Recombinant Shigella sonnei UPF0266 membrane protein yobD (yobD)

What is Shigella sonnei and why is it significant for research?

Shigella sonnei is a Gram-negative, non-spore-forming, non-motile, facultative aerobic, rod-shaped bacterium that causes disease primarily in primates, including humans. It belongs to the Shigella genus, which was first discovered in 1897 and is closely related to E. coli. Shigella species are significant as they represent one of the leading bacterial causes of diarrhea worldwide, particularly causing moderate-to-severe diarrhea in children across Africa and South Asia . Within the genus, S. sonnei is notable for its disease contribution relative to its genomic diversity, with research indicating it contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity . Understanding S. sonnei proteins is therefore critical for developing effective interventions against this pathogen.

What is the UPF0266 membrane protein yobD and what is its role in Shigella sonnei?

The UPF0266 membrane protein yobD belongs to a class of membrane-associated proteins in Shigella sonnei. The "UPF" designation (Uncharacterized Protein Family) indicates that while the protein has been identified through genomic analysis, its precise biological function remains incompletely characterized . As a membrane protein, yobD is likely involved in cellular processes such as transport, signaling, or maintenance of membrane integrity. Research into membrane proteins like yobD is valuable for understanding bacterial pathogenesis and potentially identifying novel targets for antimicrobial development.

How does recombinant yobD differ from native yobD protein?

Recombinant yobD is produced through genetic engineering techniques in expression systems like E. coli, yeast, baculovirus, or mammalian cells . Unlike native yobD, which exists within the context of the S. sonnei membrane, recombinant versions typically contain modifications such as affinity tags (commonly histidine tags) that facilitate purification and detection . While the core protein sequence is preserved, recombinant production may result in differences in post-translational modifications, protein folding, or associated lipids compared to the native form. These differences must be considered when extrapolating experimental findings to the native bacterial context.

What experimental approaches are most effective for studying the structural properties of recombinant yobD protein?

Structural analysis of recombinant yobD requires a multi-technique approach due to the challenges inherent in membrane protein characterization. For initial structural assessment, circular dichroism (CD) spectroscopy provides valuable information about secondary structure content and protein folding. For higher-resolution analysis, X-ray crystallography remains the gold standard, though it requires successful crystallization of the protein, which is often challenging for membrane proteins.

Cryo-electron microscopy (cryo-EM) has emerged as a powerful alternative that doesn't require crystallization. Nuclear magnetic resonance (NMR) spectroscopy can be valuable for analyzing dynamics and interactions, particularly for specific domains. Computational approaches like molecular dynamics simulations complement experimental data by providing insights into conformational changes and functional mechanisms. A comprehensive structural analysis would likely combine these techniques with functional assays to correlate structure with biological activity.

How can researchers overcome challenges in expressing and purifying recombinant Shigella membrane proteins?

Successful expression and purification of recombinant Shigella membrane proteins like yobD require strategic approaches to overcome their hydrophobic nature and potential toxicity to host cells. Based on established protocols for similar membrane proteins, the following methodology is recommended:

• Expression system selection: While E. coli remains common, alternative systems such as yeast, baculovirus, or mammalian cell expression may yield better results for certain membrane proteins .

• Optimization strategy:

  • Modify culture conditions (temperature, induction timing, media composition)

  • Test multiple fusion tags beyond His-tags (MBP, GST, SUMO)

  • Consider codon optimization for the expression host

  • Evaluate membrane-targeting sequences

• Extraction protocol development:

  • Compare detergent types (DDM, LDAO, OG) for solubilization efficiency

  • Implement stringent quality control via SEC-MALS and DLS

For Shigella proteins specifically, the GMMA (Generalized Modules of Membrane Antigens) approach has demonstrated high yields of membrane-associated proteins (approximately 100 mg/L) from high-density fermentation of genetically modified strains . This approach, using strains with specific genetic modifications such as ΔtolR ΔgalU, represents an alternative to traditional recombinant expression systems.

What are the implications of genetic modifications in Shigella sonnei for recombinant protein production?

Genetic modifications in Shigella sonnei significantly impact recombinant protein production and characteristics. Research has demonstrated that targeted gene deletions can enhance both yield and quality of membrane proteins. The deletion of tolR (ΔtolR) increases outer membrane vesicle release, substantially improving the yield of membrane proteins including potential recombinant yobD .

Additional modifications, such as deletion of galU (ΔgalU), affect lipopolysaccharide (LPS) structure, potentially reducing endotoxicity while maintaining immunogenicity . The deletion of msbB genes (involved in lipid A biosynthesis) further decreases LPS toxicity, though it slows growth rate (55 min duplication time compared to 28 min for the ΔtolR single mutant) .

