Recombinant Salmonella schwarzengrund Bifunctional protein aas (aas)

What is the structure and function of Salmonella schwarzengrund Bifunctional protein aas?

Salmonella schwarzengrund Bifunctional protein aas (aas) is a 719-amino acid protein that contains two functional domains. It is primarily characterized as a 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40) and also functions as an acyl-[acyl-carrier-protein]--phospholipid acyltransferase . The protein plays a crucial role in bacterial membrane phospholipid metabolism, specifically in the remodeling of membrane phospholipids. The bifunctional nature of this protein allows it to participate in multiple stages of phospholipid biosynthesis and modification, which is essential for maintaining membrane integrity and function in Salmonella species. The protein's secondary structure contains multiple domains that facilitate its enzymatic activities in phospholipid metabolism pathways .

How is Recombinant Salmonella schwarzengrund Bifunctional protein aas typically expressed and purified?

Recombinant Salmonella schwarzengrund Bifunctional protein aas is typically expressed in Escherichia coli expression systems, which provide an efficient platform for producing bacterial proteins. The full-length protein (amino acids 1-719) is commonly fused with an N-terminal histidine tag to facilitate purification . The expression is conducted under controlled conditions to optimize protein yield while maintaining proper folding and biological activity.

Following expression, the protein is purified using affinity chromatography, typically employing nickel or cobalt resins that bind the His-tag. After elution, the protein undergoes quality control measures including SDS-PAGE analysis to confirm purity levels (generally greater than 90%) . The purified protein is then formulated in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 and typically lyophilized for storage stability . This expression and purification methodology enables researchers to obtain high-quality protein suitable for various downstream applications in structural and functional studies.

What are the optimal storage conditions for Recombinant Salmonella schwarzengrund Bifunctional protein aas?

For optimal stability and activity preservation of Recombinant Salmonella schwarzengrund Bifunctional protein aas, specific storage conditions are recommended. The lyophilized protein should be stored at -20°C to -80°C upon receipt . For reconstituted protein, it's advisable to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and to aliquot the protein to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity and stability .

For short-term usage, working aliquots can be stored at 4°C for up to one week . The protein is typically reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL before use . When preparing for long-term storage, it's critical to ensure proper aliquoting in appropriate volumes to minimize the need for repeated freezing and thawing. These storage practices help maintain protein integrity and enzymatic activity for extended periods, which is essential for reproducible experimental results in research applications.

How can Recombinant Salmonella schwarzengrund Bifunctional protein aas be used in ELISA-based detection methods?

Recombinant Salmonella schwarzengrund Bifunctional protein aas can be effectively utilized in ELISA-based detection methods for both research and diagnostic applications. The protein can serve as a capture antigen when immobilized on ELISA plates, enabling the detection of anti-Salmonella antibodies in research samples . Alternatively, it can be used to generate specific antibodies that can subsequently be employed in sandwich ELISA formats for detecting Salmonella in various matrices.

For implementing such assays, researchers should consider the following methodological approach:

• Plate coating: Coat high-binding ELISA plates with optimized concentrations (typically 1-10 μg/ml) of purified recombinant aas protein in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block remaining binding sites with 1-5% BSA or casein solution to prevent non-specific binding.

• Sample addition: Add diluted test samples containing potential anti-Salmonella antibodies.

• Detection system: Utilize enzyme-conjugated secondary antibodies specific to the host species of the primary antibody.

• Visualization: Develop the assay using appropriate substrates and measure absorbance at relevant wavelengths.

This approach can be optimized for different research contexts, including surveillance studies and cross-reactivity analyses with other Salmonella species or enterobacteria . The high purity of commercially available recombinant aas protein (>90%) contributes to improved assay specificity and reduced background interference .

What are the comparative advantages of using aptamer-based detection versus antibody-based methods for Salmonella schwarzengrund research?

Aptamer-based detection systems for Salmonella offer several advantages for research applications, particularly when studying specific proteins like the bifunctional protein aas. Aptamers are single-stranded DNA or RNA molecules that can bind specifically to targets and can be generated through systematic evolution of ligands by exponential enrichment (SELEX) procedures .

For Salmonella detection, aptamers can be developed using whole-cell SELEX approaches that target surface-exposed proteins, potentially including exposed domains of the aas protein. This approach allows for higher specificity, especially when counter-selection steps against related bacteria are incorporated, as demonstrated in studies using S. Typhimurium as the target with S. Enteritidis, E. coli, and S. aureus as counter targets . The resulting aptamers can be applied in various biosensor formats for rapid detection, which is particularly valuable for time-sensitive research applications.

