Recombinant Enterococcus faecalis UPF0291 protein EF_1580 (EF_1580)

What is the genomic context of the EF_1580 gene in Enterococcus faecalis?

EF_1580 is classified as a hypothetical protein belonging to the UPF0291 protein family. Transcriptomic analyses have identified EF_1580 as one of the down-regulated genes during mammalian infection, suggesting a potential role in bacterial adaptation to changing environments . The gene appears in antisense transcript lists from RIVET (Recombination-based In Vivo Expression Technology) screens, indicating potential regulation by antisense RNA mechanisms . The genomic neighborhood analysis suggests potential co-regulation with genes involved in stress response pathways, although this requires further experimental validation.

What expression patterns does EF_1580 show under different growth conditions?

Transcriptomic data indicates that EF_1580 expression is significantly down-regulated during growth in mammalian host environments, particularly at 8 hours post-infection . This down-regulation pattern aligns with other genes that are regulated in response to changing nutrient availability, suggesting EF_1580 may be part of the adaptive response to host environments. Comprehensive expression profiling under various conditions (pH variations, nutrient limitations, temperature shifts, and antibiotic exposures) would provide valuable insights into the regulatory network controlling EF_1580 expression.

How is EF_1580 conserved across different enterococcal species and strains?

While specific conservation data for EF_1580 is not directly provided in the search results, the approach to answering this question would involve:

• Performing multiple sequence alignments of EF_1580 homologs across various enterococcal species and strains

• Calculating sequence identity and similarity percentages

• Identifying conserved domains and motifs

• Constructing phylogenetic trees to visualize evolutionary relationships

The UPF0291 protein family is found across multiple bacterial species, suggesting potential functional importance. Conservation analysis would help establish whether EF_1580 represents a core gene within the enterococcal genome or shows strain-specific variations that might correlate with pathogenicity or niche adaptation.

What are the optimal methods for recombinant expression of EF_1580?

The recombinant expression of EF_1580 requires careful consideration of several factors:

Expression System Selection:

• E. coli BL21(DE3): Suitable for initial expression attempts due to well-established protocols

• E. coli Rosetta: Preferred if EF_1580 contains rare codons

• Homologous expression in E. faecalis: Valuable for maintaining native post-translational modifications

Vector Design Considerations:

• Inclusion of appropriate affinity tags (His, GST, MBP) for purification

• Incorporation of protease cleavage sites for tag removal

• Codon optimization based on the expression host

Expression Conditions Optimization:

• Testing multiple induction temperatures (16°C, 25°C, 37°C)

• Varying IPTG concentrations (0.1-1.0 mM)

• Evaluating different media formulations (LB, TB, auto-induction)

Solubility Enhancement Strategies:

• Co-expression with molecular chaperones

• Fusion with solubility-enhancing tags

• Addition of osmolytes or mild detergents to lysis buffers

Assessment of expression levels and solubility through SDS-PAGE and Western blotting would guide optimization of these parameters for maximum yield of functional protein.

What purification strategies are most effective for obtaining high-purity EF_1580 for structural studies?

A multi-step purification approach is recommended:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Glutathione affinity chromatography for GST-fusion proteins

• Intermediate Purification:

  • Ion exchange chromatography based on theoretical pI of EF_1580

  • Tag removal using specific proteases (TEV, PreScission, thrombin)

• Polishing Steps:

  • Size exclusion chromatography to separate monomeric from aggregated forms

  • Removal of endotoxins if protein is intended for immunological studies

• Quality Control Assessment:

  • SDS-PAGE and Western blotting for purity evaluation

  • Dynamic light scattering for aggregation analysis

  • Mass spectrometry for identity confirmation

  • Thermal shift assays for stability assessment

Optimizing buffer conditions (pH, salt concentration, additives) at each purification stage is crucial for maintaining protein stability and preventing aggregation.

What structural characterization techniques are most informative for EF_1580?

Multiple complementary approaches should be employed:

For UPF0291 family proteins like EF_1580, integrating computational modeling with experimental data can provide valuable structural insights, especially when high-resolution experimental structures prove challenging to obtain.

How might EF_1580 contribute to E. faecalis virulence and pathogenesis?

The transcriptomic data showing down-regulation of EF_1580 during infection suggests several hypotheses regarding its role in virulence :

• Metabolic Adaptation Hypothesis: EF_1580 may function in metabolic pathways active during environmental growth but down-regulated during host adaptation.

