Recombinant Shigella boydii serotype 4 Uncharacterized protein yjiK (yjiK)

What is Shigella boydii serotype 4 and its significance in microbiology research?

Shigella boydii serotype 4 is one of the four species of Shigella bacteria that cause diarrheal disease. While less common than S. sonnei in the United States, S. boydii remains clinically significant, particularly in regions with limited resources . Research significance stems from its unique O-antigen structure and genetic organization. The O-antigen gene cluster of S. boydii serotype 4 contains 10 genes within a 10,551 bp sequence located between galF and gnd genes . This serotype's O-antigen is identical to the E. coli O53 antigen, suggesting evolutionary relationships between these bacterial species . Understanding S. boydii serotype 4 contributes to our broader knowledge of bacterial pathogenesis, evolution, and potential vaccine development.

What structural characteristics define uncharacterized bacterial proteins like yjiK?

Uncharacterized bacterial proteins like yjiK typically lack definitive functional annotations due to limited experimental evidence. Their structural characteristics can be predicted through computational methods including:

• Primary sequence analysis: Identifying conserved domains, motifs, and sequence homology

• Secondary structure prediction: Determining α-helices, β-sheets, and turns

• Tertiary structure modeling: Using homology modeling or ab initio prediction methods

• Functional site prediction: Identifying potential active sites, binding pockets, or protein-protein interaction surfaces

For S. boydii proteins, analysis often begins with comparison to better-characterized proteins in related enterobacterial species, particularly E. coli, given their close genetic relationship . The sequence similarity between S. boydii and other bacterial proteins can provide initial hypotheses about structural features. For example, many proteins in the S. boydii O-antigen biosynthesis pathway show 47-58% similarity to proteins in other bacterial species .

What recombinant expression systems are most effective for S. boydii yjiK protein production?

The optimal expression system for recombinant S. boydii yjiK protein depends on research objectives and protein characteristics. Based on successful approaches with other bacterial proteins, the following methodological framework is recommended:

Expression Systems Comparison for S. boydii Proteins:

Methodologically, optimization should include:

• Codon optimization for the expression host

• Testing multiple fusion tags (His6, GST, MBP) for solubility enhancement

• Screening expression temperatures (16-37°C) and induction conditions

• Evaluating periplasmic versus cytoplasmic expression targeting

When working with uncharacterized proteins like yjiK, parallel small-scale expression trials with varying conditions should precede large-scale production to determine optimal parameters for soluble, functional protein.

What purification strategies yield the highest purity recombinant yjiK protein?

Purification of recombinant yjiK protein requires a multi-step approach tailored to its biochemical properties. While specific characteristics of yjiK remain uncharacterized, the following methodological strategy is recommended based on successful purification of other Shigella proteins:

• Initial capture: Affinity chromatography using an appropriate fusion tag (His6-tag IMAC, GST-affinity)

• Intermediate purification: Ion exchange chromatography based on predicted pI

• Polishing step: Size exclusion chromatography for final purity and buffer exchange

The purification protocol should be optimized considering:

• Stability assessment at various pH values and temperatures

• Addition of stabilizing agents (glycerol, reducing agents, specific ions)

• Removal of endotoxins if protein will be used for immunological studies

• Analysis of oligomeric state

A typical purification workflow might achieve 85-95% purity after the first step, with >98% purity after the complete process. Purification success should be monitored by SDS-PAGE, Western blotting, and activity assays when available. For uncharacterized proteins like yjiK, maintaining native conformation throughout purification is critical, which may require screening multiple buffer conditions during each purification step.

How can researchers determine if recombinant yjiK maintains native conformation?

