Recombinant Salmonella schwarzengrund UPF0266 membrane protein yobD (yobD)

What is the basic structure and characteristics of Salmonella schwarzengrund UPF0266 membrane protein yobD?

Salmonella schwarzengrund UPF0266 membrane protein yobD is a 152-amino acid protein with hydrophobic regions consistent with its membrane-associated function. The complete amino acid sequence is: MTITDLVLILFIAALLAYALYDQFIMPRRNGPTLLSIALLRRGRVDSVIFVGLVAILIYNVTSHGAQMTTWLLSALALMGFYIFWIRTPRIIFKQRGFFFANVWIEYNRIKEMNLSEDGVLVMQLEQRRLLIRVRNIDDLEKIYKLLIENQ .

Analysis of this sequence reveals typical characteristics of membrane proteins, including hydrophobic domains likely to span the bacterial membrane. Its classification in the UPF0266 family indicates it belongs to a group of functionally uncharacterized proteins that share structural similarities. When working with this protein, researchers should note that its membrane-spanning regions may affect solubility and require specific extraction and purification protocols.

How does yobD protein compare structurally to other bacterial membrane proteins with known functions?

When comparing yobD to well-characterized bacterial membrane proteins, several structural similarities emerge with proteins involved in secretion systems. While yobD's function isn't fully characterized, its membrane location suggests possible involvement in transport or secretion mechanisms.

Structural comparison with Yersinia YopB protein, which contains two transmembrane helices (residues 166-188 and 228-250) that insert into host plasma membranes during type III secretion, may provide functional insights . YopB participates in forming a translocation pore for effector proteins, and although belonging to different bacterial genera, membrane proteins often share conserved functional mechanisms. Researchers should perform sequence alignments and structural prediction analyses to identify conserved domains that might indicate similar functions between yobD and other bacterial membrane proteins.

What expression systems are most effective for producing recombinant Salmonella yobD protein?

For recombinant expression of Salmonella yobD protein, Escherichia coli expression systems have proven effective as documented in commercial production . Several expression options exist with varying advantages:

For initial studies, E. coli systems with specialized membrane protein expression strains (e.g., C41(DE3) or C43(DE3)) are recommended. These strains are engineered to accommodate membrane protein overexpression better than standard strains. Including fusion tags like His-tag facilitates purification while minimizing impact on protein structure .

What are the potential functions of yobD in Salmonella virulence based on its membrane localization?

The membrane localization of yobD protein suggests several potential roles in Salmonella virulence that warrant investigation. By comparison to similar bacterial membrane proteins like YopB in Yersinia, which forms pores in host cell membranes for delivery of effector proteins , yobD may participate in a similar process for Salmonella.

Research approaches to determine virulence functions should include:

• Construction of yobD knockout strains to assess changes in:

  • Host cell invasion efficiency

  • Effector protein translocation

  • Pore formation in host membranes (measurable via dye uptake assays similar to those used for YopB/D )

  • Survival within host cells

• Protein interaction studies to identify binding partners, particularly other secretion system components

Comparing experimental results with what's known about YopB/D function could provide valuable insights, as these proteins are critical for translocation of effector proteins across host membranes and formation of pores in erythrocytes and macrophages .

How can researchers effectively analyze the membrane topology and insertion mechanism of yobD?

Analyzing membrane topology and insertion mechanisms for yobD requires multiple complementary approaches:

• Computational prediction: Begin with transmembrane domain prediction tools like TMHMM, Phobius, or MEMSAT to identify potential membrane-spanning regions.

• Biochemical mapping: Apply techniques such as:

  • Cysteine scanning mutagenesis with subsequent labeling

  • Protease protection assays

  • Reporter fusion analysis (PhoA/LacZ dual reporters)

• Structural analysis: For higher resolution data, consider:

  • Cryo-electron microscopy of membrane-embedded protein

  • X-ray crystallography (challenging but possible with appropriate detergents)

  • NMR studies of isolated domains

• Insertion mechanism: To study how yobD inserts into membranes:

  • Create mutations in hydrophobic domains analogous to the helix-disrupting mutations used for YopB (double proline substitutions)

  • Assess membrane insertion efficiency using fluorescence-based assays

  • Perform in vitro translation/translocation assays with purified membrane vesicles

The research approach used with YopB, where helix-disrupting substitutions were introduced into transmembrane domains, provides a valuable experimental template . Similar mutations in yobD's predicted membrane-spanning regions could help determine if its function also requires membrane insertion.

What functional interactions might exist between yobD and other components of Salmonella secretion systems?

Based on comparative analysis with Yersinia secretion systems, yobD may interact with multiple components in Salmonella secretion pathways. To identify and characterize these interactions:

• Protein-protein interaction screening:

  • Co-immunoprecipitation with epitope-tagged yobD

  • Bacterial two-hybrid systems

  • Proximity labeling approaches (BioID or APEX2)

  • Cross-linking mass spectrometry

• Functional cooperation assays:

  • Genetic complementation studies

  • Double knockout phenotype analysis

  • Secretion assays with various component mutations

The YopB-YopD interaction in Yersinia provides a model for potential protein partnerships . YopB functions with YopD to form translocation pores, and both are required for hemolytic activity. Researchers should investigate whether yobD requires similar partner proteins for its function in Salmonella.

What purification strategies are most effective for isolating functional recombinant yobD protein?