• Immune Evasion Strategy: Down-regulation could represent a mechanism to alter surface properties or antigenic profiles during infection, potentially evading host immune recognition.

• Stress Response Coordination: EF_1580 might participate in stress response pathways that are differentially regulated during infection.

• Regulatory Role in Virulence Gene Expression: As a potential antisense regulatory target, EF_1580 could influence expression of virulence factors through RNA-based mechanisms.

Experimental approaches to investigate these hypotheses include:

• Generation of EF_1580 deletion and overexpression mutants

• Virulence assessment in infection models, including biofilm formation capacity

• Transcriptomic and proteomic comparison of wildtype and mutant strains

• Identification of interaction partners through pull-down assays and interactome studies

The observation that EF_1580 appears in antisense transcript lists suggests potential involvement in regulatory networks controlling virulence gene expression .

What role might EF_1580 play in biofilm formation and antibiotic resistance?

Given that E. faecalis is known for its ability to form biofilms and demonstrate antibiotic resistance , EF_1580's potential role in these processes merits investigation:

• Biofilm Context: E. faecalis isolates from clinical and oral sources demonstrate enhanced biofilm formation capabilities compared to food isolates . If EF_1580 is involved in biofilm formation, its expression might correlate with biofilm development stages.

• Experimental Approach for Biofilm Studies:

  • Quantitative biofilm assays comparing wildtype and EF_1580 mutants

  • Confocal microscopy analysis of biofilm architecture

  • Transcriptomic analysis during biofilm development

  • Assessment of cell surface properties and extracellular matrix composition

• Antibiotic Resistance Connection:

  • Minimum inhibitory concentration (MIC) determination for various antibiotics

  • Stress response profiling in presence of sub-inhibitory antibiotic concentrations

  • Assessment of persister cell formation in EF_1580 mutants

The observation that clinical and plaque/saliva isolates show similar antibiotic resistance patterns suggests shared adaptative mechanisms that might involve proteins like EF_1580.

How does the antisense regulation of EF_1580 impact E. faecalis adaptation during infection?

The RIVET antisense screen identified EF_1580 as potentially regulated by antisense transcripts during infection , suggesting a complex regulatory mechanism:

• Antisense Regulation Mechanisms:

  • Transcriptional interference through RNA polymerase collision

  • Translational inhibition by blocking ribosome binding

  • Double-stranded RNA formation triggering RNase III degradation

  • Alteration of mRNA secondary structure affecting stability

• Experimental Validation Approaches:

  • Northern blot analysis to confirm antisense transcript expression

  • RT-qPCR to quantify sense and antisense transcript levels

  • RNA-seq with strand-specific library preparation

  • Reporter gene assays to assess regulatory effects

• Functional Consequences:

  • Temporal expression profiling during infection progression

  • Correlation with stress response and virulence gene expression

  • Identification of conditions triggering antisense regulation

Since approximately 9.3% of down-regulated genes at eight hours post-infection corresponded with antisense transcripts identified in the RIVET screen , this represents a significant regulatory mechanism potentially affecting E. faecalis adaptation to the host environment.

How can transcriptomic data be leveraged to understand EF_1580 regulation and function?

The available transcriptomic data provides several analytical approaches for understanding EF_1580:

• Co-expression Network Analysis:

  • Identify genes with expression patterns similar to EF_1580

  • Construct co-expression networks to predict functional associations

  • Apply gene set enrichment analysis to identify biological processes

• Comparative Transcriptomics:

  • Compare EF_1580 expression across different infection models

  • Analyze expression in various stress conditions

  • Examine regulatory patterns in different E. faecalis strains

• Regulatory Element Identification:

  • Analyze promoter regions for transcription factor binding sites

  • Search for small RNA interaction sites

  • Identify potential antisense transcription start sites

• Integration with Other Omics Data:

  • Correlate transcriptomic changes with proteomic profiles

  • Combine with metabolomic data to connect to metabolic pathways

  • Integrate with ChIP-seq data to identify regulatory interactions

The observation that EF_1580 was identified in both microarray and RIVET studies underscores its significance in E. faecalis gene regulation during infection.

What bioinformatic approaches are most useful for predicting EF_1580 function?