Verifying the native conformation of recombinant yjiK requires multiple complementary biophysical techniques:

• Circular Dichroism (CD) Spectroscopy: Analyze secondary structure content and compare with computational predictions

• Differential Scanning Fluorimetry (DSF): Determine thermal stability and identify stabilizing buffer conditions

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): Confirm oligomeric state and homogeneity

• Limited Proteolysis: Probe for well-folded domains resistant to proteolytic degradation

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For smaller proteins, obtain fingerprint spectra of well-folded structures

For uncharacterized proteins like yjiK, without a known function for activity assays, these biophysical characteristics become crucial quality control metrics. Researchers should compare the recombinant protein properties with computational predictions and, when possible, with properties of homologous proteins from related species. Significant deviations in predicted versus experimental properties may indicate non-native conformations requiring optimization of expression and purification protocols.

What computational approaches can predict potential functions of the uncharacterized yjiK protein?

Computational prediction of yjiK function requires a multi-faceted bioinformatic analysis workflow:

• Sequence-based analysis:

  • Homology detection using PSI-BLAST, HHpred, and HMMER

  • Identification of conserved domains using InterPro, CDD, and Pfam

  • Ortholog analysis across bacterial species to identify evolutionary patterns

• Structure-based prediction:

  • Ab initio or homology-based 3D structure prediction

  • Structural comparison against known protein folds using DALI or FATCAT

  • Binding site and active site prediction using CASTp, SiteMap

• Genomic context analysis:

  • Operon structure examination

  • Gene neighborhood conservation across species

  • Co-expression pattern analysis

• Systems biology approaches:

  • Protein-protein interaction network prediction

  • Metabolic pathway analysis and gap identification

  • Phenotype-based function prediction

By integrating these computational approaches, researchers can generate testable hypotheses about yjiK function. For context, this approach has successfully identified functions of previously uncharacterized proteins in Shigella species, including the YhjC transcriptional regulator, which was found to regulate virulence through activation of virF transcription .

What experimental techniques are most effective for determining yjiK interaction partners?

Identifying protein interaction partners is crucial for understanding the function of uncharacterized proteins like yjiK. The following methodological approaches are recommended:

In Vitro Methods:

• Pull-down assays: Using tagged recombinant yjiK to capture interacting proteins from S. boydii lysates

• Surface Plasmon Resonance (SPR): For quantitative binding analysis with candidate partners

• Isothermal Titration Calorimetry (ITC): For thermodynamic characterization of specific interactions

In Vivo Methods:

• Bacterial two-hybrid system: Adapted for prokaryotic protein interactions

• Co-immunoprecipitation (Co-IP): Using antibodies against yjiK or epitope tags

• Crosslinking mass spectrometry (XL-MS): To capture transient interactions in the native environment

Systems-Level Approaches:

• Proximity-dependent biotin identification (BioID): For capturing spatial proximity interactions

• Protein microarrays: Testing interactions against hundreds of potential partners simultaneously

Each technique has specific strengths and limitations, and an integrated approach using multiple complementary methods provides the most reliable results. For bacterial pathogen proteins, particular attention should be paid to potential interactions with host proteins, as these may reveal roles in virulence or immune evasion.

How can genetic knockout or knockdown studies contribute to understanding yjiK function?

Genetic manipulation studies provide critical insights into protein function through phenotype analysis. For yjiK function determination, a comprehensive genetic approach should include:

• Generation of deletion mutants:

  • CRISPR-Cas9 system adapted for S. boydii

  • Lambda Red recombination system for precise gene deletion

  • Transposon mutagenesis for high-throughput screening

• Phenotypic characterization:

  • Growth curves under various conditions

  • Stress response assays (oxidative, acid, osmotic)

  • Virulence assays in cellular and animal models

  • Transcriptomic analysis via RNA-seq

  • Metabolomic profiling to identify affected pathways

• Complementation studies:

  • Episomal expression of wild-type yjiK

  • Structure-function analysis with mutated versions

  • Heterologous complementation with homologs from related species

• Conditional systems when deletion is lethal:

  • Inducible antisense RNA

  • Degron-tagged protein for inducible degradation

  • CRISPR interference (CRISPRi) for transcriptional repression

Similar genetic approaches have been successfully employed to characterize the function of the YhjC transcriptional regulator in Shigella flexneri, revealing its role in virulence regulation through activation of virF transcription . The yhjC deletion in S. flexneri significantly reduced colonization in guinea pig colons and decreased host cell adhesion and invasion capabilities , demonstrating the power of genetic approaches for functional characterization.

