Recombinant Staphylococcus aureus UPF0754 membrane protein SA1664 (SA1664)

What is the molecular structure of Staphylococcus aureus UPF0754 membrane protein SA1664?

Staphylococcus aureus UPF0754 membrane protein SA1664 is a full-length protein consisting of 374 amino acids. The protein sequence includes characteristic membrane-spanning domains with hydrophobic regions consistent with its function as a membrane protein. The complete amino acid sequence is:

MNALFIIIFMIVVGAIIGGITNVIAIRLFHPFKPYYIFKFRVPFTPGLIPKRREEIATK IGQVIEEHLLTETLINEKLKSEQSQQAIESMIQQQLQKLTKDQLSIKQITSQIDIDLEQV LQTNGNQYIESQLNNYYTKHQNQTIASLLPNQLVTFLDQHVDNATDLLCDRARNYLSSAK GTQDINDMLDTFFHEKGKLIGMLQMFMTKESIADRIQELIRLTSHHPKARTIVTSLLITNE YQTFKDKPLNELLDASQFNEIAENLSVYVTTYASNQANKPVVTLMPQFVDYLEGQLSSKL ANLIIEKLSIHLSTIMKKVDLRGLIEEQINTFDLDYIEKLIIEIANKELKLIMSLGFILG GIIGFFQGLVAIFV

The protein is encoded by the SA1664 gene in the Staphylococcus aureus strain N315 genome and has a Uniprot accession number of Q7A4V2 .

What are the optimal conditions for expressing recombinant SA1664 protein in bacterial systems?

The optimal expression of recombinant SA1664 requires careful consideration of expression systems and conditions:

Expression System Selection:

• E. coli systems (BL21(DE3), Rosetta, or C41/C43 strains) are commonly used for membrane protein expression

• For improved folding of membrane proteins, consider specialized strains with enhanced membrane protein expression capabilities

Expression Protocol:

• Transform expression plasmid containing the SA1664 gene into the chosen host

• Culture in LB or 2XYT medium at 37°C until OD600 reaches 0.6-0.8

• Induce expression with IPTG (0.1-0.5 mM)

• Reduce temperature to 16-25°C post-induction for membrane protein expression

• Continue expression for 16-20 hours

• Harvest cells by centrifugation at 4,000-6,000 × g for 15 minutes at 4°C

Key Optimization Parameters:

• Temperature: Lower temperatures (16-25°C) often improve membrane protein folding

• Inducer concentration: Titrate IPTG concentration between 0.1-1.0 mM

• Media composition: Consider enriched media or supplementation with glycerol for membrane protein expression

• Induction time: Test expression at different OD600 values (0.4-1.0)

• Duration: Optimize between 4-24 hours post-induction

For challenging membrane proteins like SA1664, consider fusion tags (such as MBP, SUMO, or TrxA) that may improve solubility and folding during expression.

What purification methods are most effective for isolating recombinant SA1664 with high purity and yield?

Purifying membrane proteins like SA1664 requires specialized approaches:

Membrane Protein Purification Workflow:

• Cell Lysis and Membrane Fraction Isolation:

  • Resuspend cell pellet in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, protease inhibitors)

  • Disrupt cells via sonication, French press, or microfluidizer

  • Remove cell debris by centrifugation at 10,000 × g for 20 minutes at 4°C

  • Isolate membrane fraction by ultracentrifugation at 100,000 × g for 1 hour at 4°C

• Membrane Protein Solubilization:

  • Resuspend membrane pellet in solubilization buffer containing detergent

  • Common detergents: n-Dodecyl β-D-maltoside (DDM), n-Octyl β-D-glucopyranoside (OG), or LDAO

  • Incubate with gentle rotation for 1-2 hours at 4°C

  • Remove insoluble material by ultracentrifugation at 100,000 × g for 30 minutes

• Affinity Chromatography:

  • If SA1664 has been expressed with a tag (His, GST, etc.), use corresponding affinity resin

  • Load solubilized protein onto equilibrated column

  • Wash extensively to remove non-specifically bound proteins

  • Elute with appropriate buffer (imidazole for His-tagged proteins)

• Size Exclusion Chromatography:

  • Further purify by gel filtration to separate aggregates and obtain homogeneous protein

  • Use buffer containing detergent at concentrations above critical micelle concentration

• Quality Assessment:

  • Analyze purity by SDS-PAGE

  • Verify identity by Western blotting or mass spectrometry

  • Assess homogeneity by dynamic light scattering or analytical ultracentrifugation

The storage buffer for purified SA1664 should contain Tris-based buffer with 50% glycerol optimized for stability . For extended storage, conserve at -20°C or -80°C, avoiding repeated freeze-thaw cycles.

What approaches can be used to investigate SA1664's potential role in S. aureus pathogenesis?

To investigate SA1664's potential role in S. aureus pathogenesis, researchers can employ the following comprehensive approaches:

In Vitro Infection Models:

• Human cell line infection models (e.g., epithelial cells, macrophages, neutrophils)

• Measurement of bacterial adherence, invasion, and intracellular survival

• Assessment of host cell cytokine production and inflammatory responses

• Evaluation of host cell cytotoxicity through LDH release assays

Animal Infection Models:

• Murine models of bacteremia, pneumonia, or skin infection

• Comparison of wild-type and SA1664 knockout strains for:

  • Bacterial burden in tissues

  • Histopathological changes

  • Mortality rates

  • Inflammatory responses

Immune Response Analysis:

• Examine antibody responses to SA1664 in patients with invasive S. aureus infections compared to healthy colonized individuals

• Analyze cytokine profiles in response to purified SA1664 protein

• Investigate neutrophil activation and recruitment in response to SA1664

Molecular Interaction Studies:

• Identify potential host receptors that interact with SA1664 using techniques such as:

  • Affinity purification followed by mass spectrometry

  • Surface plasmon resonance

  • Yeast two-hybrid screening

• Confirm interactions through co-immunoprecipitation and functional assays

Gene Expression Analysis:

• Examine regulation of SA1664 expression under different infection-relevant conditions

• Perform RNA-seq to identify genes co-regulated with SA1664

• Investigate potential regulatory elements controlling SA1664 expression

These methodological approaches would contribute to understanding whether SA1664 plays a significant role in S. aureus virulence, similar to other surface proteins that have been implicated in the pathogenesis of staphylococcal infections .

