Recombinant Brucella suis Elongation factor G (fusA), partial

What is Elongation factor G (fusA) and what is its role in Brucella suis?

Elongation factor G (EF-G) is a GTPase that facilitates the translocation of peptidyl-tRNA from the A-site to the P-site of the ribosome during protein synthesis. In Brucella suis, this protein is essential for bacterial survival and replication. The fusA gene encodes EF-G and has been identified as one of the critical genes for both the functioning and growth of Brucella, making it a potential target for antimicrobial development. Like other essential translation factors, interfering with EF-G function can inhibit bacterial growth, as demonstrated in studies using peptide nucleic acids (PNAs) targeting translation factors in Brucella .

What expression systems are typically used to produce recombinant Brucella suis EF-G?

Recombinant B. suis EF-G is typically produced using heterologous expression systems including E. coli, yeast, baculovirus, or mammalian cell systems. The choice of expression system depends on research needs, with E. coli being most common for basic structural and functional studies. For studies requiring post-translational modifications or improved folding, eukaryotic expression systems may be preferred. Commercial recombinant EF-G preparations, such as those for Brucella abortus (which shares high sequence identity with B. suis), are produced in systems like E. coli and purified to >90% purity, supplied in liquid form containing glycerol .

How should researchers optimize experimental conditions when studying B. suis EF-G inhibition?

When designing experiments to study inhibition of B. suis EF-G, researchers should consider the following parameters:

When testing potential inhibitors, researchers should monitor both optical density (OD₅₅₀) in real-time and perform CFU counts at various timepoints to assess bactericidal effects, similar to methodologies used for other essential gene targets in Brucella .

What are the key considerations when designing antisense molecules targeting fusA in Brucella suis?

When designing antisense molecules such as peptide nucleic acids (PNAs) targeting the fusA gene in B. suis, researchers should:

• Target the start codon region of the gene to effectively block translation initiation

• Design sequences with high specificity to the fusA mRNA

• Consider linking the antisense molecules to cell-penetrating peptides (CPPs) for improved cellular uptake

• Test concentration-dependent effects (typically in the micromolar range)

• Evaluate sequence specificity using control PNAs with scrambled sequences

• Assess both growth inhibition in pure culture and intracellular replication within macrophages

Studies with PNAs targeting other essential genes in Brucella have shown that antimicrobial effects can be achieved at micromolar concentrations through sequence-specific and dose-dependent inhibition of gene expression .

How can researchers effectively evaluate the impact of fusA inhibition on Brucella suis pathogenesis?

To evaluate the impact of fusA inhibition on B. suis pathogenesis, researchers should employ a multi-tiered approach:

• Pure culture growth assays to establish direct antimicrobial effects

• Macrophage infection models (e.g., J774.A1 murine macrophage cell line) to assess intracellular replication

• Cell viability assays to ensure antimicrobial effects are not due to host cell toxicity

• Microscopy to visualize bacterial trafficking and replication within host cells

• Gene expression analysis to confirm specific inhibition of target gene

• Comparative studies with other essential gene targets to contextualize the importance of fusA

This approach allows researchers to distinguish between direct bactericidal effects and interference with virulence mechanisms. Studies with other essential genes in Brucella have demonstrated that inhibitors effective in pure culture may not always work intracellularly, and vice versa, highlighting the importance of testing in both contexts .

How does inhibition of EF-G affect the Unfolded Protein Response (UPR) during Brucella infection?

Inhibition of EF-G in Brucella likely disrupts bacterial protein synthesis, which could consequently affect the bacterial ability to manipulate host UPR pathways. Brucella is known to induce a UPR in infected cells, which appears to support intracellular replication. This UPR induction involves all three primary signaling axes (PERK, IRE1, and ATF6), as evidenced by downstream induction of CHOP, ERdj4, and BiP mRNAs, respectively .

When EF-G function is compromised, Brucella would likely have reduced capacity to produce the proteins needed for UPR manipulation, such as TcpB, which has been implicated in UPR induction. Chemical inhibition of the UPR using compounds like TUDCA significantly decreases recoverable Brucella CFUs, typically by a log or more by 24-36 hours post-infection . This suggests that interference with bacterial protein synthesis through targeting EF-G could have similar effects by indirectly preventing UPR manipulation, thereby compromising intracellular replication.