How does the expression profile of Bifunctional protein aas change during Salmonella infection models?

While specific expression data for Salmonella schwarzengrund Bifunctional protein aas during infection is limited in the provided search results, research on Salmonella Typhimurium provides valuable insights into how similar proteins behave during infection. Studies using dual RNA-seq approaches have revealed that many Salmonella proteins, including membrane-associated proteins like aas, undergo significant expression changes during host cell infection .

During epithelial cell infection, membrane remodeling enzymes like aas likely experience upregulation as the bacteria adapt to the intracellular environment. This is part of a coordinated virulence response that involves modifications to the bacterial membrane composition to resist host defense mechanisms. The dual functionality of aas in phospholipid metabolism would be particularly important during these adaptation processes.

In macrophage infection models, Transposon-directed insertion sequencing (TraDIS) studies have identified genes contributing to bacterial fitness inside host cells . While not specifically mentioned for aas in the provided references, similar bifunctional proteins involved in membrane metabolism are often critical for survival in the hostile macrophage environment, where bacteria must withstand oxidative stress and antimicrobial peptides.

These expression changes reflect the importance of membrane composition regulation during different stages of infection, highlighting the potential significance of bifunctional proteins like aas in Salmonella pathogenesis and adaptation to host environments .

What role might the Bifunctional protein aas play in Salmonella virulence and pathogenesis?

The Bifunctional protein aas likely plays significant roles in Salmonella virulence through multiple mechanisms related to membrane homeostasis and adaptation. As a bifunctional enzyme involved in phospholipid metabolism, the aas protein contributes to membrane remodeling during infection processes, which is crucial for adapting to host environments and resisting host defense mechanisms .

In research examining small proteins and their roles in Salmonella virulence, membrane-associated proteins have been shown to contribute to bacterial survival under stress conditions encountered during infection. The integration of data from dual RNA-seq, Transposon-directed insertion sequencing (TraDIS), and molecular interaction studies (Grad-seq) has revealed connections between membrane protein function and virulence phenotypes .

By analogy with other bifunctional membrane proteins such as MgrB in S. Typhimurium, which has demonstrated effects on virulence, motility, and transcriptome regulation under infection-relevant conditions, the aas protein may similarly influence Salmonella schwarzengrund's pathogenic capabilities . The protein's acyltransferase activity could be particularly important for maintaining membrane integrity when exposed to host antimicrobial factors or for modulating outer membrane composition to evade immune recognition.

Research targeting the functional characterization of aas through gene deletion studies, complementation assays, and infection models would be needed to fully elucidate its specific contributions to Salmonella schwarzengrund virulence and host-pathogen interactions.

How do post-translational modifications affect the function of Recombinant Salmonella schwarzengrund Bifunctional protein aas?

Post-translational modifications (PTMs) of Recombinant Salmonella schwarzengrund Bifunctional protein aas can significantly impact its structural integrity, enzymatic activity, and biological functions. Although specific PTM data for this particular protein is not directly provided in the search results, general principles of bacterial protein modifications can be applied to understand potential impacts.

Several types of PTMs that may affect the bifunctional protein aas include:

• Phosphorylation: May regulate the enzymatic activity of both functional domains, particularly in response to environmental signals during infection.

• Acetylation: Could affect protein stability and interaction with membrane components, altering the efficiency of phospholipid remodeling.

• Proteolytic processing: Might generate functional fragments with distinct activities or localizations within the bacterial cell.

• Lipidation: May influence protein localization within membrane microdomains, affecting substrate accessibility.

When working with recombinant versions of the protein, researchers should be aware that E. coli expression systems might not replicate all native PTMs present in Salmonella . This limitation could lead to functional differences between native and recombinant proteins. Mass spectrometry-based proteomics approaches would be valuable for mapping the PTM landscape of native aas protein and comparing it with recombinant versions to identify critical modifications that should be preserved or introduced in experimental systems.

Understanding these modifications could provide insights into regulatory mechanisms controlling aas activity during different stages of Salmonella infection and reveal potential targets for intervention strategies aimed at disrupting membrane homeostasis in pathogenic bacteria.

What structural and functional differences exist between Bifunctional protein aas in different Salmonella strains?

The Bifunctional protein aas exhibits both conservation and variation across different Salmonella strains, reflecting evolutionary adaptations to diverse ecological niches and host environments. While specific comparative data for Salmonella schwarzengrund aas versus other strains is limited in the provided search results, several important structural and functional differences can be inferred based on general principles of bacterial protein evolution.

Key areas of potential variation include:

• Sequence polymorphisms: While catalytic residues tend to be highly conserved, variations in non-catalytic regions may affect substrate specificity, protein-protein interactions, or regulatory properties.

