Recombinant Yersinia pseudotuberculosis serotype O:3 Translation initiation factor IF-2 (infB), partial

What is the biological significance of Translation Initiation Factor IF-2 in Yersinia pseudotuberculosis?

Translation Initiation Factor IF-2 (infB) in Y. pseudotuberculosis serves as an essential protein involved in bacterial translation initiation, but emerging research indicates additional non-canonical functions. Beyond its primary role in protein synthesis initiation, IF-2 isoforms have been implicated in DNA transactions and homologous recombination (HR). Specifically, IF2 exists in multiple isoforms (IF2-1, IF2-2, and IF2-3) synthesized from three in-frame initiation codons within the infB gene. These isoforms differentially affect HR functions, with IF2-1 identified as a novel facilitator of RecA's function during DNA recombination events . This dual functionality positions IF-2 at the intersection of translation and DNA metabolism, making it both a potential virulence regulator and an intriguing target for understanding bacterial adaptation mechanisms.

How does Y. pseudotuberculosis serotype O:3 differ from other serotypes in pathogenicity and molecular characteristics?

Y. pseudotuberculosis serotype O:3 exhibits distinct epidemiological patterns and virulence characteristics compared to other serotypes. This serotype has been implicated in widespread foodborne outbreaks, notably through contaminated produce like iceberg lettuce . Serotype O:3 strains possess the full complement of core virulence determinants found on both the bacterial chromosome and the approximately 70-kb virulence plasmid .

The pathogenicity profile of serotype O:3 includes:

Molecular studies have demonstrated that serotype O:3 strains employ the YopJ effector protein to limit macrophage responses by targeting prostaglandin E2 (PGE2) biosynthesis pathways, representing a sophisticated immune evasion strategy .

What are the optimal expression systems for producing recombinant Y. pseudotuberculosis IF-2?

The optimal expression of recombinant Y. pseudotuberculosis IF-2 requires careful selection of expression systems based on research objectives. While the search results don't specifically address expression systems for Y. pseudotuberculosis IF-2, we can extrapolate methodological approaches from similar bacterial recombinant proteins:

E. coli-based expression systems are typically preferred due to:

• High expression yields for bacterial proteins

• Compatibility with bacterial translation machinery

• Ability to generate sufficient quantities for structural and functional studies

• Established protocols for codon optimization

For functional studies requiring proper folding and minimal interference, consider:

• Using BL21(DE3) or Rosetta strains to address codon bias issues

• Employing temperature-controlled induction (16-18°C) to improve solubility

• Implementing auto-induction media to maximize yield while minimizing toxicity

• Testing multiple affinity tags (His6, GST, MBP) for optimal solubility and activity

Advanced researchers should consider cell-free expression systems when studying interactions between IF-2 isoforms and RecA-mediated processes, as these systems can minimize interference from host proteins involved in homologous recombination .

What purification challenges are specific to recombinant Y. pseudotuberculosis IF-2, and how can they be addressed?

Purification of recombinant Y. pseudotuberculosis IF-2 presents several challenges stemming from its biochemical properties and multiple isoforms. While specific purification data for Y. pseudotuberculosis IF-2 is not directly provided in the search results, methodological approaches can be derived from related recombinant protein purification protocols and IF-2 characteristics:

Common challenges and solutions include:

For isoform-specific studies, researchers should consider developing an isoform-specific purification strategy, potentially using ion exchange chromatography to separate IF2-1, IF2-2, and IF2-3 based on their slightly different charge profiles. This approach is particularly important when investigating the differential effects of IF2 isoforms on homologous recombination .

How does Y. pseudotuberculosis IF-2 interact with the bacterial type III secretion system (T3SS)?

Although direct interactions between IF-2 and the T3SS in Y. pseudotuberculosis are not explicitly detailed in the search results, we can analyze potential relationships based on the available data:

Y. pseudotuberculosis utilizes its T3SS to deliver effector Yop proteins into host cells, which are critical for virulence and colonization . Translation regulation through IF-2 likely influences the expression of T3SS components and effectors, creating an indirect but important relationship.

