Recombinant Staphylococcus epidermidis UPF0382 membrane protein SE_0353 (SE_0353)

What is the structural characterization of UPF0382 membrane protein SE_0353?

UPF0382 membrane protein SE_0353 is a 122-amino acid transmembrane protein from Staphylococcus epidermidis strain ATCC 12228. The protein has a predominantly hydrophobic amino acid sequence (MKVFIILGALNAMMAVGTGAFGAHGLEDKLSDKYMSIWEKATTYQMYHGLGLLVIGLISGTTSINVNWAGWLLFFGIVFFSGSLYFLALTQVRILGAITPIGGVLFIIGWLVLVIATLKFAG) consistent with its membrane-spanning function . Based on sequence analysis, this protein likely contains multiple transmembrane domains with hydrophobic regions that anchor it within the bacterial cell membrane. The "UPF" designation indicates it belongs to the Uncharacterized Protein Family, suggesting its precise biological function remains to be fully elucidated through structural and functional studies.

What are the optimal storage conditions for recombinant UPF0382 membrane protein SE_0353?

The recombinant UPF0382 membrane protein SE_0353 should be stored at -20°C in a Tris-based buffer containing 50% glycerol that has been optimized specifically for this protein's stability . For extended storage periods, conservation at -80°C is recommended to maintain protein integrity. To minimize protein degradation caused by freeze-thaw cycles, it is advisable to prepare single-use aliquots prior to freezing, as repeated freezing and thawing is not recommended. Working aliquots may be stored at 4°C for up to one week, but longer periods at this temperature may lead to protein degradation or loss of activity .

How does the expression of membrane proteins in S. epidermidis compare to other staphylococcal species?

While the search results don't directly compare expression patterns between different staphylococcal species, research on S. epidermidis membrane proteins suggests that, like the SdrG protein, expression of membrane proteins may be regulated by environmental conditions. For example, SdrG expression increases significantly following exposure to bloodstream conditions . This pattern may extend to other membrane proteins like UPF0382 SE_0353, suggesting that expression of staphylococcal membrane proteins is dynamically regulated in response to specific host environmental cues. Unlike S. aureus, which expresses numerous toxins, S. epidermidis relies more heavily on surface proteins for colonization and biofilm formation, potentially giving its membrane proteins distinct expression profiles compared to other staphylococcal species.

What computational methods are most effective for predicting interactions between UPF0382 and other membrane proteins?

For predicting interactions between UPF0382 and other membrane proteins, computational docking methods specifically designed for membrane proteins would be most effective. Mem-LZerD has demonstrated superior performance for transmembrane protein complex modeling, successfully performing unbound docking on approximately 62% of transmembrane complexes in established benchmarks . This algorithm would likely provide reliable predictions for UPF0382 interactions.

For optimal results, a multi-stage approach is recommended:

• Initial sequence-based analysis using algorithms like TMHMM to predict transmembrane regions

• Homology modeling if structural templates exist

• Membrane protein-specific docking using Mem-LZerD

• Refinement through molecular dynamics simulations in a lipid bilayer environment

This computational pipeline would help identify potential interaction partners and binding interfaces that could then be validated experimentally through techniques such as co-immunoprecipitation or crosslinking studies .

How might post-translational modifications affect the function of UPF0382 membrane protein SE_0353?

Post-translational modifications (PTMs) could significantly impact UPF0382 membrane protein function through several mechanisms:

• Phosphorylation: May regulate protein-protein interactions or conformational changes

• Glycosylation: Could affect protein stability or recognition by host immune factors

• Lipidation: Might enhance membrane anchoring or localization to specific membrane domains

While specific PTMs of UPF0382 have not been characterized in the provided search results, membrane proteins often undergo modifications that regulate their localization, stability, and function. By analogy with other bacterial membrane proteins like SdrG, which shows regulated expression in response to environmental cues , UPF0382 may undergo conditional PTMs that affect its functional properties during different growth phases or environmental conditions.

To investigate potential PTMs, mass spectrometry analysis of the native protein isolated from bacteria grown under various conditions would be the recommended approach, followed by site-directed mutagenesis of putative modification sites to assess functional consequences.

What role might UPF0382 membrane protein play in S. epidermidis biofilm formation and pathogenicity?