How might yjiK contribute to S. boydii serotype 4 pathogenesis and virulence?

Understanding yjiK's potential role in S. boydii serotype 4 pathogenesis requires integrating knowledge of Shigella virulence mechanisms with specific investigations of this uncharacterized protein. While direct evidence for yjiK's function is limited, methodological approaches to investigate its role in virulence include:

• Comparative virulence studies:

  • Infection assays comparing wild-type and yjiK-deleted strains

  • Tissue culture invasion and replication studies

  • Animal model comparison (guinea pig colon colonization model)

• Virulence gene expression analysis:

  • qRT-PCR quantification of key virulence genes (virF, virB, ipaA, ipaB, ipaC)

  • Proteomic analysis of type III secretion system (T3SS) components

  • Reporter gene assays for virulence promoter activity

• Regulatory network analysis:

  • Chromatin immunoprecipitation (ChIP) to identify potential DNA binding

  • Electrophoretic mobility shift assays (EMSA) for specific DNA interactions

  • RNA immunoprecipitation (RIP) for RNA binding assessment

In other Shigella species, novel regulators like YhjC have been shown to significantly impact virulence by regulating virF expression, which controls the expression of multiple virulence factors . The yhjC mutant showed significantly reduced colonization in guinea pig colons and decreased cell adhesion and invasion capabilities . Similarly, investigating whether yjiK affects established virulence pathways or represents a novel virulence mechanism would contribute significantly to understanding S. boydii pathogenesis.

What is the evolutionary significance of yjiK conservation across Shigella and related Enterobacteriaceae?

The evolutionary context of yjiK can provide important insights into its functional significance. A comprehensive evolutionary analysis would include:

• Phylogenetic analysis:

  • Construction of phylogenetic trees based on yjiK sequences

  • Comparison with species trees to identify incongruences indicating horizontal gene transfer

  • Calculation of selection pressures (dN/dS ratios) to determine evolutionary constraints

• Comparative genomics:

  • Synteny analysis to examine gene order conservation

  • Investigation of genetic linkage to mobile genetic elements

  • Assessment of presence/absence patterns across bacterial species

• Structural evolution:

  • Analysis of domain architecture conservation

  • Identification of critical conserved residues

  • Mapping of variable regions that might indicate species-specific functions

This evolutionary perspective is particularly relevant given the complex evolutionary history of Shigella species, which are in reality clones of E. coli that have emerged relatively recently . The O-antigen gene clusters of S. boydii serotype 4, for example, show significant similarity to those of E. coli O53 . Determining whether yjiK follows similar patterns of conservation and horizontal transfer would contribute to understanding both its function and the broader evolutionary history of Shigella pathogens.

How can structural biology approaches contribute to understanding yjiK function?

Structural biology provides powerful insights into protein function, particularly for uncharacterized proteins like yjiK. A comprehensive structural biology approach would include:

Experimental Structure Determination Methods:

The methodological workflow should include:

• High-throughput screening for crystallization conditions

• Structure determination and refinement

• Structure-based functional annotation through comparison with known protein folds

• Identification of potential active sites or binding pockets

• Computational docking with potential ligands

• Structure-guided mutagenesis to validate functional predictions

Structural information can reveal unexpected similarities to functionally characterized proteins, as observed with other bacterial proteins. For example, in S. boydii O9, structural analysis revealed that WbgS shares 55% similarity with AceP of A. xylinum, suggesting it catalyzes the α glucosyl 1-4 glucuronic acid linkage . Similar structural insights for yjiK could provide crucial functional clues.