Research methodologies to investigate this relationship should include:

• Co-immunoprecipitation assays to detect direct protein-protein interactions between IF-2 and T3SS components

• Ribosome profiling to examine translational efficiency of T3SS mRNAs in the presence of wild-type versus mutant IF-2

• Conditional expression systems to modulate IF-2 levels and observe effects on T3SS function

• Fluorescence microscopy with tagged proteins to visualize potential co-localization

A significant experimental approach would involve creating Y. pseudotuberculosis strains with mutations in specific IF-2 isoforms and assessing the impact on T3SS assembly, effector secretion, and ultimately virulence in cellular and animal models .

What is the functional relationship between IF-2 isoforms and DNA metabolism in Y. pseudotuberculosis?

The functional relationship between IF-2 isoforms and DNA metabolism represents an emerging area of bacterial physiology research. Based on studies in E. coli, we can infer similar mechanisms may exist in Y. pseudotuberculosis:

IF-2 isoforms have been identified as novel modulators of homologous recombination (HR) in bacteria. Specifically:

• IF2-1 functions as a facilitator of RecA activity during HR processes

• Loss of IF2-1 phenocopies the loss of RecA or the RecFORQ presynaptic pathway

• IF2-1 deficiency has been shown to suppress synthetic lethalities in cells with defects in transcription termination factor Rho and Ruv proteins

• Conversely, deficiency in IF2-2 and IF2-3 exacerbates these synthetic defects

To investigate this relationship in Y. pseudotuberculosis, researchers should employ:

• Genetic complementation assays with individual IF-2 isoforms in infB mutant backgrounds

• DNA damage sensitivity assays comparing wild-type and isoform-specific mutants

• Biochemical assays measuring RecA-mediated DNA strand exchange in the presence of purified IF-2 isoforms

• ChIP-seq analysis to identify potential DNA binding sites of IF-2 isoforms

These functional studies would help establish whether the role of IF-2 in DNA metabolism is conserved in Y. pseudotuberculosis and potentially reveal pathogen-specific adaptations of this system .

How does IF-2 contribute to Y. pseudotuberculosis virulence during infection?

While direct evidence linking IF-2 to Y. pseudotuberculosis virulence is not explicitly presented in the search results, we can propose mechanistic hypotheses based on known functions of IF-2 and Y. pseudotuberculosis pathogenesis:

IF-2's primary role in translation initiation positions it as a central regulator of virulence factor expression. During infection, Y. pseudotuberculosis must rapidly adapt to changing host environments, which requires precise translational control. Given the demonstrated role of IF-2 isoforms in DNA metabolism and recombination , IF-2 likely contributes to virulence through:

• Temperature-responsive gene expression regulation - Y. pseudotuberculosis expresses different virulence factors at environmental (25°C) versus host (37°C) temperatures

• Stress adaptation - Translation regulation during host-imposed stresses

• DNA damage repair - Supporting genomic integrity during oxidative stress encountered in macrophages

• Fine-tuning of virulence factor expression - Potentially prioritizing translation of specific mRNAs

Methodologically, researchers could investigate these hypotheses by:

• Creating temperature-sensitive IF-2 mutants and assessing virulence in infection models

• Performing ribosome profiling of wild-type versus IF-2 mutant strains during infection conditions

• Comparing transcriptome and proteome profiles to identify post-transcriptional regulation by IF-2

• Analyzing host cell responses to infection with IF-2 mutant strains

What role might IF-2 play in Y. pseudotuberculosis survival within macrophages?

Y. pseudotuberculosis can survive and multiply within macrophage phagosomes, which represents a critical aspect of its pathogenesis . While specific roles for IF-2 in this process are not directly documented in the search results, we can propose potential mechanisms based on bacterial adaptation requirements:

Y. pseudotuberculosis modifies macrophage function by inhibiting phagosome acidification and production of nitric oxide . These processes require precisely timed expression of bacterial factors, which would necessitate translational control potentially involving IF-2. Additionally, the demonstrated connection between IF-2 isoforms and DNA recombination suggests potential roles in genome maintenance during the oxidative stress encountered within phagosomes .

