Recombinant Brucella abortus biovar 1 Putative peptide permease protein BruAb2_0794 (BruAb2_0794)

What is BruAb2_0794 and what is its functional classification?

BruAb2_0794 is a putative peptide permease protein from Brucella abortus biovar 1. The protein consists of 302 amino acids and functions as part of nutrient transport systems in Brucella species . Based on its sequence characterization, BruAb2_0794 belongs to the ABC (ATP-Binding Cassette) transporter family, which plays crucial roles in the import and export of various substrates across bacterial membranes .

The protein's putative function as a peptide permease suggests its involvement in the uptake of peptides from the extracellular environment, which is essential for bacterial nutrition and potentially for pathogenesis. Within the Brucella genus, transport systems like BruAb2_0794 may contribute to the organism's ability to survive within host cells during infection, as ABC transporters are known to play roles in bacterial virulence and adaptation to host environments .

How does BruAb2_0794 relate to the broader ATP-Binding Cassette systems in Brucella?

BruAb2_0794 is one component of the extensive ATP-Binding Cassette (ABC) systems found across Brucella species. Research has demonstrated that Brucella species possess numerous ABC systems, with B. melitensis having 79, B. suis 72, B. abortus 64, B. canis 74, and B. ovis 59 . These variations in ABC system counts among species may relate to their differing host ranges and pathogenic potential.

ABC systems in Brucella typically consist of multiple components:

• ATP-binding proteins (ABC) that provide energy through ATP hydrolysis

• Permease proteins (IM) that form transmembrane channels

• Substrate-binding proteins (LPP) that recognize and bind specific substrates

BruAb2_0794, as a permease protein, forms the transmembrane component of its specific ABC system. It likely works in conjunction with ATP-binding proteins and substrate-binding proteins to facilitate the import of specific peptides across the bacterial membrane . The permease component is crucial as it forms the channel through which substrates move.

Comparative genomic analysis suggests that Brucella species have particularly high numbers of ABC systems dedicated to nutrient import, reflecting their adaptation to various ecological niches and host environments .

What is the evolutionary conservation of BruAb2_0794 across Brucella species?

Evolutionary analysis of ABC transporters across Brucella species reveals interesting patterns of conservation and divergence. While detailed conservation data specific to BruAb2_0794 is limited in the provided search results, broader analysis of ABC systems in Brucella species provides context for understanding its potential conservation.

The comprehensive inventory of ABC systems across five Brucella species (B. melitensis, B. abortus, B. suis, B. canis, and B. ovis) reveals that many ABC components are conserved across species, though some systems show species-specific presence or absence . For example, certain ABC systems are absent in B. ovis, which is not known to cause human brucellosis, suggesting their potential role in human pathogenesis.

From the comparative table in the research:

This example shows how components of ABC systems may be conserved across some Brucella species but absent in others (as shown by the absence of the LPP component in B. ovis and B. canis for this particular system) .

How can BruAb2_0794 contribute to understanding virulence mechanisms in Brucella?

BruAb2_0794, as a putative peptide permease, may play a significant role in Brucella virulence through nutrient acquisition within the host environment. Research on ABC transporters in Brucella suggests that these systems can be directly linked to virulence . The comparative genomic analysis of ABC systems across Brucella species provides evidence that certain transport systems are present in species that cause human brucellosis but absent in B. ovis, which does not infect humans.

To investigate BruAb2_0794's role in virulence, researchers could employ several approaches:

• Comparative virulence studies: Creating isogenic mutants lacking BruAb2_0794 and comparing their virulence to wild-type strains in cellular and animal models.

• Nutrient uptake analysis: Determining what specific peptides are transported by this system and whether they are essential for survival within host cells.

• Expression profiling: Analyzing whether BruAb2_0794 expression changes during different phases of infection, particularly within macrophages where Brucella typically resides.

• Host response studies: Investigating whether BruAb2_0794 activity influences host cell responses, such as cytokine production or autophagy pathways.

The significance of peptide permeases in bacterial pathogenesis lies in their ability to acquire essential nutrients in restrictive host environments, making them potential targets for therapeutic intervention .

What experimental strategies are optimal for functional characterization of BruAb2_0794?