How can insights from yjiK research contribute to development of new diagnostics for S. boydii infections?

Translating yjiK research into diagnostic applications requires systematic evaluation of its utility as a biomarker and development of detection methodologies. A comprehensive approach includes:

• Biomarker validation studies:

  • Determination of yjiK expression during infection

  • Assessment of immunogenicity and antibody responses

  • Evaluation of specificity for S. boydii serotype 4 versus other enteric pathogens

  • Sensitivity analysis in clinical sample matrices

• Diagnostic platform development:

  • ELISA-based detection using specific antibodies

  • Nucleic acid amplification tests targeting yjiK gene

  • Aptamer-based detection systems

  • Rapid immunochromatographic tests for field use

• Clinical validation:

  • Retrospective studies using banked clinical samples

  • Prospective studies in endemic settings

  • Comparison with gold standard diagnostics

  • Determination of positive and negative predictive values

While phage-based diagnostics have been described for S. boydii type 1 , serotype-specific diagnostics for S. boydii serotype 4 remain limited. If yjiK shows serotype specificity or is expressed during infection, it could serve as a valuable biomarker for improved diagnostics, addressing the current challenges in Shigella detection and serotyping.

What potential does the yjiK protein offer as a therapeutic target for treating S. boydii infections?

Evaluating yjiK as a potential therapeutic target requires systematic assessment of its druggability and essentiality. The research roadmap includes:

• Target validation:

  • Essentiality screening through conditional knockdown systems

  • Virulence contribution assessment in infection models

  • Conservation analysis across clinical isolates

  • Homology assessment with human proteins to predict off-target effects

• Druggability assessment:

  • Structural analysis of potential binding pockets

  • Fragment-based screening for initial chemical matter

  • Virtual screening against compound libraries

  • Development of biochemical assays for high-throughput screening

• Lead compound development:

  • Structure-activity relationship studies

  • Medicinal chemistry optimization

  • ADME/T property assessment

  • In vitro and in vivo efficacy testing

While specific information about yjiK's function is limited, other uncharacterized proteins in pathogens have successfully been developed as therapeutic targets. The methodological framework for target-based drug discovery remains applicable regardless of the current knowledge gaps, with the understanding that additional functional characterization would proceed in parallel with early-stage drug discovery efforts.

What are the most critical knowledge gaps remaining in yjiK research?

Despite advances in S. boydii genomics and proteomics, several critical knowledge gaps remain regarding the yjiK protein:

• Functional characterization: The basic biochemical function and cellular role of yjiK remain undefined

• Structural information: Three-dimensional structure and active site architecture are unknown

• Regulatory networks: The placement of yjiK within bacterial regulatory circuits is not established

• Expression patterns: The conditions under which yjiK is expressed during infection or environmental stress are undetermined

• Host interactions: Potential interactions with host cellular components have not been characterized

Addressing these knowledge gaps requires an integrated research approach combining genomics, proteomics, structural biology, and infection models. Given the relatively recent emergence of Shigella as clones of E. coli , comparative studies with E. coli homologs could provide valuable insights into yjiK function.

What emerging technologies might accelerate characterization of proteins like yjiK?

Several cutting-edge technologies show promise for accelerating the characterization of uncharacterized proteins like yjiK:

• AI-powered structure prediction: Tools like AlphaFold2 and RoseTTAFold can predict protein structures with unprecedented accuracy

• Single-cell techniques: Single-cell RNA-seq and proteomics can reveal expression patterns in heterogeneous bacterial populations

• CRISPR-based functional genomics: High-throughput screening of gene function using CRISPR interference or activation

• Microfluidic approaches: Droplet-based screening for protein function and interactions

• Native mass spectrometry: Characterization of protein complexes in their native state

• Cryo-electron tomography: Visualization of proteins in their cellular context

• Spatial transcriptomics and proteomics: Mapping expression patterns during infection