UPF0382 membrane protein SE_0353 may contribute to S. epidermidis biofilm formation and pathogenicity through several potential mechanisms:

• Membrane integrity: As a membrane protein, UPF0382 likely contributes to cell envelope stability during biofilm development

• Adhesion mediator: By analogy with other staphylococcal membrane proteins like SdrG , UPF0382 might mediate adherence to host tissues or implanted medical devices

• Environmental sensing: The protein could function in signal transduction pathways that regulate biofilm formation in response to environmental cues

Drawing parallels with SdrG, which is expressed during bloodstream infection and contributes to pathogenicity , UPF0382 may similarly respond to host environmental signals and contribute to S. epidermidis virulence. The protein's transmembrane structure suggests it could serve as a receptor or transporter involved in sensing environmental conditions relevant to biofilm formation.

To investigate this role, knockout mutants lacking the SE_0353 gene could be assessed for biofilm formation capacity, adhesion to various surfaces, and virulence in animal infection models.

What are the most effective methods for expressing and purifying recombinant UPF0382 membrane protein SE_0353?

The expression and purification of recombinant UPF0382 membrane protein SE_0353 requires specialized techniques due to its hydrophobic nature. Based on successful approaches with other membrane proteins, the following protocol is recommended:

Expression System Selection:

• E. coli C41(DE3) or C43(DE3) strains: These strains are engineered to tolerate membrane protein overexpression

• pET vector system with tunable promoter: Allows controlled expression to prevent toxicity

Expression Protocol:

• Transform expression plasmid into selected E. coli strain

• Grow culture at 37°C until OD600 = 0.6-0.8

• Induce with low IPTG concentration (0.1-0.5 mM)

• Shift to lower temperature (16-25°C) for 16-20 hours for expression

Purification Strategy:

• Cell lysis via French press or sonication in buffer containing:

  • 50 mM Tris-HCl pH 7.5

  • 150 mM NaCl

  • Protease inhibitors

• Membrane isolation by ultracentrifugation (100,000 × g)

• Solubilization with mild detergents (DDM, LDAO, or C12E8)

• Purification via:

  • Nickel affinity chromatography (for His-tagged protein)

  • Size exclusion chromatography for final polishing

Storage:
Store in Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage .

This protocol may need optimization based on protein-specific characteristics and experimental requirements.

What approaches should be used to study UPF0382 protein interactions with host cellular components?

To study interactions between UPF0382 and host cellular components, multiple complementary approaches should be employed:

In vitro Binding Assays:

• Surface Plasmon Resonance (SPR): Immobilize purified UPF0382 on a sensor chip and flow potential host interaction partners to measure binding kinetics.

• ELISA-based binding assays: Similar to methods used for studying PfEMP1 domains , coat plates with potential host receptors and detect binding of labeled UPF0382.

• Pull-down assays with host cell lysates: Use immobilized UPF0382 to capture interacting host proteins, followed by mass spectrometry identification.

Cellular Assays:

• Fluorescence microscopy: Label UPF0382 and monitor binding to various host cell types, similar to approaches used for PfEMP1 .

• Flow cytometry: Quantify binding of fluorescently labeled UPF0382 to host cells.

Mutagenesis Studies:

• Generate domain deletions or point mutations in UPF0382

• Assess impact on binding to identify critical interaction interfaces

Computational Prediction:
Use Mem-LZerD or similar algorithms to predict potential interaction interfaces .

A comprehensive study would integrate these approaches to identify and characterize host cellular components that interact with UPF0382, potentially revealing its role in S. epidermidis colonization or pathogenesis.

How can researchers effectively monitor the expression levels of UPF0382 under different environmental conditions?

To effectively monitor UPF0382 expression under different environmental conditions, researchers should employ a multi-faceted approach:

Transcriptional Analysis:

• Quantitative PCR (qPCR): Design primers specific to the SE_0353 gene to quantify mRNA levels under various conditions, similar to the approach used for monitoring sdrG transcripts .

• RNA-Seq: For genome-wide expression analysis to identify co-regulated genes and regulatory networks.

Protein Level Analysis:

• Western Blotting: Develop specific antibodies against UPF0382 for semi-quantitative protein detection.

• Immunofluorescence Microscopy: Visualize UPF0382 expression and localization within bacterial cells under different conditions, similar to the approach used for SdrG .

• Flow Cytometry: Quantify surface expression levels when antibodies to UPF0382 are available.

Reporter Systems:

• Transcriptional Fusions: Create fusions of the SE_0353 promoter with reporter genes (GFP, luciferase) to monitor promoter activity in real-time.

• Translational Fusions: Generate C-terminal tagged versions of UPF0382 to track protein levels without disrupting function.