Functional characterization of BruAb2_0794 requires a multi-faceted experimental approach:

• Substrate identification:

  • Radiolabeled peptide transport assays using purified protein reconstituted in liposomes

  • Competitive binding assays with potential peptide substrates

  • Metabolomic profiling comparing wild-type and BruAb2_0794 mutant strains

• Structure-function analysis:

  • Site-directed mutagenesis of conserved residues

  • Chimeric protein construction with permease domains from other species

  • Crystallization and structural determination of the protein alone and in complex with substrates

• In vivo functional studies:

  • Creation of deletion mutants using homologous recombination

  • Complementation studies to confirm phenotype specificity

  • Conditional expression systems to study essentiality

• Interaction studies:

  • Co-immunoprecipitation to identify partner proteins

  • Bacterial two-hybrid assays to confirm protein-protein interactions with other ABC system components

  • Cross-linking studies to capture transient interactions

For optimal results, researchers should use the recombinant protein (RFL4126BF) expressed with the N-terminal His-tag in E. coli , which allows for efficient purification and subsequent functional studies. The purified protein should be reconstituted according to recommended protocols to maintain its native conformation and functionality .

How does the peptide permease function of BruAb2_0794 potentially relate to Brucella pathogenesis?

The peptide permease function of BruAb2_0794 may contribute to Brucella pathogenesis through several mechanisms:

• Nutritional adaptation: As intracellular pathogens, Brucella species must adapt to the nutrient-limited environment inside host cells. Peptide permeases allow for the uptake of peptides as nitrogen and carbon sources, which is critical for bacterial survival and replication within host cells.

• Modulation of host responses: Some bacterial transporters have been shown to import host-derived signaling molecules or export bacterial factors that modulate host immune responses. BruAb2_0794 could potentially be involved in such processes.

• Stress response: During infection, bacteria encounter various stresses including oxidative stress and nutrient limitation. ABC transporters can contribute to stress tolerance by importing protective peptides or exporting toxic compounds.

• Persistence and chronic infection: Brucella is known for establishing chronic infections. Nutrient acquisition systems like BruAb2_0794 may be essential for long-term persistence within host tissues.

The fact that B. ovis, which does not cause human brucellosis, lacks certain ABC systems present in other Brucella species suggests that specific transporters like BruAb2_0794 might be directly involved in host-pathogen interactions relevant to human disease . Comparative analysis of ABC systems across Brucella species with different host ranges and virulence profiles provides insights into their potential roles in pathogenesis.

What structural analysis approaches are most informative for studying BruAb2_0794?

Several structural analysis approaches can provide valuable insights into BruAb2_0794's function and mechanism:

• X-ray crystallography and cryo-electron microscopy:

  • Determination of high-resolution 3D structure

  • Co-crystallization with substrates or inhibitors to identify binding sites

  • Visualization of conformational changes during transport cycle

• Circular dichroism (CD) spectroscopy:

  • Assessment of secondary structure elements

  • Monitoring conformational changes upon substrate binding

  • Thermal stability analysis

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Analysis of protein dynamics

  • Identification of substrate binding residues

  • Study of protein-protein interactions with other ABC system components

• Molecular modeling and simulations:

  • Homology modeling based on related permease structures

  • Molecular dynamics simulations to predict substrate translocation pathways

  • Docking studies to identify potential inhibitors

• Limited proteolysis coupled with mass spectrometry:

  • Identification of flexible regions and domains

  • Mapping of transmembrane topology

  • Detection of conformational changes upon substrate binding

When working with recombinant BruAb2_0794, researchers should carefully consider protein preparation methods. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage . Repeated freeze-thaw cycles should be avoided to maintain protein integrity .

What gene knockout and complementation strategies are most effective for studying BruAb2_0794?

Effective gene knockout and complementation strategies for studying BruAb2_0794 include:

• Targeted gene deletion:

  • Homologous recombination using suicide vectors containing BruAb2_0794 flanking regions

  • CRISPR-Cas9 system adapted for Brucella to create precise deletions

  • Insertional inactivation using antibiotic resistance cassettes

• Conditional knockout systems:

  • Tetracycline-responsive promoters to control expression

  • Temperature-sensitive replicons for conditional expression

  • Degradation tag systems for protein-level depletion

• Complementation approaches:

  • Chromosomal integration at neutral sites

  • Plasmid-based complementation with native promoters

  • Complementation with orthologous genes from other Brucella species

• Reporter fusion strategies:

  • Transcriptional fusions to monitor expression patterns

  • Translational fusions to track protein localization

  • Split protein complementation to identify interaction partners

When designing knockout experiments, researchers should consider the potential polar effects on downstream genes, especially if BruAb2_0794 is part of an operon with other ABC transporter components. Additionally, complementation studies should include controls with the empty vector and with site-directed mutants to validate functional domains.

Phenotypic analysis should examine growth in various media, survival within macrophages, virulence in animal models, and specific transport functions to fully characterize the role of BruAb2_0794 in Brucella biology and pathogenesis.

What are the optimal protocols for expression and purification of recombinant BruAb2_0794?

Optimal expression and purification of recombinant BruAb2_0794 involves several critical steps:

• Expression system selection:

  • E. coli is the recommended expression host for BruAb2_0794

  • BL21(DE3) or similar strains are typically preferred for membrane protein expression

  • pET vector systems with N-terminal His-tags provide good expression levels and purification options

• Culture conditions optimization:

  • Induction at lower temperatures (16-25°C) improves proper folding

  • Lower IPTG concentrations (0.1-0.5 mM) for gentler induction

  • Supplementing media with glucose to reduce basal expression

  • Addition of glycylbetaine and sorbitol as chemical chaperones

• Membrane protein extraction:

  • Cell disruption by sonication or French press

  • Gentle detergent solubilization (DDM, LDAO, or Triton X-100)

  • Centrifugation to remove insoluble material

• Purification strategy:

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

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for further purification if needed

• Quality assessment:

  • SDS-PAGE to confirm >90% purity

  • Western blotting to verify identity

  • Circular dichroism to assess proper folding

  • Dynamic light scattering to check homogeneity

The product information indicates that recombinant BruAb2_0794 can be successfully expressed in E. coli with an N-terminal His-tag, resulting in >90% purity as determined by SDS-PAGE . Following purification, the protein is typically provided as a lyophilized powder in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

What are the recommended storage and reconstitution procedures for recombinant BruAb2_0794?

Proper storage and reconstitution are crucial for maintaining the functionality of recombinant BruAb2_0794:

Storage recommendations:

• Store the lyophilized protein at -20°C/-80°C upon receipt

• For long-term storage, aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% (or between 5-50% as needed)

• Aliquot into small volumes for single use and store appropriately

Important considerations:

• Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity

• The storage buffer (Tris/PBS-based buffer with 6% trehalose, pH 8.0) is designed to maintain protein stability

• For functional studies, the reconstitution buffer may need to be adjusted depending on the specific experimental requirements

Following these storage and reconstitution guidelines will help maintain the structural integrity and functional activity of the recombinant protein for experimental use.

How can binding assays be designed to identify substrates of BruAb2_0794?

Designing effective binding assays to identify substrates of BruAb2_0794 requires multiple complementary approaches:

• Direct binding assays:

  • Microscale thermophoresis (MST) to measure affinity for potential peptide substrates

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Fluorescence anisotropy with labeled peptides to assess binding

• Transport assays:

  • Liposome reconstitution with purified BruAb2_0794 and associated ABC components

  • Fluorescent substrate accumulation assays

  • Radioactive substrate uptake measurements

  • Membrane vesicle preparation from expressing cells

• Competition assays:

  • Displacement of known ligands by potential substrates

  • Structure-activity relationship studies with peptide libraries

  • Inhibition studies with transport blockers

• In silico prediction approaches:

  • Homology-based substrate prediction based on related permeases

  • Molecular docking of peptide libraries

  • Phylogenetic profiling to identify substrates based on evolutionary relationships

When designing these assays, it's important to consider that BruAb2_0794 is a putative peptide permease protein , suggesting its substrates are likely peptides of specific sequences or characteristics. The assays should include positive controls (known substrates of related ABC transporters) and negative controls (non-substrate peptides or proteins) to validate the specificity of binding.

Additionally, as BruAb2_0794 is part of an ABC system, some assays may require reconstitution with partner proteins (ATP-binding and substrate-binding components) to observe full transport functionality.

What protein interaction studies can reveal BruAb2_0794's role in ABC transport systems?

