Recombinant Staphylococcus haemolyticus UPF0060 membrane protein SH0717 (SH0717)

What is the basic structure and localization of the SH0717 protein in Staphylococcus haemolyticus?

SH0717 is a UPF0060 class membrane protein found in Staphylococcus haemolyticus strain JCSC1435. The full-length protein consists of 108 amino acids with the sequence: mLYSIFIFLLAGLCEIGGGYLIWLWLREGQSSWLGFIGGVILMMYGVIATFQSFPTFGRVYAAYGGVFIVMSLIWAYIVDKQAPDKYDLIGACICIIGVCVMILPSRT. Structural analysis suggests it contains multiple transmembrane domains with hydrophobic regions typical of integral membrane proteins. The protein is encoded by the SH0717 gene in the S. haemolyticus genome and has been assigned the UniProt identifier Q4L8J9 .

To investigate membrane localization, researchers typically employ fractionation techniques to separate cellular components followed by Western blotting with specific antibodies against SH0717. Fluorescence microscopy using tagged variants of the protein can also provide visual confirmation of membrane association in living cells.

How does the UPF0060 protein family classification inform our understanding of SH0717's potential functions?

The UPF0060 designation (Uncharacterized Protein Family 0060) indicates that this protein belongs to a group of functionally uncharacterized membrane proteins conserved across various bacterial species. While the specific function remains undetermined, comparative analysis with other UPF0060 family members suggests potential roles in membrane integrity, transport processes, or signal transduction.

Methodologically, researchers can employ evolutionary trace analysis and structural comparison with better-characterized UPF0060 family members to identify conserved domains that may indicate functional significance. Computational approaches like protein-protein interaction prediction can guide experimental design to elucidate function through targeted mutagenesis of conserved residues.

What are the optimal conditions for expressing and purifying recombinant SH0717 protein for structural studies?

For structural studies of SH0717, expression in E. coli systems using specialized vectors designed for membrane proteins is typically recommended. The following protocol has shown efficacy:

• Clone the SH0717 gene into an expression vector containing a strong inducible promoter (e.g., pET series) with an appropriate fusion tag (His6 or MBP tags often improve solubility).

• Transform into E. coli strains optimized for membrane protein expression (C41(DE3) or C43(DE3)).

• Grow cultures at lower temperatures (16-20°C) after induction to reduce inclusion body formation.

• Extract membrane fractions using detergent solubilization (commonly n-dodecyl-β-D-maltoside or DDM at 1-2%).

• Purify using affinity chromatography followed by size exclusion chromatography.

For structural preservation during purification, researchers should avoid repeated freeze-thaw cycles, maintaining purified protein at 4°C for short-term storage or at -80°C (with 50% glycerol) for extended storage . The addition of specific lipids during purification may help maintain native protein conformation.

What methods are most effective for studying SH0717 protein interactions with other bacterial proteins and host factors?

To investigate SH0717 protein interactions, researchers should consider a multi-faceted approach:

• Co-immunoprecipitation (Co-IP): Using antibodies against SH0717 to pull down potential protein partners from S. haemolyticus lysates, followed by mass spectrometry identification.

• Bacterial two-hybrid systems: Modified for membrane proteins to detect potential interactions in vivo.

• Surface plasmon resonance (SPR): For quantifying binding kinetics between purified SH0717 and candidate interacting proteins.

• Cross-linking studies: Using membrane-permeable cross-linking agents followed by mass spectrometry to identify proximal proteins in their native environment.

• Fluorescence resonance energy transfer (FRET): For studying interactions in living cells when using fluorescently tagged proteins.

For host-pathogen interaction studies, researchers can employ infection models with human cell lines expressing fluorescently tagged cellular components, followed by co-localization analysis with labeled SH0717 protein.

How is the SH0717 gene positioned within the Staphylococcus haemolyticus genome, and what can we learn from comparative genomics?

The SH0717 gene is identified in the Staphylococcus haemolyticus JCSC1435 genome. Comparative genomic analysis reveals that the gene is located outside the "oriC environ" region, which typically contains species-specific genes contributing to the unique biological features of staphylococcal species . This positioning suggests the protein may serve a conserved function across staphylococcal species.

Methodologically, researchers can perform synteny analysis of the genomic region containing SH0717 across multiple staphylococcal species to identify conserved gene neighborhoods that might indicate functional relationships or operonic organization. Whole-genome sequencing of multiple S. haemolyticus clinical isolates can reveal polymorphisms in the SH0717 gene that might correlate with phenotypic variations or pathogenicity.

What techniques are recommended for analyzing the expression regulation of SH0717 under different environmental conditions?

To analyze SH0717 expression regulation, researchers should implement:

• Quantitative RT-PCR: For precise measurement of SH0717 mRNA levels under different conditions (varying pH, temperature, antibiotic exposure, host cell contact).

• Promoter-reporter fusion assays: Constructing fusions between the SH0717 promoter region and reporter genes (like GFP or luciferase) to monitor promoter activity in real-time.

• RNA-Seq: For transcriptome-wide analysis to identify co-regulated genes and potential regulatory networks involving SH0717.

• ChIP-Seq: To identify transcription factors binding to the SH0717 promoter region.

• CRISPR interference (CRISPRi): For targeted repression of regulatory elements to assess their impact on SH0717 expression.

Environmental conditions to test should include those relevant to clinical scenarios, such as biofilm formation conditions, antibiotic exposure, and host cell proximity, as S. haemolyticus is known to adapt to hospital environments through genomic plasticity and frequent rearrangements .

What is the potential role of SH0717 in Staphylococcus haemolyticus pathogenicity and biofilm formation?