Experimental approaches to investigate this relationship should include:

• Intracellular survival assays comparing wild-type and IF-2 mutant strains within macrophages

• Phagosome pH measurements to assess whether IF-2 mutations affect the bacterium's ability to inhibit acidification

• Transcriptome analysis of intracellular bacteria to identify IF-2-dependent gene expression patterns

• Fluorescence microscopy to track IF-2 localization during intracellular infection stages

Since Y. pseudotuberculosis expresses YopJ to inhibit macrophage PGE2 biosynthesis , researchers should also investigate whether IF-2 influences the expression or secretion of this critical virulence factor.

What potential exists for targeting Y. pseudotuberculosis IF-2 in antimicrobial development?

The essential nature of IF-2 for bacterial translation initiation makes it a promising but challenging antimicrobial target. Several characteristics make Y. pseudotuberculosis IF-2 particularly interesting for therapeutic development:

• Structural divergence from host translation factors - Bacterial IF-2 differs significantly from eukaryotic translation initiation factors, potentially allowing selective targeting

• Multiple functional roles - Beyond translation, IF-2's involvement in DNA metabolism provides opportunities for multi-modal inhibition

• Isoform-specific functions - The differential roles of IF-2 isoforms in homologous recombination suggest the possibility of targeting specific bacterial adaptations

Methodological approaches for antimicrobial development should include:

Researchers should be aware that targeting IF-2 may lead to rapid resistance development due to its essential nature. Therefore, combination approaches targeting multiple aspects of Y. pseudotuberculosis physiology would be advisable .

How can recombinant Y. pseudotuberculosis IF-2 be utilized in diagnostic assay development?

Recombinant Y. pseudotuberculosis IF-2 offers several opportunities for developing improved diagnostic assays for Y. pseudotuberculosis infections. While direct applications are not specified in the search results, methodological approaches can be extrapolated from standard recombinant protein applications in diagnostics:

Potential diagnostic applications include:

• Serological assays - Recombinant IF-2 could serve as an antigen in ELISA or similar assays to detect anti-IF-2 antibodies in patient samples

• Nucleic acid amplification test (NAAT) standards - Recombinant protein or encoding plasmids could function as positive controls in PCR-based diagnostics

• Aptamer selection - IF-2 could be used to develop nucleic acid aptamers for sensitive detection systems

• Immunohistochemistry reagents - Labeled anti-IF-2 antibodies raised against the recombinant protein could identify bacteria in tissue samples

When developing such assays, researchers should consider:

• The serotype specificity of IF-2 variants across Y. pseudotuberculosis strains

• Potential cross-reactivity with IF-2 from closely related species like Y. enterocolitica or Y. pestis

• The relative abundance and accessibility of IF-2 compared to other diagnostic targets

• The stability of recombinant IF-2 under various storage and assay conditions

Notably, diagnostic development should address the challenge of distinguishing Y. pseudotuberculosis infections from similar clinical presentations, particularly appendicitis, which is a common differential diagnosis .

What structural biology approaches are most effective for studying Y. pseudotuberculosis IF-2 isoforms?

Characterizing the structure of Y. pseudotuberculosis IF-2 isoforms requires a multi-technique approach to address challenges related to protein size, flexibility, and functional states:

X-ray crystallography remains the gold standard for high-resolution protein structures but faces challenges with flexible proteins like IF-2. Researchers should consider:

• Using truncation constructs focusing on individual domains

• Co-crystallization with binding partners (GTP, tRNA, ribosomal components)

• Surface entropy reduction mutations to enhance crystal packing

Cryo-electron microscopy (cryo-EM) offers advantages for studying IF-2 in functional complexes:

• Sample preparation using mild conditions preserves native conformations

• Single-particle analysis can capture multiple conformational states

• Sub-3Å resolution is achievable for ribosome-bound IF-2

Nuclear Magnetic Resonance (NMR) provides valuable information about dynamics:

• Suitable for individual domains rather than full-length protein

• Can identify flexible regions and conformational changes

• Enables studies of protein-ligand interactions in solution

Small-angle X-ray scattering (SAXS) complements high-resolution techniques:

For studying the distinct functions of IF-2 isoforms in homologous recombination, researchers should combine structural approaches with biochemical assays to correlate structural features with their roles in RecA-mediated processes .