• Domain organization: The relative position and orientation of the two functional domains may vary slightly between strains, potentially affecting their cooperative activity.

• Expression regulation: Promoter differences might lead to strain-specific expression patterns during infection or stress response.

• Subcellular localization: Variations in membrane-targeting sequences could result in different spatial distributions within the bacterial cell envelope.

Comparative genomic and structural biology approaches would be valuable for characterizing these differences. Techniques such as homology modeling based on the amino acid sequence provided for S. schwarzengrund aas compared with sequences from other Salmonella strains could reveal structural variations. Functional assays comparing enzymatic activities across recombinant proteins from different strains would complement these structural insights.

Understanding these differences could provide valuable insights into strain-specific adaptations and potentially explain variations in virulence or host specificity among Salmonella strains.

How can protein-protein interaction studies with Bifunctional protein aas reveal new aspects of Salmonella pathogenesis?

Protein-protein interaction (PPI) studies involving Bifunctional protein aas can uncover novel insights into Salmonella pathogenesis by revealing its functional networks and regulatory relationships. Advanced methodologies for studying these interactions can significantly enhance our understanding of how aas contributes to bacterial virulence and survival during infection.

Approaches for investigating aas protein interactions include:

• Affinity purification-mass spectrometry (AP-MS): Using His-tagged recombinant aas protein as bait to capture interacting proteins from Salmonella lysates, followed by identification via mass spectrometry.

• Bacterial two-hybrid assays: For detecting binary interactions between aas and candidate partners.

• Grad-seq analysis: This technique, as applied to Salmonella Typhimurium, separates macromolecular complexes based on their size and composition, enabling the identification of protein-protein and protein-RNA interactions . Similar analysis with S. schwarzengrund could reveal aas association with other macromolecules.

• Cross-linking mass spectrometry: For capturing transient or weak interactions under physiologically relevant conditions.

• Proximity-dependent biotin identification (BioID): For identifying proteins in close spatial proximity to aas in living bacteria.

These studies could reveal interactions with:

• Other membrane-remodeling enzymes in coordinated pathways

• Regulatory proteins controlling expression or activity

• Virulence factors dependent on membrane composition

• Components of stress response systems

Integration of PPI data with other 'omics' approaches, similar to that done for small proteins in S. Typhimurium , would provide a systems-level understanding of aas function within the context of Salmonella pathogenesis networks.

What are the key considerations for optimizing recombinant expression of Salmonella schwarzengrund Bifunctional protein aas?

Optimizing recombinant expression of Salmonella schwarzengrund Bifunctional protein aas requires careful consideration of multiple parameters to achieve high yield, proper folding, and preserved functionality. Based on established expression protocols, the following factors are crucial for successful production:

For membrane-associated proteins like aas, the addition of detergents or phospholipids during purification may be necessary to maintain proper folding and activity. The final formulation in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been established as effective for maintaining stability .

When assessing expression success, functional assays for both enzymatic activities should be performed alongside standard purity checks to ensure that the recombinant protein maintains its bifunctional capabilities and is suitable for downstream research applications.

What analytical methods are most effective for characterizing the dual functionality of Recombinant Salmonella schwarzengrund Bifunctional protein aas?

Characterizing the dual functionality of Recombinant Salmonella schwarzengrund Bifunctional protein aas requires a comprehensive analytical approach targeting both its 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40) activity and its acyl-[acyl-carrier-protein]--phospholipid acyltransferase function . The following analytical methods are particularly effective for assessing these distinct enzymatic activities:

For acyltransferase activity:

• Radiometric assays using 14C-labeled acyl donors to measure the transfer of acyl groups to phospholipid acceptors

• HPLC-based assays monitoring the disappearance of substrate and appearance of product

• Coupled enzyme assays that link product formation to a spectrophotometrically detectable reaction

For structural characterization:

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Limited proteolysis combined with mass spectrometry to identify domain boundaries

• Thermal shift assays to evaluate protein stability and ligand binding

• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

For substrate specificity profiling:

• High-throughput screening against libraries of potential acyl donors and phospholipid acceptors

• Kinetic analysis to determine Km and Vmax values for various substrates

• Competition assays to identify preferred substrates when multiple options are available

These analytical approaches should be performed under conditions that mimic the bacterial membrane environment, possibly using liposomes or nanodiscs to reconstitute the protein in a membrane-like context. Comparing wild-type activity with site-directed mutants targeting predicted catalytic residues would further validate functional assignments and deepen our understanding of this bifunctional enzyme's mechanisms.

How can researchers address challenges in antibody production against Recombinant Salmonella schwarzengrund Bifunctional protein aas?