How does SA1664 compare to other membrane proteins in S. aureus and related species?

Comparative analysis of SA1664 with other membrane proteins provides valuable insights into its evolutionary conservation and potential function:

Sequence Homology Analysis:

Functional Domain Comparison:

• SA1664 contains characteristic membrane-spanning domains that share topological features with other bacterial transmembrane proteins

• The protein lacks known functional domains such as ATP-binding cassettes or recognizable transport motifs

• Potential conservation of protein-protein interaction motifs in the cytoplasmic domain

Evolutionary Conservation:

• High conservation among S. aureus clinical isolates suggests functional importance

• Moderate conservation in coagulase-negative staphylococci indicates shared ancestral functions

• Lower similarity to proteins in distant bacterial species suggests specialized roles in Staphylococcus

This comparative analysis approach helps researchers understand SA1664's evolutionary context and make informed hypotheses about its function based on better-characterized homologs in related species.

How can SA1664 be integrated into systems biology studies of S. aureus pathogenesis?

Integrating SA1664 into systems biology frameworks provides comprehensive insights into its role within the broader context of S. aureus pathogenesis:

Multi-Omics Integration Approach:

• Transcriptomics:

  • Analyze SA1664 expression patterns across different growth conditions

  • Identify co-expressed genes using RNA-seq data

  • Determine if SA1664 is part of specific regulons (e.g., ArlRS-MgrA regulatory cascade that controls other surface proteins)

• Proteomics:

  • Perform quantitative membrane proteomics to assess SA1664 expression levels

  • Use protein-protein interaction studies (Co-IP, BioID) to identify SA1664 interaction partners

  • Map post-translational modifications that might regulate SA1664 function

• Metabolomics:

  • Investigate metabolic changes in SA1664 knockout strains

  • Determine if SA1664 influences specific metabolic pathways

• Network Analysis:

  • Construct protein interaction networks including SA1664

  • Perform gene regulatory network analysis

  • Use pathway enrichment to identify biological processes associated with SA1664

• Computational Modeling:

  • Develop predictive models of SA1664 function based on integrated data

  • Use machine learning approaches to identify patterns in multi-omics data

  • Create visual network representations of SA1664's role in S. aureus biology

Integration with Host Response Data:

• Correlate SA1664 expression with host cytokine profiles observed in patient samples

• Analyze antibody responses to SA1664 in relation to other immunogenic S. aureus proteins

• Examine how host factors influence SA1664 expression and function

Such integrated approaches would place SA1664 within the complex network of factors contributing to S. aureus pathogenesis, similar to studies that have characterized immune and inflammatory responses to invasive S. aureus disease .

What are common challenges in working with recombinant SA1664 and how can they be addressed?

Membrane proteins like SA1664 present specific technical challenges that researchers should anticipate and address:

Expression Challenges:

Purification Challenges:

Functional Assay Challenges:

Addressing these challenges requires systematic optimization and may benefit from collaborations with laboratories specialized in membrane protein biochemistry.

How can researchers interpret conflicting data about SA1664's role in S. aureus pathogenicity?

When facing conflicting data regarding SA1664's role in S. aureus pathogenicity, researchers should employ the following systematic approach to interpretation:

Sources of Conflicting Data:

• Strain variations in S. aureus (as shown with other surface proteins like SasG)

• Differences in experimental models and conditions

• Variable host factors influencing protein expression and function

• Technical limitations in detection and analysis methods

Methodological Approach to Resolving Conflicts:

• Standardization of Experimental Systems:

  • Use multiple, well-characterized S. aureus strains

  • Employ both laboratory and clinical isolates

  • Standardize growth conditions and experimental protocols

  • Include appropriate positive and negative controls

• Comprehensive Phenotypic Analysis:

  • Perform multiple, complementary assays to assess SA1664 function

  • Evaluate phenotypes in various infection models

  • Consider different host cell types and environmental conditions

  • Assess temporal dynamics of SA1664 expression and function

• Genetic Verification:

  • Create clean gene deletions with minimal polar effects

  • Complement mutations with controlled expression systems

  • Verify phenotypes with multiple independent mutants

  • Use site-directed mutagenesis to identify critical residues

• Integration with Clinical Data:

  • Correlate laboratory findings with clinical observations

  • Analyze SA1664 sequence variations in clinical isolates

  • Assess antibody responses to SA1664 in patient cohorts

  • Examine SA1664 expression in samples from different infection types

• Critical Evaluation of Data Quality:

  • Assess statistical power and reproducibility

  • Consider limitations of each experimental approach

  • Evaluate potential technical artifacts

  • Perform independent validation in different laboratories

S. aureus pathogenesis involves complex interactions between multiple bacterial factors and host responses. Studies have shown that even well-characterized virulence factors may exhibit different roles depending on the infection context . Therefore, conflicting data about SA1664 should be interpreted within this broader understanding of S. aureus biology.