How can systems biology approaches integrate IF-2 function into the broader network of Y. pseudotuberculosis virulence mechanisms?

Systems biology offers powerful frameworks for understanding how IF-2 functions within the complex network of Y. pseudotuberculosis virulence mechanisms. Given the dual roles of IF-2 in translation and DNA metabolism , integration of multiple data types is essential.

Methodological approaches should include:

• Multi-omics integration

  • Combine transcriptomics, proteomics, and metabolomics data from IF-2 mutant strains

  • Implement network analysis to identify regulatory hubs connected to IF-2 function

  • Use correlation analysis to detect co-regulated pathways

• Mathematical modeling

  • Develop kinetic models of translation initiation incorporating IF-2 isoform dynamics

  • Create Boolean networks representing virulence factor expression control

  • Implement agent-based models of host-pathogen interactions

• Genome-scale analyses

  • Perform Tn-seq or CRISPRi screens to identify genetic interactions with IF-2

  • Implement ChIP-seq to map genome-wide binding profiles of IF-2 isoforms

  • Use ribosome profiling to identify translationally regulated genes

• Infection dynamics visualization

  • Employ single-cell tracking to monitor bacterial adaptation during infection

  • Implement tissue-specific infection models to capture organ-specific dynamics

  • Use intravital microscopy to observe bacteria-host cell interactions in real-time

A particularly promising direction is examining how IF-2-mediated translational control intersects with the expression timing of T3SS components and effectors like YopJ, which inhibits macrophage PGE2 biosynthesis . This approach could reveal how Y. pseudotuberculosis coordinates its virulence mechanisms across different infection stages and host environments .

What are the most promising future research directions for understanding Y. pseudotuberculosis IF-2 functions?

Based on the current knowledge gaps and emerging findings about IF-2 functions, several high-priority research directions can be identified:

• Isoform-specific functions in pathogenesis

  • Develop isoform-specific mutants to assess their distinct roles during infection

  • Investigate potential tissue-specific expression patterns of IF-2 isoforms

  • Examine environmental triggers that modulate isoform ratios

• Translation-DNA metabolism crosstalk

  • Further characterize the mechanisms by which IF-2 isoforms affect RecA-mediated processes

  • Investigate potential direct interactions between IF-2 and DNA repair machinery

  • Examine how these functions contribute to bacterial fitness during host cell invasion

• Structure-function relationships

  • Solve high-resolution structures of Y. pseudotuberculosis IF-2 isoforms

  • Map interaction surfaces with translation and DNA metabolism partners

  • Identify regions responsible for isoform-specific functions

• Host response modulation

  • Investigate whether IF-2-dependent translation affects evasion of host immunity

  • Examine potential roles in regulating YopJ and other effectors that modulate host cell functions

  • Study IF-2's influence on bacterial adaptation to macrophage environments

• Therapeutic targeting

  • Develop isoform-specific inhibitors to dissect functions pharmacologically

  • Explore combination approaches targeting translation and virulence mechanisms

  • Investigate potential for attenuated strains with modified IF-2 function as vaccine candidates

The unique interface between translation initiation and DNA metabolism represented by IF-2 isoforms offers exciting opportunities to better understand how bacterial pathogens coordinate core cellular processes with virulence mechanisms.

What contradictions or knowledge gaps exist in the current understanding of Y. pseudotuberculosis IF-2?

Current research on Y. pseudotuberculosis IF-2 presents several notable contradictions and knowledge gaps that warrant further investigation:

Conceptual contradictions:

• Dual functionality paradox - How does IF-2 balance its essential role in translation with its involvement in DNA metabolism? This dual functionality raises questions about protein moonlighting and evolutionary adaptations .

• Isoform antagonism - The observation that IF2 isoforms have opposing effects on homologous recombination remains mechanistically unexplained. How can proteins with such similar sequences produce contrary phenotypes?

• Selective pressure conundrum - If IF-2 isoforms affect bacterial fitness and virulence, what selective pressures maintain the ratio of isoforms across strains and species?

Methodological knowledge gaps: