

This serotype demonstrates notable virulence characteristics, including the ability to effectively colonize the gastrointestinal tract and cause symptoms ranging from acute gastroenteritis to more severe complications such as bacteremia. The clinical significance of O:3 is highlighted by the fact that in the documented Finnish outbreak, one patient died from bacteremia, and five required appendectomies .

When studying this serotype in the laboratory, researchers should note its distinct growth requirements and colony morphology on selective media compared to other Y. pseudotuberculosis serotypes.

How does Y. pseudotuberculosis serotype O:3 persist in environmental reservoirs?

Y. pseudotuberculosis serotype O:3 demonstrates remarkable environmental persistence, allowing it to contaminate food products such as fresh produce. Research evidence indicates that this serotype can survive in various environmental niches, with a documented case linking it to iceberg lettuce in a large-scale outbreak .

The persistence mechanism involves several factors:

• Ability to grow at refrigeration temperatures (psychrotrophic properties)

• Resistance to environmental stressors

• Capacity to form biofilms on plant surfaces

Methodologically, when investigating environmental persistence, researchers should employ both culture-based techniques and molecular methods such as pulsed-field gel electrophoresis (PFGE). In the Finnish outbreak study, PFGE analysis revealed that all 27 tested isolates from case patients had indistinguishable patterns, confirming a common source despite the nationwide distribution of cases . This demonstrates the importance of molecular subtyping in epidemiological investigations of this pathogen.

How does the Type III Secretion System (T3SS) in Y. pseudotuberculosis O:3 contribute to pathogenesis?

The Type III Secretion System (T3SS) represents a critical virulence mechanism in Y. pseudotuberculosis O:3, functioning as a molecular syringe that injects bacterial effector proteins directly into host cells. This sophisticated apparatus allows the pathogen to manipulate host cell signaling and defense mechanisms.

Y. pseudotuberculosis has evolved a remarkable control mechanism for T3SS-mediated effector delivery. Research has demonstrated that the bacterium selectively modulates host Rho GTPase activity to induce cellular changes that control pore formation and effector translocation . This process involves:

• Adhesin-mediated high-affinity binding to β1 integrins on the host cell surface

• Interaction of T3SS components with the host cell membrane

• Coordinated delivery of effector proteins that manipulate host cell functions

Experimental approaches to study this process include:

• In vitro infection models using cell lines such as HeLa cells

• Measurement of LDH release as an indicator of pore formation

• Use of specific inhibitors to dissect the mechanisms of pore formation

Notably, research has shown that YopB/D-mediated LDH release occurs by a process distinct from pyroptosis, as demonstrated by experiments showing that glycine treatment (5 mM) had no effect on LDH release from infected cells . This finding highlights the unique nature of the T3SS-mediated membrane disruption in Yersinia.

What roles do YopK and YopJ play in the virulence of Y. pseudotuberculosis, and how can they be experimentally manipulated?

YopK and YopJ are crucial virulence factors associated with the pathogenicity of Y. pseudotuberculosis. These Yersinia outer proteins (Yops) are delivered into host cells via the Type III Secretion System and perform distinct functions:

YopK: Functions as a regulator of the T3SS, controlling the translocation of other effector proteins and potentially preventing excessive pore formation that could lead to cell death before effective manipulation of host defenses.

YopJ: Acts as an acetyltransferase that inhibits MAPK and NF-κB signaling pathways, thereby suppressing pro-inflammatory cytokine production and inducing apoptosis in macrophages.

Experimental approaches to study these proteins include:

• Generation of deletion mutants (ΔyopK, ΔyopJ) to assess their individual and combined contributions to virulence

• Complementation studies to confirm phenotypes

• Protein secretion assays under calcium-deprived conditions (a trigger for T3SS activation)

Research has demonstrated the significance of these virulence factors through the development of attenuated vaccine strains. For example, the χ10069 strain with ΔyopK ΔyopJ Δasd triple mutations has been successfully used to deliver protective antigens against Yersinia infections . The deletion of these virulence factors reduces pathogenicity while maintaining immunogenicity, making this approach valuable for vaccine development.

To experimentally analyze Yop secretion, researchers can use Western blot analysis to detect proteins in both bacterial lysates and culture supernatants, as demonstrated in studies examining the synthesis and T3SS-mediated secretion of native LcrV and recombinant fusion proteins .

What are the methodological considerations when expressing recombinant Y. pseudotuberculosis glycine dehydrogenase (gcvP) for biochemical characterization?

Expressing recombinant glycine dehydrogenase [decarboxylating] (gcvP) from Y. pseudotuberculosis requires careful consideration of several methodological aspects:

Expression System Selection:

• Prokaryotic systems: E. coli BL21(DE3) is often preferred for bacterial protein expression due to its reduced protease activity and high expression levels

• Protein tags: His-tag or GST-tag fusion systems facilitate purification while minimizing interference with enzymatic activity

Optimization Parameters:

• Induction conditions: IPTG concentration (typically 0.1-1.0 mM), temperature (16-37°C), and duration (4-24 hours)

• Growth media: Enhanced expression often occurs in rich media such as TB (Terrific Broth) or auto-induction media

• Codon optimization: Adaptation to E. coli codon usage may improve expression levels

Purification Strategy:

• Initial capture: Affinity chromatography (Ni-NTA for His-tagged proteins)

• Intermediate purification: Ion exchange chromatography

• Polishing: Size exclusion chromatography

Activity Assessment:
For gcvP enzyme activity, the standard assay measures NAD+ reduction to NADH spectrophotometrically at 340 nm as glycine is oxidized and decarboxylated. The specific activity is typically expressed as μmol NADH produced per minute per mg of protein.

Sample Enzymatic Assay Conditions:

• Buffer: 100 mM potassium phosphate buffer (pH 7.5)

• Substrates: 5 mM glycine, 0.5 mM NAD+

• Cofactors: 0.1 mM pyridoxal phosphate, 0.5 mM tetrahydrofolate

• Temperature: 37°C

• Monitoring: Continuous measurement at 340 nm for 5 minutes

These methodological considerations ensure proper expression, purification, and characterization of the recombinant gcvP enzyme for further biochemical and structural studies.

How can glycine dehydrogenase (gcvP) be incorporated into attenuated Y. pseudotuberculosis vaccine strategies?

The integration of glycine dehydrogenase (gcvP) into Y. pseudotuberculosis vaccine development represents an innovative approach with several potential advantages. Based on established recombinant Yersinia vaccine research, the following methodological considerations apply:

Antigen Design Strategies:

• Whole protein expression: Incorporating the complete gcvP coding sequence into an expression plasmid

• Epitope fusion: Creating fusion proteins by linking immunogenic epitopes of gcvP with established immunodominant antigens

• Multi-antigen constructs: Combining gcvP with other protective antigens

The fusion protein approach has proven successful in Yersinia vaccine development. For example, the YopE Nt138-LcrV fusion protein delivered by attenuated Y. pseudotuberculosis induced significant protection (80% survival) against intranasal challenge with Y. pestis . This suggests that creating a gcvP-based fusion protein could similarly enhance immunogenicity.

Delivery System Development:
The attenuated Y. pseudotuberculosis strain χ10069 with ΔyopK ΔyopJ Δasd triple mutations represents an effective delivery platform. This strain maintains the ability to synthesize and secrete proteins via the T3SS under calcium-deprived conditions at 37°C . For gcvP incorporation:

• Design expression plasmid containing the gcvP gene under appropriate regulatory control

• Transform the attenuated Y. pseudotuberculosis strain

• Verify protein expression and secretion via Western blot analysis

Verification Protocol:
To confirm proper expression and secretion of recombinant gcvP:

• Culture bacteria in calcium-sufficient and calcium-deprived media at 37°C

• Collect both bacterial lysates and culture supernatants

• Perform Western blot analysis using anti-gcvP antibodies

• Verify protein molecular weight against predicted values using prestained protein markers

• Compare expression patterns with control strains

This approach allows researchers to develop and optimize innovative gcvP-based vaccine candidates for protection against Yersinia infections.

What are the optimal methods for isolating and identifying Y. pseudotuberculosis serotype O:3 from environmental samples?