Developing high-quality antibodies against Recombinant Salmonella schwarzengrund Bifunctional protein aas presents several challenges due to its membrane association, potential conformational complexity, and conservation with homologous proteins in other bacteria. Researchers can implement the following strategies to overcome these challenges:

• Antigen design optimization:

  • Use full-length His-tagged recombinant protein for antibodies targeting conformational epitopes

  • Design synthetic peptides from unique, surface-exposed regions for highly specific antibodies

  • Create domain-specific constructs to generate antibodies targeting either functional domain

• Host selection considerations:

  • Use phylogenetically distant hosts (e.g., rabbits, goats) to improve immunogenicity

  • Consider chickens for IgY production, which may respond better to bacterial proteins conserved in mammals

  • Implement customized immunization schedules with gradual antigen dose escalation

• Purification approaches:

  • Employ affinity purification using immobilized recombinant protein

  • Implement negative selection against homologous proteins from related bacteria

  • Use epitope-specific purification for antibodies raised against specific domains

• Validation protocols:

  • Test for cross-reactivity against related Salmonella proteins

  • Verify recognition of both native and denatured forms

  • Confirm antibody functionality in multiple applications (Western blot, immunoprecipitation, immunofluorescence)

• Alternative approaches:

  • Consider phage display technologies for selecting high-affinity antibodies

  • Explore camelid single-domain antibodies (nanobodies) for accessing epitopes in folded proteins

  • Develop aptamer alternatives when antibody approaches prove challenging

By implementing these strategies, researchers can develop specific antibodies that distinguish Salmonella schwarzengrund Bifunctional protein aas from homologous proteins in other bacterial species, enabling more precise studies of this protein's expression, localization, and function during infection processes.

What experimental designs are most appropriate for investigating the role of Bifunctional protein aas in Salmonella virulence?

Investigating the role of Bifunctional protein aas in Salmonella virulence requires multilevel experimental approaches that integrate genetic, biochemical, and infection-based methodologies. Drawing inspiration from studies on other Salmonella virulence factors, the following experimental designs would be particularly effective:

• Genetic manipulation strategies:

  • Gene deletion mutants (Δaas) created using lambda Red recombination system

  • Complementation strains expressing wild-type aas from controlled promoters

  • Domain-specific mutants to dissect the contribution of each functional domain

  • Conditional expression systems to study temporal requirements during infection

• In vitro phenotypic characterization:

  • Membrane lipid composition analysis by mass spectrometry

  • Stress resistance assays (antimicrobial peptides, pH, oxidative stress)

  • Motility assays on semi-solid agar

  • Biofilm formation capacity assessment

• Cell culture infection models:

  • Epithelial cell invasion assays

  • Macrophage survival studies

  • Dual RNA-seq to monitor host and bacterial transcriptional responses

  • Fluorescence microscopy to track subcellular localization during infection

• Animal infection models:

  • Competitive index assays comparing wild-type and Δaas mutants

  • Virulence assessment in multiple host species

  • Transposon-directed insertion sequencing (TraDIS) to identify genetic interactions

  • In vivo imaging to track infection progression

• Systems biology integration:

  • Proteomics to identify changes in protein expression patterns in Δaas mutants

  • Transcriptomics to reveal regulatory networks affected by aas deletion

  • Grad-seq analysis to identify molecular interaction partners

  • Metabolomics to assess changes in phospholipid profiles

This comprehensive approach, similar to that used for studying small proteins like MgrB in Salmonella Typhimurium , would provide mechanistic insights into how Bifunctional protein aas contributes to Salmonella schwarzengrund pathogenesis through its roles in membrane remodeling, stress adaptation, and host-pathogen interactions.

How might Recombinant Salmonella schwarzengrund Bifunctional protein aas be utilized in vaccine development?

Recombinant Salmonella schwarzengrund Bifunctional protein aas presents multiple opportunities for novel vaccine development strategies against Salmonella infections. As a conserved bacterial protein with enzymatic functions critical for membrane homeostasis, aas could serve as an effective vaccine antigen through several approaches:

• Subunit vaccine development:

  • The purified recombinant protein with His-tag could be formulated with appropriate adjuvants to stimulate protective immunity

  • Domain-specific constructs might elicit more targeted immune responses against functional epitopes

  • Combination with other Salmonella antigens could provide broader protection across strains

• Epitope-based vaccine design:

  • Computational prediction and experimental validation of B-cell and T-cell epitopes within the aas sequence

  • Construction of multi-epitope vaccines incorporating immunodominant regions

  • Strategic modification of epitopes to enhance immunogenicity while maintaining specificity