Environmental Conditions to Test:
Based on research with SdrG , conditions that might affect UPF0382 expression include:

• Growth in human serum or blood

• Exposure to varying CO₂ levels

• Iron limitation

• Biofilm versus planktonic growth

• Exposure to antimicrobial peptides

• Different growth phases

This comprehensive approach will help identify the environmental cues that regulate UPF0382 expression and provide insights into its biological function.

How should researchers interpret contradictory functional data regarding UPF0382 membrane protein?

When faced with contradictory functional data regarding UPF0382 membrane protein, researchers should implement the following systematic approach:

1. Methodological Analysis:

• Compare experimental conditions across studies (growth conditions, strain backgrounds, purification methods)

• Assess protein integrity and proper folding in each study

• Evaluate the sensitivity and specificity of the assays used

2. Context-Dependent Function Assessment:

• Consider that membrane proteins may exhibit different functions under different conditions

• Analyze whether contradictions might represent condition-specific behaviors rather than true contradictions

• Examine whether protein modifications or interaction partners differ between studies

3. Resolution Strategies:

• Conduct side-by-side experiments using standardized protocols

• Employ multiple complementary techniques to assess the same function

• Consider whether observed contradictions might reflect different aspects of a multifunctional protein

4. Statistical Rigor:

• Re-analyze data using appropriate statistical methods

• Ensure adequate sample sizes and proper controls

• Consider meta-analysis approaches when multiple datasets are available

Similar to the investigation of SdrG, which showed environment-dependent expression patterns , UPF0382 may exhibit context-dependent functions that appear contradictory when studied under different conditions. By carefully analyzing methodological differences and considering biological context, researchers can often reconcile apparently contradictory results.

What bioinformatic tools are most valuable for predicting the function of uncharacterized membrane proteins like UPF0382?

For predicting the function of uncharacterized membrane proteins like UPF0382, the following bioinformatic tools and approaches offer the greatest value:

Sequence-Based Analysis:

• BLAST/PSI-BLAST: Identify distant homologs with known functions

• HMMER/HHpred: Detect remote homology through profile-based searches

• InterPro/Pfam: Identify conserved domains and functional motifs

• TMHMM/TOPCONS: Predict transmembrane topology and orientation

Structural Prediction and Analysis:

• AlphaFold2/RoseTTAFold: Generate accurate structural models

• ProFunc/COFACTOR: Predict function from structural features

• Mem-LZerD: Model potential protein-protein interactions

• molecular dynamics simulations: Assess structural dynamics in membrane environments

Genomic Context Analysis:

• Gene neighborhood analysis: Identify functionally related genes

• Co-expression networks: Find genes with similar expression patterns

• Phylogenetic profiling: Determine co-occurrence patterns across species

Integrative Approaches:

• STRING database: Combine multiple lines of evidence for functional prediction

• PSICQUIC: Integrate protein interaction data from multiple databases

• Gene Ontology enrichment: Identify statistically overrepresented functions

By integrating results from these complementary approaches, researchers can generate testable hypotheses about UPF0382 function, prioritizing experimental validation of the most consistent predictions.

How can researchers distinguish between direct and indirect effects when studying UPF0382 knockout phenotypes?

Distinguishing between direct and indirect effects when studying UPF0382 knockout phenotypes requires a systematic approach:

Genetic Complementation Analysis:

• Reintroduce the wild-type SE_0353 gene to confirm phenotype reversal

• Create point mutants affecting specific domains/functions to identify critical regions

• Use inducible expression systems to establish temporal relationships between gene expression and phenotype manifestation

Molecular Interaction Studies:

• Identify direct interaction partners using techniques like:

  • Co-immunoprecipitation

  • Bacterial two-hybrid assays

  • Cross-linking followed by mass spectrometry

• Validate direct interactions using purified components in vitro

Temporal Analysis:

• Use time-course experiments to establish the sequence of events following UPF0382 deletion

• Employ pulsed expression of UPF0382 to determine immediate versus delayed effects

Comparative Multi-omics:

Spatial Localization:

• Use fluorescence microscopy to determine if knockout affects localization of other proteins

• Examine membrane domain organization in wild-type versus knockout strains

This multi-faceted approach, similar to methodologies used in studying other membrane proteins, will help distinguish the direct consequences of UPF0382 absence from secondary effects that arise from disruption of cellular homeostasis.

How might UPF0382 contribute to antibiotic resistance mechanisms in S. epidermidis?