Isolation and identification of Y. pseudotuberculosis serotype O:3 from environmental samples requires a systematic approach combining selective enrichment, differential plating, and confirmatory testing:

Sample Collection and Processing:

• Collect representative samples (soil, water, plant material)

• Homogenize solid samples in peptone water (1:10 dilution)

• Pre-enrich in non-selective broth (TSB) at 25°C for 24h

• Perform cold enrichment at 4°C for 14-21 days to select for psychrotrophic Yersinia

Isolation Protocol:

• Transfer enrichment culture to Cefsulodin-Irgasan-Novobiocin (CIN) agar

• Incubate at 28-30°C for 24-48h

• Identify suspect colonies (bull's eye appearance with red centers)

• Pick 5-10 characteristic colonies for further testing

Biochemical Identification:

• API 20E or Vitek 2 systems for biochemical profiling

• Key tests: Urease (+), Citrate (-), Ornithine decarboxylase (+)

Molecular Confirmation and Serotyping:

• PCR targeting the inv (invasin) gene for species confirmation

• Multiplex PCR for simultaneous detection of pathogenic Yersinia species

• Serotype-specific PCR or slide agglutination with O:3 antiserum

Subtyping Methods for Outbreak Investigations:
Pulsed-field gel electrophoresis (PFGE) with NotI or XbaI restriction enzymes has been successfully used to establish genetic relatedness among isolates in outbreak investigations. In the Finnish outbreak study, all 27 isolates from case patients showed indistinguishable PFGE patterns, confirming a common source .

Data Analysis Approach:
When analyzing environmental sampling data, statistical methods such as regression analysis can help establish associations between isolation rates and environmental parameters. For outbreak investigations, geographic information systems (GIS) can be employed to map the spatial distribution of cases and potential environmental sources.

How should researchers design experiments to evaluate the efficacy of recombinant Y. pseudotuberculosis-based vaccines?

Designing robust experiments to evaluate recombinant Y. pseudotuberculosis-based vaccines requires careful consideration of multiple factors:

Animal Model Selection:

• Mouse models are standard for initial efficacy studies

• Swiss Webster mice have been successfully used in Y. pseudotuberculosis vaccine research

• Consider both immunocompetent and immunocompromised models to evaluate safety profile

Immunization Protocol Design:

• Route of administration: Oral route mimics natural infection and induces both mucosal and systemic immunity

• Dosing schedule: Primary immunization followed by booster doses (typically 2-3 weeks apart)

• Control groups: Include vector-only control, wild-type strain control, and unvaccinated control

Sample Immunization Study Design:

Immune Response Assessment:

• Humoral immunity:

  • Serum IgG by ELISA

  • Mucosal IgA in intestinal lavage and fecal samples

• Cell-mediated immunity:

  • T-cell proliferation assays

  • Cytokine profiles (IFN-γ, IL-4, IL-17)

• Functional assays:

  • Serum bactericidal activity

  • Opsonophagocytic assays

Challenge Studies:

• Select appropriate challenge strain (homologous and heterologous challenges)

• Determine challenge dose based on preliminary LD50 studies

• Use multiple challenge routes (intranasal, intraperitoneal)

• Monitor survival, bacterial burden, histopathology, and clinical parameters

Data Analysis and Reporting:

• Use Kaplan-Meier survival analysis for challenge studies

• Apply appropriate statistical tests (t-test, ANOVA with post-hoc analysis)

• Report detailed methodologies including strain construction, growth conditions, and immunization protocols

Previous research has demonstrated that Y. pseudotuberculosis strain χ10069 engineered with ΔyopK ΔyopJ Δasd triple mutations effectively delivered the YopE Nt138-LcrV fusion protein as a protective antigen against Yersinia infections . This approach induced significant protection (80% survival) against intranasal challenge with Y. pestis, establishing a methodological foundation for future vaccine candidates.

What molecular typing methods are most effective for tracking Y. pseudotuberculosis serotype O:3 in outbreak investigations?