These genetic approaches provide researchers flexibility in designing expression systems with specific characteristics:

The GMMA approach leveraging these modifications has proven scalable from laboratory to industrial production, supporting feasible manufacturing processes for membrane protein production .

How can researchers evaluate potential functional interactions between yobD and other Shigella proteins?

Investigating functional interactions between yobD and other Shigella proteins requires a systematic approach combining in silico prediction with experimental validation. Recommended methodologies include:

• Computational prediction phase:

  • Implement protein-protein interaction network analysis

  • Apply gene neighborhood and co-expression analysis

  • Conduct homology-based inference from related bacteria

• Experimental validation phase:

  • Perform co-immunoprecipitation with anti-yobD antibodies followed by mass spectrometry

  • Develop bacterial two-hybrid or split-GFP assays for targeted interaction testing

  • Implement FRET-based approaches for in vivo interaction dynamics

• Functional confirmation:

  • Generate knockout mutants of predicted interaction partners

  • Assess phenotypic changes in membrane integrity, stress response, or virulence

  • Perform comparative proteomics between wild-type and mutant strains

When designing interaction studies, researchers should consider the membrane localization of yobD, which necessitates appropriate detergent conditions to maintain protein stability while enabling protein-protein interactions. Cross-validation using multiple independent techniques is essential for confirming genuine interactions versus experimental artifacts.

What expression systems are optimal for producing recombinant Shigella sonnei membrane proteins?

The optimal expression system for recombinant Shigella sonnei membrane proteins depends on downstream applications, required yield, and protein characteristics. Based on established protocols, the following expression systems offer distinct advantages:

For yobD specifically, E. coli systems typically provide sufficient yield for most applications , though researchers should evaluate multiple expression systems if functional studies reveal unexpected results. The GMMA approach using genetically modified S. sonnei (ΔtolR ΔgalU) represents a specialized alternative that maintains the protein in its native membrane environment, yielding approximately 100 mg of membrane-associated proteins per liter of fermentation at optical densities of 30-45 .

What purification strategies maximize yield while preserving structural integrity of yobD?

Purifying membrane proteins like yobD requires specialized strategies that balance efficient extraction with structural preservation. A comprehensive protocol typically includes:

• Initial extraction optimization:

  • Screen multiple detergents (DDM, LDAO, OG) at varying concentrations

  • Test solubilization efficiency at different temperatures (4°C, room temperature)

  • Optimize buffer components (salt concentration, pH, glycerol percentage)

• Purification workflow:

  • Initial capture: IMAC (immobilized metal affinity chromatography) for His-tagged constructs

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography in appropriate detergent

• Quality assessment:

  • SEC-MALS to confirm monodispersity and oligomeric state

  • Thermal stability assays (DSF/nanoDSF) to identify stabilizing conditions

  • Circular dichroism to verify secondary structure integrity

For membrane proteins specifically, researchers should verify protein orientation and folding using limited proteolysis paired with mass spectrometry. Throughout purification, maintaining a consistent detergent concentration above the critical micelle concentration is essential to prevent protein aggregation.

How can researchers effectively analyze the membrane topology of yobD?

Determining the membrane topology of yobD requires a combination of computational prediction and experimental validation. Computational approaches include transmembrane prediction algorithms (TMHMM, MEMSAT, Phobius) and hydropathy analysis to identify potential membrane-spanning regions and their orientation.

For experimental validation, several complementary techniques are recommended:

• Substituted cysteine accessibility method (SCAM): Introduce cysteine residues at predicted loop regions and assess their accessibility to membrane-impermeable sulfhydryl reagents.

• Protease protection assays: Treat intact bacterial cells or proteoliposomes with proteases, then analyze protected fragments by mass spectrometry to determine which regions were shielded by the membrane.

• Reporter fusion approach: Generate fusions of yobD fragments with reporter proteins like GFP or alkaline phosphatase at different positions, then analyze cellular localization and activity.

• Site-directed fluorescence labeling: Introduce fluorescent probes at specific positions and analyze their accessibility or fluorescence properties in the membrane environment.

The integration of these approaches provides a comprehensive topological map that can inform structural models and functional hypotheses about yobD's role in the bacterial membrane.

What techniques can be used to investigate potential post-translational modifications of yobD?