As a membrane protein, UPF0382 could potentially contribute to antibiotic resistance in S. epidermidis through several mechanisms:

Membrane Permeability Modulation:
UPF0382 might influence membrane structure and permeability, potentially reducing antibiotic penetration into the bacterial cell. Its hydrophobic sequence (MKVFIILGALNAMMAVGTGAFGAHGLEDKLSDKYMSIWEKATTYQMYHGLGLLVIGLISGTTSINVNWAGWLLFFGIVFFSGSLYFLALTQVRILGAITPIGGVLFIIGWLVLVIATLKFAG) suggests it could alter membrane fluidity or organization .

Efflux Pump Cooperation:
While not an efflux pump itself, UPF0382 might interact with or stabilize efflux pump complexes that actively export antibiotics from the cell.

Biofilm Formation Enhancement:
By potentially contributing to biofilm formation, similar to other S. epidermidis surface proteins like SdrG , UPF0382 could indirectly promote antibiotic tolerance by facilitating the formation of biofilms where antibiotics penetrate poorly.

Stress Response Signaling:
UPF0382 might participate in membrane-associated stress response signaling pathways that upregulate resistance mechanisms in response to antibiotic exposure.

To investigate these possibilities, researchers should compare antibiotic susceptibility profiles between wild-type and UPF0382 knockout strains under both planktonic and biofilm growth conditions, and assess changes in membrane permeability using fluorescent dye uptake assays.

What immunogenic potential does UPF0382 have as a vaccine candidate against S. epidermidis infections?

The immunogenic potential of UPF0382 as a vaccine candidate against S. epidermidis infections can be evaluated through several considerations:

Immunogenicity Assessment:
Recombinant UPF0382 could be tested for its ability to elicit antibody responses in animal models, similar to studies with SdrG that demonstrated robust antibody production following immunization with recombinant protein domains .

Conservation Analysis:
Sequence conservation of UPF0382 across clinical S. epidermidis isolates would need to be assessed to determine its potential as a broadly protective antigen. High conservation would support its utility as a vaccine target.

Surface Accessibility:
While UPF0382 is a membrane protein, the degree to which epitopes are exposed on the bacterial surface would determine antibody accessibility. Computational topology prediction and experimental epitope mapping would be essential.

Protective Efficacy:
Similar to studies with SdrG where vaccination with recombinant protein domains reduced bacteremia in mouse models , UPF0382 vaccination would need to demonstrate protection in relevant infection models.

Potential Challenges:

• Limited surface exposure of membrane-embedded regions

• Possible cross-reactivity with commensal staphylococci

• Variable expression levels under different conditions

A comprehensive evaluation would include immunization studies with different protein domains, assessment of antibody functionality (opsonization, neutralization), and protection studies in relevant animal models of S. epidermidis infection.

How can structural information about UPF0382 inform the development of novel antimicrobial compounds?

Structural information about UPF0382 can guide antimicrobial development through several strategic approaches:

Structure-Based Drug Design:
Detailed structural information, potentially generated through methods like those used by Mem-LZerD , could reveal binding pockets suitable for small molecule targeting. These pockets might be identified at:

• Protein-protein interaction interfaces

• Channel or pore regions if UPF0382 functions in transport

• Sites critical for conformational changes

Functional Inhibition Strategies:
If UPF0382 plays a critical role in S. epidermidis virulence or survival, compounds that disrupt its function could be developed based on:

• Competitive inhibitors that mimic natural ligands

• Allosteric modulators that prevent conformational changes

• Covalent modifiers that irreversibly bind to critical residues

Peptide-Based Approaches:
Based on UPF0382's sequence (MKVFIILGALNAMMAVGTGAFGAHGLEDKLSDKYMSIWEKATTYQMYHGLGLLVIGLISGTTSINVNWAGWLLFFGIVFFSGSLYFLALTQVRILGAITPIGGVLFIIGWLVLVIATLKFAG) , researchers could design:

• Antimicrobial peptides that disrupt membrane integrity

• Peptidomimetics that interfere with protein-protein interactions

• Peptide conjugates for targeted delivery of antimicrobial agents

Rational Design Pipeline:

• Generate structural models using computational methods

• Identify druggable sites through virtual screening

• Design compound libraries targeting these sites

• Test compounds for inhibition of UPF0382 function

• Optimize lead compounds for antimicrobial efficacy

This approach parallels strategies used for other bacterial membrane proteins and could lead to novel antimicrobials that specifically target S. epidermidis through interference with UPF0382 function.