Molecular typing of Y. pseudotuberculosis serotype O:3 during outbreak investigations requires sophisticated approaches to establish genetic relatedness between isolates from different sources:

Gold Standard Methods:

• Pulsed-Field Gel Electrophoresis (PFGE):

  • Still considered highly discriminatory for Yersinia

  • Restriction enzymes: NotI, XbaI, and SpeI provide good discrimination

  • Standardized protocols allow inter-laboratory comparison

  • Successfully applied in the Finnish outbreak investigation where all 27 isolates showed indistinguishable patterns

• Whole Genome Sequencing (WGS):

  • Provides highest resolution for strain discrimination

  • Enables detection of single nucleotide polymorphisms (SNPs)

  • Allows identification of virulence genes and mobile genetic elements

  • Can detect presence of plasmids, such as the ~70 kb plasmid identified in Y. pseudotuberculosis isolates

Complementary Techniques:

• MLST (Multi-Locus Sequence Typing): Targets 7 housekeeping genes

• MLVA (Multi-Locus Variable Number Tandem Repeat Analysis): Highly discriminatory for closely related isolates

• CRISPR Analysis: Provides insights into evolutionary relationships

Data Analysis Framework:

• Generate dendrograms using appropriate algorithms (UPGMA, neighbor-joining)

• Establish genetic relatedness thresholds for epidemiological linkage

• Integrate molecular data with epidemiological information

• Apply appropriate statistical methods to establish significance of clusters

Implementation Strategy for Outbreak Scenarios:

The Finnish outbreak investigation demonstrated the effectiveness of laboratory-based surveillance coupled with serotype analysis for rapid detection of Y. pseudotuberculosis O:3 infections that might otherwise appear sporadic . This approach, combined with case-control studies and molecular typing, successfully implicated iceberg lettuce as the vehicle of transmission.

How can researchers distinguish between pathogenic and non-pathogenic strains of Y. pseudotuberculosis in environmental and clinical samples?

Distinguishing between pathogenic and non-pathogenic Y. pseudotuberculosis strains requires a multifaceted approach targeting virulence determinants at both phenotypic and genotypic levels:

Virulence Factor Detection:

• pYV Plasmid Screening:

  • PCR targeting key virulence genes located on pYV (e.g., yadA)

  • The yadA gene encodes a collagen-binding protein important for autoagglutination, adherence to epithelial cells, and serum resistance

  • Size determination of plasmids (~70 kb for typical pYV)

• Type III Secretion System Components:

  • PCR detection of structural (yscF, yscN) and effector (yopE, yopH) genes

  • Western blot analysis for YopE and LcrV proteins

  • Calcium-dependent growth restriction test (growth inhibition at 37°C in calcium-depleted media indicates functional T3SS)

Functional Assays:

• In Vitro Virulence Assays:

  • Cell invasion assays using epithelial cell lines

  • Cytotoxicity assessment measuring LDH release from infected cells

  • Pore formation assays with fluorescent dyes

• T3SS Functionality Tests:

  • Secretion of Yops under calcium-depleted conditions

  • Western blot analysis of culture supernatants for secreted effectors

  • Cell rounding assay (indicates functional YopE delivery)

Molecular Characterization Protocol:

Research has shown that recombinant proteins like YopE Nt138-LcrV are constitutively synthesized in Y. pseudotuberculosis and secreted via T3SS only under calcium-deprived conditions at 37°C . This characteristic can be exploited for differentiating pathogenic strains with functional T3SS from non-pathogenic variants.

When analyzing potential pathogens, researchers should employ both phenotypic and genotypic methods to comprehensively characterize the virulence potential of isolated strains.

How can the glycine cleavage system in Y. pseudotuberculosis be targeted for antimicrobial development?

The glycine cleavage system (GCS), including glycine dehydrogenase [decarboxylating] (gcvP), represents a promising target for antimicrobial development against Y. pseudotuberculosis due to its critical role in bacterial metabolism and potential differences from the human homolog:

Target Validation Approach:

• Gene Essentiality Assessment:

  • Generate conditional mutants (inducible promoter systems)

  • Transposon mutagenesis with next-generation sequencing (Tn-Seq)

  • Growth analysis under different nutrient conditions

• Structural Characterization:

  • X-ray crystallography or cryo-EM of purified recombinant gcvP

  • In silico modeling and identification of druggable pockets

  • Comparison with human homologs to identify bacterial-specific features

Drug Discovery Strategy:

• High-Throughput Screening (HTS):

  • Enzymatic assays measuring NAD+ reduction

  • Fragment-based screening

  • Structure-based virtual screening

• Rational Design Approach:

  • Design transition state analogs

  • Target allosteric sites

  • Develop covalent inhibitors

Candidate Compound Evaluation:

Combination Therapy Potential:
The inhibition of gcvP could be particularly effective when combined with conventional antibiotics that target cell wall synthesis or protein translation. This multi-target approach may reduce the emergence of resistance and enhance bactericidal activity.