Investigating post-translational modifications (PTMs) of yobD requires sensitive analytical methods due to the typically substoichiometric nature of many modifications. A systematic approach includes:

• Mass spectrometry-based workflows:

  • Bottom-up proteomics: Enzymatic digestion followed by LC-MS/MS with PTM-specific enrichment strategies

  • Top-down proteomics: Analysis of intact proteins to preserve modification patterns

  • Targeted approaches: MRM/PRM methods for quantitative analysis of specific modifications

• Biochemical detection:

  • Immunoblotting with modification-specific antibodies (phosphorylation, glycosylation)

  • Enzymatic treatments (phosphatase, glycosidase) followed by mobility shift analysis

  • Chemical labeling strategies (e.g., PHOS-tag for phosphorylation)

• Site-specific analysis:

  • Site-directed mutagenesis of putative modification sites

  • Functional assays comparing wild-type and mutant proteins

  • In vitro modification assays with purified enzymes

Given the bacterial origin of yobD, researchers should prioritize investigation of prokaryotic modifications such as phosphorylation, acetylation, and methylation rather than eukaryotic-specific modifications like complex glycosylation. Comparative analysis between recombinant and native protein can reveal modifications that might be absent in heterologous expression systems.

How can recombinant yobD contribute to vaccine development against Shigella sonnei?

Recombinant yobD represents a potential component in next-generation vaccine strategies against Shigella sonnei. As a membrane protein, yobD may contribute to protective immunity through specific antibody responses. The GMMA approach, which uses genetically modified Shigella strains to produce outer membrane particles containing multiple membrane proteins including potentially yobD, has demonstrated high immunogenicity in mice .

Several advantages make membrane proteins like yobD attractive vaccine candidates:

• Surface accessibility increases likelihood of antibody recognition

• Relatively high conservation compared to variable surface antigens

• Potential role in bacterial virulence or survival mechanisms

Research utilizing recombinant yobD in vaccine development should consider:

• Epitope mapping to identify immunodominant regions

• Conservation analysis across clinical isolates

• Formulation strategies to maintain native conformation

• Combination with other antigens for broader protection

The high-yield production process developed for Shigella outer membrane particles (yielding approximately 100 mg of membrane-associated proteins per liter) supports the feasibility of scaling up production for vaccine manufacturing . This approach allows for additional genetic manipulations such as LPS modifications that can reduce reactogenicity while maintaining immunogenicity.

What role might yobD play in antimicrobial resistance mechanisms in Shigella?

While specific data on yobD's role in antimicrobial resistance is limited, membrane proteins often contribute to resistance mechanisms. Given the increasing antimicrobial resistance in Shigella strains, particularly against ciprofloxacin (the WHO-recommended treatment for shigellosis) , investigating yobD's potential involvement is valuable.

Research approaches should include:

• Comparative expression analysis between susceptible and resistant strains

• Generation of knockout or overexpression mutants to assess impact on antibiotic susceptibility

• Structural analysis to identify potential antibiotic binding sites

• Investigation of potential interaction with known resistance mechanisms (efflux pumps, membrane permeability)

The convergent evolution of resistance against ciprofloxacin among Shigella isolates suggests common mechanisms that may involve membrane proteins like yobD, either directly or indirectly through membrane integrity maintenance.

How can structural information about yobD inform drug discovery efforts?

Structural characterization of yobD could significantly advance drug discovery efforts by enabling structure-based design approaches. Membrane proteins represent approximately 60% of current drug targets, making yobD a potentially valuable target if its structure and function can be fully characterized.

A systematic approach to leverage structural information includes:

• Structure-based virtual screening:

  • Computational docking of compound libraries against identified binding pockets

  • Pharmacophore modeling based on natural ligands or substrates

  • Fragment-based screening to identify chemical starting points

• Structure-activity relationship development:

  • Design of focused compound libraries based on identified hits

  • Iterative optimization guided by structural data

  • Biophysical characterization of binding interactions (SPR, ITC, MST)

• Target validation:

  • Generation of binding site mutants to confirm specificity

  • Cellular assays to correlate binding with functional outcomes

  • In vivo validation in infection models

The genetic modifications established for producing recombinant Shigella proteins provide valuable tools for validating potential drug candidates by enabling the generation of strains with modified or deleted yobD for comparative studies.

What insights can comparative genomics provide about yobD conservation and variation across Shigella species?

A comprehensive comparative genomics approach should include:

• Sequence conservation analysis:

  • Multiple sequence alignment across Shigella species and E. coli

  • Identification of highly conserved residues/domains indicating functional importance

  • Detection of positive selection signatures suggesting adaptive evolution

• Structural variation mapping:

  • Prediction of impact of sequence variations on protein structure

  • Comparison of transmembrane topology across variants

  • Correlation of structural differences with phenotypic characteristics

• Genomic context analysis:

  • Examination of gene neighborhood conservation

  • Identification of associated regulatory elements

  • Detection of horizontal gene transfer or recombination events

This comparative approach can inform both fundamental understanding of yobD function and applied research on strain-specific interventions, particularly important given the observed diversity and adaptive capacity among Shigella species that may generate vaccine escape variants .