Purifying functional membrane proteins presents challenges due to their hydrophobicity. For recombinant yobD, a multi-step purification strategy is recommended:

• Expression optimization:

  • Use His-tagged constructs expressed in E. coli

  • Induce at lower temperatures (16-20°C) to improve folding

  • Consider fusion partners that enhance solubility

• Membrane extraction:

  • Isolate bacterial membranes by ultracentrifugation

  • Screen detergents for optimal extraction (typical starting panel: DDM, LDAO, OG)

  • Use a gentle detergent concentration just above CMC

• Purification protocol:

  • Initial capture: IMAC (immobilized metal affinity chromatography) using His-tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography in appropriate detergent

• Quality assessment:

  • SDS-PAGE with Western blotting confirmation

  • Circular dichroism to verify secondary structure

  • Thermal stability assays to optimize buffer conditions

Storage of purified yobD should be at -20°C or -80°C for extended periods, with working aliquots maintained at 4°C for up to one week to avoid freeze-thaw cycles that could compromise structural integrity .

How can researchers assess the membrane insertion and pore-forming capabilities of yobD?

To evaluate membrane insertion and potential pore-forming capabilities of yobD, researchers should adapt methods used for studying similar bacterial proteins like YopB:

• Liposome association assays:

  • Prepare fluorescently labeled liposomes with varying lipid compositions

  • Incubate with purified yobD

  • Measure protein-liposome association through co-sedimentation or fluorescence changes

• Pore formation assessment:

  • Dye-loaded liposome leakage assays (using calcein or BCECF)

  • Planar lipid bilayer electrophysiology to characterize channel properties

  • Osmotic protection assays with molecules of different sizes to estimate pore diameter

• Cellular pore formation:

  • Hemolysis assays using sheep erythrocytes (similar to those used for YopB/D)

  • Macrophage dye uptake studies with molecules of varying sizes (BCECF at 623 Da vs. larger molecules)

  • Cell flattening observations in macrophages as seen with pore-forming proteins

The methods used to study YopB/D-mediated pore formation provide an excellent framework, as they demonstrated size-selective pore formation allowing passage of small molecules (<623 Da) but not larger ones (>1490 Da) . These approaches would help determine if yobD forms pores of similar characteristics.

What imaging techniques are most informative for studying yobD localization and function?

Multiple imaging approaches can provide valuable insights into yobD localization and function:

• Fluorescence microscopy:

  • Express fluorescently tagged yobD (e.g., GFP fusion) to track localization

  • Use super-resolution techniques (STORM, PALM) to achieve sub-diffraction resolution

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to assess membrane mobility

• Electron microscopy:

  • Immunogold labeling with TEM to precisely localize yobD in bacterial membranes

  • Cryo-EM of recombinant protein in nanodiscs or liposomes

  • Electron tomography for 3D visualization of membrane structures

• Correlative approaches:

  • Combine live-cell fluorescence with electron microscopy (CLEM)

  • Use click chemistry with minimally disruptive tags for pulse-chase studies

• Host-pathogen interface imaging:

  • Confocal microscopy during infection to track potential reorganization

  • Live-cell imaging with simultaneous pathogen and host membrane labeling

For optimal results, fluorescent protein fusions should be validated to ensure they don't disrupt membrane insertion or protein function. Small epitope tags or click chemistry-compatible amino acids often provide less disruption than bulky fluorescent proteins.

What are the most promising research directions for understanding yobD function in Salmonella pathogenesis?

The most promising research directions for elucidating yobD function center on comparative analysis with better-characterized bacterial membrane proteins and systematic functional characterization. Based on the available information about yobD and related proteins like YopB/D, researchers should prioritize:

• Systematic mutagenesis studies: Creating transmembrane domain disruptions similar to those used for YopB to determine the importance of membrane insertion for yobD function.

• Infection models: Developing yobD knockout and complementation systems to assess its role during actual infection, measuring effects on bacterial internalization, survival, and host response.

• Structural biology: Determining the three-dimensional structure of yobD to identify functional domains and potential interaction interfaces.

• Secretion system relationships: Investigating whether yobD functions as part of a known secretion system or represents a component of a novel system in Salmonella.

• Host-pathogen interaction studies: Identifying potential host cell targets or receptors for yobD, similar to the interaction between Shigella IpaB and the hyaluronan receptor CD44 .

By combining these approaches, researchers can build a comprehensive understanding of how this membrane protein contributes to Salmonella biology and potentially discover new mechanisms of bacterial pathogenesis that could inform therapeutic strategies.

How can findings from yobD research contribute to broader understanding of bacterial membrane proteins and pathogenesis mechanisms?

Research on yobD has significant potential to enhance our understanding of bacterial membrane proteins and pathogenesis mechanisms in several ways:

• Expanding the functional repertoire of UPF0266 proteins: As a member of an uncharacterized protein family (UPF0266), defining yobD function would illuminate the roles of this entire group of proteins across bacterial species.

• Revealing conserved mechanisms: Identifying functional parallels between yobD and better-characterized proteins like YopB/D could reveal evolutionarily conserved strategies employed by diverse pathogenic bacteria.

• Novel therapeutic targets: If yobD proves important for Salmonella virulence, it could represent a new target for antimicrobial development, particularly valuable given the increasing resistance to current antibiotics in Salmonella strains .

• Methodological advances: Technical approaches developed to study challenging membrane proteins like yobD can benefit research on other membrane proteins, advancing the broader field of membrane protein biology.

• Cross-species comparison: Comparative analysis of yobD across different Salmonella species (such as S. schwarzengrund and S. choleraesuis ) could reveal species-specific adaptations and explain differences in host range or virulence.