Recombinant Staphylococcus aureus UPF0382 membrane protein SAV0583 (SAV0583)

What is SAV0583 and how does it relate to other UPF0382 family proteins?

SAV0583 is a membrane protein belonging to the UPF0382 protein family in Staphylococcus aureus. It shares significant homology with similar proteins like MW0538 (from S. aureus MW2 strain), which consists of 122 amino acids forming a membrane-associated structure. The protein is characterized by its hydrophobic regions that facilitate membrane anchoring and potential scaffold functions within bacterial membrane microdomains . SAV0583 represents one of several strain-specific variants of UPF0382 family proteins in S. aureus, with approximately 84% sequence identity to homologous proteins in related bacterial species such as B. subtilis FloA .

What are the primary structural features of SAV0583?

Based on comparative analysis with similar UPF0382 family proteins, SAV0583 likely exhibits the following structural characteristics:

• A full protein sequence of approximately 122 amino acids

• Hydrophobic transmembrane domains that anchor the protein to the bacterial membrane

• Structural motifs consistent with scaffold protein functionality

• Potential oligomerization domains that facilitate protein-protein interactions

• Association with detergent-resistant membrane (DRM) fractions of S. aureus

The amino acid sequence would be similar to that of related proteins like MW0538, which has the sequence: MKLFIILGALNAMMAVGTGAFGAHGLQGKISDHYLSVWEKATTYQMYHGLALLIIGVISGTTSINVNWAGWLIFAGIIFFSGSLYILVLTQIKVLGAITPIGGVLFIIGWIMLIIATFKFAG .

How does the membrane localization of SAV0583 impact its function?

The membrane localization of SAV0583 is essential for its biological function as it allows the protein to serve as a scaffold within functional membrane microdomains (FMMs). This positioning enables SAV0583 to facilitate the spatial organization and oligomerization of various protein complexes involved in critical cellular processes . Similar to flotillin proteins, SAV0583 likely promotes the concentration of specific proteins within distinct membrane regions, enhancing their interaction efficiency and stabilizing multi-protein complexes. This compartmentalization within the membrane is particularly important for processes requiring coordinated protein assembly, such as RNA degradation machinery and virulence factor regulation .

What expression systems are most effective for recombinant SAV0583 production?

For optimal expression of recombinant SAV0583, E. coli-based expression systems have proven effective for similar membrane proteins. The recommended methodology includes:

• Cloning the SAV0583 gene into an expression vector with an N-terminal His-tag for purification

• Transforming the construct into E. coli expression strains optimized for membrane protein production (e.g., BL21(DE3), C41(DE3), or C43(DE3))

• Inducing protein expression under controlled conditions (temperature, inducer concentration)

• Harvesting cells and performing membrane fraction isolation

Expression levels should be monitored through SDS-PAGE and Western blot analysis using antibodies against the His-tag or the protein itself. Modifications to growth media, induction temperature (typically lowered to 16-25°C), and induction duration may be necessary to optimize the yield of properly folded protein .

What purification strategy yields the highest purity SAV0583 protein?

A multi-step purification protocol is recommended for obtaining high-purity SAV0583:

• Cell lysis using appropriate detergents that maintain membrane protein integrity

• Membrane fraction isolation through differential centrifugation

• Solubilization of membrane proteins using mild detergents (e.g., DDM, CHAPS)

• Immobilized metal affinity chromatography (IMAC) using the N-terminal His-tag

• Size exclusion chromatography to remove aggregates and further purify the protein

• Optional ion exchange chromatography for removal of remaining contaminants

This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE . For experimental applications requiring exceptionally high purity, additional chromatography steps may be implemented.

How should purified SAV0583 be stored to maintain stability?

Based on protocols for similar membrane proteins:

• Lyophilization in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 is recommended for long-term storage

• Store lyophilized protein at -20°C/-80°C

• For reconstituted protein, add 5-50% glycerol (final concentration) and aliquot in small volumes

• Store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

For reconstitution, centrifuge the vial briefly before opening and dissolve in deionized sterile water to a concentration of 0.1-1.0 mg/mL . The addition of glycerol (optimally 50%) helps prevent protein denaturation during freezing.

What is the hypothesized functional role of SAV0583 in S. aureus?

Based on studies of homologous proteins, SAV0583 likely functions as a scaffold protein within functional membrane microdomains (FMMs) of S. aureus. Its primary roles may include:

• Facilitating the oligomerization of membrane protein complexes

• Promoting the spatial organization of virulence-associated protein assemblies

• Stabilizing protein-protein interactions within the membrane environment

• Contributing to the compartmentalization of cell signaling and metabolic processes

• Potentially interacting with RNA degradation machinery (degradosome)

Similar to FloA, SAV0583 may physically interact with RNase components such as Rny, influencing RNA processing and consequently affecting virulence factor expression .

What experimental evidence links SAV0583 to S. aureus virulence?

While direct evidence for SAV0583 is limited in the provided search results, studies on homologous proteins suggest:

• Deletion of similar scaffold proteins results in altered virulence factor expression

• The protein likely stabilizes degradosome components that regulate sRNA transcripts

• These sRNA transcripts (such as rsaA and sau63) negatively regulate toxin expression

• Disruption of this regulation pathway affects the virulence potential of S. aureus

• Small molecules targeting similar proteins reduce S. aureus virulence in both in vitro and in vivo infection models

Research with mutant strains lacking these scaffold proteins has demonstrated reduced virulence in murine and invertebrate infection models, suggesting potential therapeutic applications targeting these membrane proteins .