• Live attenuated vaccine platforms:

  • Engineering of controlled aas expression in attenuated Salmonella strains

  • Development of strains with modified aas functionality to maintain immunogenicity while reducing virulence

  • Creation of heterologous antigen delivery systems based on aas secretion mechanisms

• Nucleic acid vaccine approaches:

  • DNA or mRNA vaccines encoding optimized aas sequences

  • Prime-boost strategies combining different delivery platforms

  • Co-delivery with genetic adjuvants to enhance immune responses

Research would need to address several critical questions, including the conservation of protective epitopes across Salmonella serovars, the potential for cross-protection against related pathogens, and the most effective delivery platforms for stimulating both mucosal and systemic immunity. Preclinical studies would need to evaluate protection efficacy in relevant animal models while assessing safety profiles and immunological correlates of protection.

The availability of well-characterized recombinant protein with established expression and purification protocols provides a solid foundation for initiating these vaccine development efforts.

What potential exists for developing inhibitors targeting Salmonella schwarzengrund Bifunctional protein aas for antimicrobial applications?

The bifunctional nature of Salmonella schwarzengrund aas protein presents compelling opportunities for novel antimicrobial development. As a protein with dual enzymatic functions critical for bacterial membrane homeostasis and potential virulence, aas represents an attractive target for inhibitor development through several strategic approaches:

• Structure-based drug design potential:

  • Virtual screening campaigns targeting the active sites of both functional domains

  • Fragment-based approaches to identify initial chemical scaffolds with activity against either domain

  • Rational design of dual-targeting inhibitors that simultaneously block both enzymatic functions

• Assay development considerations:

  • High-throughput enzymatic assays for each functional domain

  • Whole-cell assays measuring membrane integrity in the presence of inhibitors

  • Thermal shift assays to identify compounds that destabilize protein structure

• Inhibitor classes with therapeutic potential:

  • Small molecule competitive inhibitors of substrate binding

  • Covalent inhibitors targeting catalytic residues

  • Allosteric modulators affecting conformational dynamics between domains

  • Peptide-based inhibitors derived from interaction partners

• Target validation strategies:

  • Genetic studies confirming essentiality under infection-relevant conditions

  • Phenotypic studies of chemical inhibition compared to genetic deletion

  • Assessment of resistance development frequency and mechanisms

The development of such inhibitors would require detailed structural information beyond what is currently available in the search results. Techniques such as X-ray crystallography or cryo-electron microscopy would be valuable for determining the three-dimensional structure of the protein, particularly in complex with substrates or initial inhibitors. Successful development of aas inhibitors could lead to novel therapeutics with activity against multidrug-resistant Salmonella strains, potentially with broader activity against related enterobacterial pathogens if targeting conserved features of this bifunctional enzyme.

How can integrated multi-omics approaches advance our understanding of Bifunctional protein aas function in Salmonella pathogenesis?

Integrated multi-omics approaches offer powerful frameworks for comprehensively understanding the role of Bifunctional protein aas in Salmonella pathogenesis. Building on methodologies applied to study small proteins in Salmonella Typhimurium , the following integrated approaches would be particularly valuable:

• Multi-level 'omics' integration:

  • Genomics: Comparative analysis of aas gene context and variation across Salmonella strains

  • Transcriptomics: RNA-seq to identify genes differentially expressed in aas mutants

  • Proteomics: Global protein expression changes and post-translational modifications

  • Lipidomics: Comprehensive analysis of membrane lipid composition alterations

  • Metabolomics: Metabolic pathway perturbations resulting from aas dysfunction

• Infection-relevant contextual analyses:

  • Dual RNA-seq during infection to capture both host and bacterial transcriptional responses

  • Transposon-directed insertion sequencing (TraDIS) to identify genetic interactions under infection conditions

  • Temporal profiling across infection stages to reveal dynamic roles

• Molecular interaction mapping:

  • Grad-seq analysis to identify protein-protein and protein-RNA interaction networks

  • Crosslinking mass spectrometry to capture transient interactions

  • Proximity labeling to identify spatial relationships in the bacterial cell

• Data integration frameworks:

  • Network analysis to construct functional relationship maps

  • Machine learning approaches to identify predictive signatures of aas activity

  • Pathway enrichment to contextualize findings within bacterial physiology

By integrating these diverse data types, researchers could develop comprehensive models of how aas contributes to membrane remodeling, stress responses, and virulence factor regulation during Salmonella infection. This systems-level understanding would reveal the broader impacts of aas function beyond its immediate enzymatic activities, potentially identifying unexpected connections to other virulence mechanisms and revealing new targets for therapeutic intervention.