The molecular mechanisms of Y. pseudotuberculosis infection, including the role of Type III secretion systems in host cell manipulation , provide additional context for understanding how metabolic inhibitors might affect pathogenicity. Targeting metabolic pathways like the glycine cleavage system could potentially attenuate virulence expression in addition to inhibiting growth.

What are the current challenges and future directions in developing multivalent vaccines using recombinant Y. pseudotuberculosis strains?

The development of multivalent vaccines using recombinant Y. pseudotuberculosis strains presents both significant challenges and promising opportunities for advancement:

Current Technical Challenges:

• Antigen Selection and Optimization:

  • Identifying protective antigens across multiple pathogens

  • Optimizing epitope presentation without compromising immunogenicity

  • Balancing expression levels of multiple antigens

• Vaccine Strain Stability:

  • Ensuring genetic stability of multiple attenuating mutations

  • Preventing plasmid loss during manufacturing and storage

  • Maintaining consistent antigen expression levels

• Safety Considerations:

  • Achieving sufficient attenuation without compromising immunogenicity

  • Preventing reversion to virulence through recombination

  • Addressing safety concerns for immunocompromised individuals

Methodological Solutions:

• Advanced Genetic Engineering Approaches:

  • CRISPR/Cas9-mediated precise genome editing

  • Chromosomal integration of antigen genes for stability

  • Balanced promoter systems for controlled expression

• Improved Delivery Systems:

  • Development of temperature-sensitive strains for controlled replication

  • T3SS-mediated delivery optimization through YopK and YopJ modifications

  • Dual plasmid systems for separate antigen and attenuating gene control

Future Research Directions:

Promising Preliminary Results:
Research has demonstrated that attenuated Y. pseudotuberculosis strain χ10069 with ΔyopK ΔyopJ Δasd triple mutations effectively delivered the YopE Nt138-LcrV fusion protein, inducing significant protection (80% survival) against intranasal challenge with Y. pestis . This establishes a foundation for expanding this platform to deliver multiple antigens.

The future of this field lies in combining advances in structural vaccinology, systems biology, and synthetic biology to develop next-generation multivalent vaccines with enhanced safety, efficacy, and manufacturability profiles. Integration of glycine dehydrogenase (gcvP) or its immunogenic epitopes into these platforms could potentially enhance protection against multiple Yersinia species.

What are the most significant recent advances in Y. pseudotuberculosis serotype O:3 research that impact vaccine and therapeutic development?

Recent advances in Y. pseudotuberculosis serotype O:3 research have significantly impacted vaccine and therapeutic development through multiple interconnected breakthroughs:

Vaccine Platform Development:
The development of recombinant attenuated Y. pseudotuberculosis strains as vaccine delivery vehicles represents a major advance. The triple-mutant strain χ10069 (ΔyopK ΔyopJ Δasd) has demonstrated capacity to effectively deliver Y. pestis fusion proteins, inducing significant protection against challenge . This establishes a platform technology that could be adapted for various antigens, including those from Y. pseudotuberculosis serotype O:3.

Pathogenesis Mechanisms:
Elucidation of how Y. pseudotuberculosis controls Type III effector delivery by modulating Rho activity has provided critical insights into host-pathogen interactions . This understanding enables more rational approaches to attenuating virulence while maintaining immunogenicity, a critical balance in live vaccine development.

Epidemiological Understanding:
The identification of fresh produce, particularly iceberg lettuce, as a vehicle for Y. pseudotuberculosis O:3 outbreaks has significant implications for public health interventions and provides insights into environmental persistence mechanisms that may inform therapeutic targeting .

Molecular Characterization:
Advanced genomic analysis has revealed the presence and variation of virulence plasmids (~70 kb) in clinical isolates, enhancing our understanding of strain-to-strain variation that must be addressed in vaccine design .