How does SAV0583 interact with the RNA degradation machinery?

Based on studies of similar proteins, SAV0583 likely:

• Physically interacts with RNase Rny and helps concentrate this enzyme within specific membrane regions

• Stabilizes the degradosome complex through scaffold activity

• Influences the processing of specific RNA targets, including regulatory sRNAs

• Affects the half-life and concentration of sRNA transcripts (e.g., rsaA and sau63)

• Indirectly modulates virulence factor expression through these RNA regulatory mechanisms

Cell fractionation experiments with similar proteins have shown that RNase components preferentially concentrate in detergent-resistant membrane fractions where scaffold proteins like SAV0583 are located. Deletion of these scaffold proteins results in altered RNase distribution and function .

What are the critical controls for studying SAV0583 function in vitro?

When designing experiments to investigate SAV0583 function, the following controls are essential:

• Empty vector controls for expression studies

• Complementation experiments using wild-type SAV0583 to confirm phenotypes of deletion mutants

• Non-functional SAV0583 mutants (e.g., site-directed mutagenesis of key residues)

• Appropriate strain backgrounds (clinical isolates vs. laboratory strains)

• Time-series measurements to capture dynamic processes

• Multiple measurement techniques to confirm observations (e.g., combining microscopy, biochemical assays, and genetic approaches)

For quasi-experimental designs where complete control is not possible, time-series experiments and multiple measurement methods become particularly important to establish causality .

How should researchers design experiments to study membrane localization of SAV0583?

To effectively investigate the membrane localization of SAV0583:

• Generate chromosomally integrated tagged constructs (e.g., SAV0583-FLAG) expressed under native promoter control

• Perform cell fractionation followed by immunodetection to determine subcellular localization

• Use detergent-resistance membrane (DRM) isolation to assess microdomain association

• Compare distribution in wild-type vs. mutant backgrounds lacking other scaffold proteins

• Employ fluorescence microscopy with tagged proteins to visualize localization patterns

• Utilize co-immunoprecipitation to identify interacting partners within the membrane

It is critical to verify that any protein tags do not interfere with membrane localization or protein function through complementation studies with untagged proteins .

What experimental design is most appropriate for assessing SAV0583's role in virulence?

A comprehensive experimental design for investigating SAV0583's role in virulence should include:

• Generation of clean deletion mutants (ΔSAV0583) and complemented strains

• In vitro virulence factor production assays (toxins, enzymes)

• Transcriptomic and proteomic analysis of wild-type vs. mutant strains

• RNA stability and degradation assays focused on known virulence-associated transcripts

• Multiple in vivo infection models with varying complexity:

  • Invertebrate models (e.g., Galleria mellonella)

  • Murine infection models (systemic and localized infections)

• Time-series tracking of infection progression in each model

For robust causal inference, a multiple time-series design with appropriate controls is recommended, as it addresses many potential threats to validity in biological systems .

How can SAV0583 be targeted for antimicrobial development?

Given the potential role of SAV0583 in virulence regulation, several approaches for antimicrobial development could be explored:

• High-throughput screening for small molecules that inhibit SAV0583 oligomerization

• Structure-based drug design targeting key functional domains of SAV0583

• Peptide inhibitors designed to disrupt SAV0583 interactions with partner proteins

• Development of compounds that alter membrane microdomain organization

• Combination therapies targeting SAV0583 alongside conventional antibiotics

Small molecules identified through these approaches could effectively reduce S. aureus virulence without exerting direct bactericidal effects, potentially reducing selective pressure for resistance development . This anti-virulence approach is particularly promising for addressing infections caused by multidrug-resistant S. aureus strains, including MRSA, which have approximately 20% mortality rates in clinical settings .

What techniques can be used to investigate SAV0583's protein-protein interaction network?

Advanced methodologies for mapping SAV0583's interaction network include:

• Bacterial two-hybrid systems adapted for membrane proteins

• Pull-down assays using tagged SAV0583 followed by mass spectrometry

• Proximity labeling approaches (e.g., BioID, APEX) to identify neighboring proteins

• Förster resonance energy transfer (FRET) to study interactions in living cells

• Cross-linking mass spectrometry to capture transient interactions

• Quantitative proteomics comparing wild-type and ΔSAV0583 membrane compositions

These techniques should be used in combination to build a comprehensive interaction map, as each method has specific strengths and limitations for detecting different types of protein-protein interactions, particularly for membrane-associated proteins .

How do post-translational modifications affect SAV0583 function?

Investigation of post-translational modifications (PTMs) of SAV0583 requires:

• Mass spectrometry-based identification of PTMs (phosphorylation, acetylation, etc.)

• Site-directed mutagenesis of modified residues to assess functional importance

• Identification of enzymes responsible for adding or removing modifications

• Temporal analysis of modification patterns under various growth conditions

• Correlation of modification states with protein activity and localization

Understanding how PTMs regulate SAV0583 could reveal additional mechanisms for fine-tuning scaffold protein function and potentially identify new targets for therapeutic intervention. Changes in modification patterns during infection could be particularly informative regarding the protein's role in pathogenesis.