Multiple computational strategies can provide functional insights:

For hypothetical proteins like EF_1580, combining structural predictions with evolutionary analysis often provides the most reliable functional hypotheses.

How should researchers address conflicting data regarding EF_1580 function?

When faced with contradictory results:

• Methodological Reconciliation:

  • Carefully examine experimental conditions across studies

  • Consider strain differences and genetic backgrounds

  • Evaluate methodology variations and their potential impact

  • Assess statistical approaches and significance thresholds

• Biological Explanation Exploration:

  • Consider context-dependent functions

  • Evaluate potential pleiotropic effects

  • Explore post-translational modifications

  • Examine protein interaction networks in different conditions

• Validation Strategies:

  • Perform independent replication studies

  • Use complementary methodological approaches

  • Develop in vitro systems that recapitulate in vivo conditions

  • Employ genetic approaches (knockouts, complementation, point mutations)

• Integration Framework:

  • Develop testable models that accommodate apparently conflicting results

  • Design critical experiments to distinguish between alternative hypotheses

  • Consider mathematical modeling to integrate diverse datasets

The observation that different experimental approaches (microarray vs. RIVET) identified distinct but complementary sets of genes highlights the importance of methodological diversity in building a comprehensive understanding of bacterial gene function.

What are promising approaches for defining EF_1580's role in E. faecalis pathogenesis?

Several strategic research directions could advance understanding of EF_1580:

• Genetic Manipulation Strategies:

  • CRISPR-Cas9 genome editing for precise genetic modifications

  • Conditional expression systems to study essential functions

  • Single-cell analysis to examine population heterogeneity

• In Vivo Infection Models:

  • Comparison across multiple infection models (abscess, endocarditis, UTI)

  • Host-specific adaptation patterns

  • In vivo competition assays between wildtype and mutant strains

• Host-Pathogen Interaction Studies:

  • Identification of host cell targets or receptors

  • Immune response profiling

  • Visualization of EF_1580 localization during infection

• Multi-Species Interactions:

  • Role in polymicrobial infections

  • Contribution to competitive fitness against other microbes

  • Function in mixed biofilm communities

The observation that E. faecalis can persist in specific host niches like kidneys while also being found in diverse environments including food and oral sites suggests adaptative mechanisms potentially involving proteins like EF_1580.

How can advanced structural biology techniques enhance understanding of EF_1580?

Cutting-edge structural approaches offer new opportunities:

• Integrative Structural Biology:

  • Combining multiple data sources (X-ray, NMR, cryo-EM, SAXS)

  • Hybrid modeling approaches

  • In-cell structural determination

• Dynamic Structural Studies:

  • Time-resolved crystallography

  • Single-molecule FRET

  • Nuclear magnetic resonance relaxation dispersion

  • Hydrogen-deuterium exchange mass spectrometry

• Structure-Guided Functional Analysis:

  • Alanine scanning mutagenesis of predicted functional sites

  • Chimeric protein construction

  • Structure-based inhibitor design

  • Computational simulations of conformational changes

• Protein Interaction Mapping:

  • Structural characterization of protein complexes

  • Identification of interaction interfaces

  • Allosteric regulation mechanisms

Advanced structural information would complement the transcriptomic data by providing molecular-level insights into how EF_1580 functions within the cell.

What potential does EF_1580 have as a therapeutic target or biomarker?

Evaluating EF_1580's potential clinical applications requires:

• Target Validation Studies:

  • Essentiality assessment in different infection models

  • Virulence contribution quantification

  • Conservation analysis across clinical isolates

  • Absence of human homologs

• Drug Discovery Approaches:

  • High-throughput screening for inhibitors

  • Fragment-based drug design

  • Structure-based virtual screening

  • Peptide inhibitor development

• Biomarker Development:

  • Detection of EF_1580 or antibodies against it in clinical samples

  • Correlation with infection severity or antibiotic resistance

  • Development of rapid diagnostic tests

• Resistance Development Assessment:

  • Frequency of resistance emergence

  • Fitness cost of resistance mutations

  • Alternative pathways that might circumvent inhibition

Given E. faecalis's significant role in nosocomial infections and its concerning antibiotic resistance profiles , novel therapeutic targets like EF_1580 merit thorough investigation, especially if they contribute to virulence or antibiotic resistance mechanisms.