While the specific function of SH0717 remains uncharacterized, membrane proteins in pathogenic bacteria often contribute to virulence through roles in adhesion, invasion, or immune evasion. S. haemolyticus is known to form biofilms involved in catheter-associated infections and secretes factors for bacterial adherence including enterotoxins, hemolysins, and fibronectin-binding proteins .

To investigate SH0717's potential role in pathogenicity, researchers should:

• Generate SH0717 knockout strains using homologous recombination or CRISPR-Cas9 editing.

• Compare wild-type and knockout strains for:

  • Biofilm formation capacity using crystal violet staining and confocal microscopy

  • Adherence to relevant cell lines and medical device materials

  • Survival in human serum and response to antimicrobial peptides

  • Virulence in appropriate infection models

Complementation studies with the wild-type SH0717 gene would confirm phenotypic changes are specifically due to the absence of this protein.

How does the expression of SH0717 correlate with antibiotic resistance patterns in clinical isolates?

S. haemolyticus is notorious for its multidrug resistance and early acquisition of resistance to methicillin and glycopeptide antibiotics . To investigate potential correlations between SH0717 expression and antibiotic resistance:

• Collect diverse clinical isolates of S. haemolyticus with varying antibiotic resistance profiles.

• Quantify SH0717 expression levels using qRT-PCR or Western blotting.

• Determine minimum inhibitory concentrations (MICs) for relevant antibiotics.

• Perform statistical analysis to identify correlations between expression levels and resistance patterns.

• For mechanistic insights, assess whether upregulation or downregulation of SH0717 (using inducible expression systems) affects antibiotic susceptibility.

This approach could reveal whether SH0717 contributes to the remarkable antibiotic resistance of S. haemolyticus, potentially through membrane permeability changes or interactions with other resistance mechanisms.

What advanced biophysical techniques are recommended for elucidating the membrane topology and structural dynamics of SH0717?

For advanced structural characterization of SH0717, researchers should consider:

• Cryo-electron microscopy: Particularly suitable for membrane proteins that resist crystallization, providing near-atomic resolution structures.

• NMR spectroscopy: Solution NMR with detergent-solubilized protein or solid-state NMR in lipid bilayers can provide detailed structural information and dynamics.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To identify solvent-accessible regions and conformational changes upon ligand binding.

• Single-particle analysis: For structural heterogeneity assessment.

• Molecular dynamics simulations: To model protein behavior in membrane environments and predict conformational changes.

For topology mapping, combining experimental approaches yields the most reliable results:

• Cysteine scanning mutagenesis with membrane-impermeable labeling reagents

• Protease protection assays

• Fluorescence quenching experiments with lipid-embedded quenchers

These methods together can generate a comprehensive topology model indicating transmembrane segments, extracellular loops, and cytoplasmic domains.

How can researchers investigate potential post-translational modifications of SH0717 and their functional significance?

Post-translational modifications (PTMs) can significantly impact protein function. To investigate PTMs in SH0717:

• Mass spectrometry-based proteomics: Using high-resolution LC-MS/MS to identify modifications. Sample preparation should preserve modifications (phosphorylation, glycosylation, lipidation).

• Site-directed mutagenesis: Mutating potential modification sites followed by functional assays to determine significance.

• PTM-specific antibodies: For Western blotting to detect specific modifications.

• Metabolic labeling: Using modified amino acids or sugars that can be incorporated into proteins and detected through click chemistry.

• In vitro modification assays: Exposing purified SH0717 to relevant bacterial modification enzymes to assess susceptibility.

For bacterial membrane proteins, lipidation (particularly at cysteine residues) and phosphorylation are common modifications that affect membrane association and signaling functions, respectively.

What is the potential of SH0717 as a target for novel antimicrobial strategies against multidrug-resistant S. haemolyticus?

S. haemolyticus poses a significant clinical challenge due to its high level of antibiotic resistance . If SH0717 proves essential for bacterial viability or virulence, it could represent a promising target for novel antimicrobials.

Research approaches to evaluate SH0717 as a therapeutic target include:

• Essentiality screening: Using conditional knockdown systems to determine if SH0717 is required for bacterial growth or survival under relevant conditions.

• High-throughput screening: Developing assays to identify small molecules that bind to SH0717 or interfere with its function.

• Structure-based drug design: If structural data becomes available, in silico screening and rational design of inhibitors targeting SH0717.

• Combination therapy evaluation: Testing whether SH0717 inhibitors sensitize resistant strains to conventional antibiotics.

• Bacteriocin-based approaches: Evaluating whether bacteriocins like the recently developed Hybrid 1 (H1) bacteriocin interact with or require SH0717 for their antimicrobial activity against S. haemolyticus.

How can researchers develop effective immunodetection methods for SH0717 in clinical and research applications?

For developing sensitive and specific immunodetection methods for SH0717:

• Epitope mapping and antibody development:

  • Identify immunogenic epitopes using computational prediction tools

  • Synthesize peptides corresponding to exposed regions of SH0717

  • Immunize animals (typically rabbits or mice) to generate polyclonal antibodies

  • Develop monoclonal antibodies for higher specificity

• Validation of antibody specificity:

  • Western blotting against recombinant SH0717

  • Testing against SH0717 knockout strains (negative control)

  • Cross-reactivity assessment with related staphylococcal proteins

• Assay development:

  • ELISA protocols optimized for sensitivity and specificity

  • Immunofluorescence protocols for localization studies

  • Flow cytometry methods for quantification in mixed populations

• Clinical application considerations:

  • Sample preparation methods for clinical specimens

  • Multiplexed detection platforms for simultaneous identification of multiple staphylococcal markers

  • Point-of-care adaptations for rapid diagnostics

These immunodetection tools would facilitate both basic research on SH0717 function and potential clinical applications in S. haemolyticus identification and characterization.