Protein interaction studies can provide crucial insights into how BruAb2_0794 functions within its ABC transport system:

• Co-immunoprecipitation (Co-IP):

  • Pull-down assays using anti-His antibodies against the tagged BruAb2_0794

  • Mass spectrometry identification of co-precipitated proteins

  • Reverse Co-IP to confirm interactions

• Bacterial two-hybrid system:

  • Testing interactions with known ABC components

  • Screening genomic libraries to identify novel interaction partners

  • Mapping interaction domains

• Cross-linking studies:

  • Chemical cross-linking followed by mass spectrometry (XL-MS)

  • Photo-activatable cross-linkers for capturing transient interactions

  • In vivo cross-linking to preserve physiologically relevant complexes

• Förster resonance energy transfer (FRET):

  • Fluorescent protein fusions to BruAb2_0794 and potential partners

  • Live-cell imaging to visualize interactions

  • Quantitative FRET measurements to assess binding affinities

• Split-protein complementation assays:

  • Bacterial adenylate cyclase two-hybrid system (BACTH)

  • Split-GFP reassembly to visualize interaction sites

  • Protein-fragment complementation assays (PCA)

ABC systems in Brucella typically consist of multiple components that work together, including ATP-binding proteins, permease proteins, and substrate-binding proteins . Identifying which specific proteins interact with BruAb2_0794 will help determine its complete functional system and substrate specificity.

The search results indicate that comparative genomic analysis has been used to map ABC systems in Brucella species , providing a framework for identifying potential interaction partners of BruAb2_0794. This approach can guide targeted interaction studies to confirm predicted functional relationships.

How can site-directed mutagenesis be applied to understand BruAb2_0794 function?

Site-directed mutagenesis is a powerful approach for understanding structure-function relationships in BruAb2_0794:

• Target selection for mutation:

  • Conserved residues identified through multiple sequence alignment with homologous proteins

  • Predicted transmembrane regions that may form the substrate channel

  • Potential substrate-binding residues identified through modeling or docking

  • Interface residues that may interact with other ABC system components

• Mutation strategies:

  • Alanine scanning of transmembrane regions

  • Conservative substitutions to probe specific interactions

  • Charge reversal mutations to test electrostatic interactions

  • Cysteine substitutions for accessibility studies or cross-linking

• Functional analysis of mutants:

  • Transport assays to measure effects on substrate specificity or kinetics

  • ATPase activity measurements to assess coupling with ATP-binding components

  • Protein interaction assays to determine effects on complex formation

  • In vivo complementation to assess biological relevance

• Structural impact assessment:

  • Circular dichroism to confirm proper folding

  • Thermal stability assays to detect structural perturbations

  • Limited proteolysis to probe conformational changes

  • Crystallization of key mutants to visualize structural alterations

When designing mutation experiments, researchers should consider the complete amino acid sequence of BruAb2_0794 and focus on regions that are most likely involved in substrate binding or transport. The amino acid sequence suggests multiple transmembrane domains characteristic of permease proteins, which would be prime targets for mutation studies.

Mutations should be introduced into expression constructs similar to the one used for recombinant protein production (His-tagged in E. coli) , allowing for consistent purification and analysis protocols across wild-type and mutant variants.

What in vivo experimental models are appropriate for studying BruAb2_0794's role in Brucella infection?

Several in vivo experimental models can be employed to study BruAb2_0794's role in Brucella infection:

• Cellular infection models:

  • Macrophage infection assays (RAW264.7, J774A.1, or primary macrophages)

  • Trophoblast cell models for reproductive tract tropism

  • Dendritic cell infection to assess immunological interactions

  • Comparative survival assays between wild-type and BruAb2_0794 mutants

• Small animal models:

  • Mouse models (BALB/c, C57BL/6) for systemic infection

  • Guinea pig models for reproductive pathology

  • Pregnant mouse models to study vertical transmission

  • Genetic knockout mice lacking specific immune components

• Large animal models:

  • Goats and sheep as natural hosts for certain Brucella species

  • Cattle models for B. abortus specifically

  • Reproductive pathology assessment in natural hosts

• Ex vivo tissue models:

  • Placental explant cultures

  • Spleen and liver slice cultures

  • Precision-cut lung slices

When using these models, researchers should compare wild-type Brucella strains with isogenic mutants lacking or overexpressing BruAb2_0794. Key parameters to assess include:

• Bacterial load in various tissues

• Survival within professional phagocytes

• Inflammatory responses (cytokine profiles, histopathology)

• Ability to establish chronic infection

• Reproductive pathology in pregnant animal models

The search results indicate that B. ovis, which lacks certain ABC systems, does not cause human brucellosis . This suggests that comparative studies between Brucella species with and without specific transporters could provide insights into the role of these systems in host specificity and virulence.