What are the differences in efficacy when targeting fusA versus other essential genes in Brucella?

Comparative efficacy of targeting different essential genes in Brucella reveals interesting patterns:

This variation in efficacy suggests that different sets of genes become conditionally essential depending on the growth environment. The intracellular environment of macrophages is nutritionally restricted compared to rich laboratory media, potentially explaining why some targets are more effective in one context versus the other .

How does targeting EF-G compare with traditional antibiotics for inhibiting intracellular Brucella?

Targeting EF-G presents several distinct advantages and challenges compared to traditional antibiotics:

• Specificity: EF-G-targeted inhibitors can be designed with high specificity for bacterial translation machinery

• Intracellular penetration: Unlike some traditional antibiotics, antisense molecules targeting EF-G can be conjugated with cell-penetrating peptides to improve delivery into macrophages

• Reduced resistance development: Target-specific inhibitors may face different resistance mechanisms than broad-spectrum antibiotics

• Synergistic potential: EF-G inhibitors could potentially synergize with antibiotics that target other aspects of protein synthesis

What are the optimal storage and handling conditions for recombinant B. suis EF-G to maintain functionality?

For optimal storage and handling of recombinant B. suis EF-G:

• Store stock solutions at -20°C for routine use or -80°C for long-term storage

• Prepare working aliquots and store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they can compromise protein structure and function

• Maintain the protein in buffer containing glycerol (typically 10-20%) to prevent freezing damage

• When thawing, keep the protein on ice and use quickly to minimize degradation

• For functional assays, verify activity periodically using GTPase activity assays

These recommendations are based on standard protocols for similar recombinant proteins, including commercial preparations of Brucella abortus EF-G, which shares high homology with B. suis EF-G .

What techniques can researchers use to assess the interaction between B. suis EF-G and potential inhibitors?

Researchers can employ several techniques to assess interactions between B. suis EF-G and potential inhibitors:

• In vitro GTPase assays: Measure the inhibition of EF-G's GTPase activity in the presence of potential inhibitors using colorimetric or fluorometric methods to detect inorganic phosphate release

• Ribosome binding assays: Evaluate how inhibitors affect EF-G binding to ribosomes using techniques such as:

  • Filter binding assays with radiolabeled components

  • Surface plasmon resonance (SPR)

  • Microscale thermophoresis (MST)

• Structural studies:

  • X-ray crystallography of EF-G in complex with inhibitors

  • Cryo-electron microscopy to visualize EF-G-ribosome-inhibitor complexes

  • NMR studies for smaller fragments

• Thermal shift assays: Determine changes in protein thermal stability upon inhibitor binding

• Bacterial growth inhibition: Assess sequence-specific and dose-dependent inhibition of bacterial growth when targeting the fusA gene, similar to approaches used for testing PNAs against other essential genes in Brucella

How can researchers effectively measure the impact of fusA inhibition on Brucella protein synthesis?

To measure the impact of fusA inhibition on Brucella protein synthesis, researchers can use the following methods:

• Metabolic labeling: Incorporate radioactive amino acids (e.g., ³⁵S-methionine) or non-radioactive analogs (e.g., azidohomoalanine) into newly synthesized proteins followed by detection and quantification

• Polysome profiling: Analyze the distribution of ribosomes on mRNA using sucrose gradient centrifugation to detect translation defects; inhibition of EF-G typically results in characteristic changes in polysome profiles

• Reporter systems: Utilize reporter constructs (e.g., luciferase or fluorescent proteins) under the control of Brucella promoters to quantify changes in protein synthesis rates

• Quantitative proteomics:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture)

  • iTRAQ (Isobaric Tags for Relative and Absolute Quantification)

  • Label-free quantitative proteomics

• Ribosome footprinting: Map the positions of ribosomes on mRNAs with nucleotide resolution to identify translation stalling sites

When testing fusA-targeted inhibitors, these methods can be complemented by growth inhibition assays in both pure culture and intracellular settings, as has been done with PNAs targeting other essential Brucella genes .

How should researchers interpret differences in efficacy between in vitro and intracellular inhibition of B. suis EF-G?

When interpreting differences in efficacy between in vitro and intracellular inhibition studies targeting B. suis EF-G, researchers should consider several factors:

• Metabolic state differences: Brucella adapts its metabolism to the intracellular environment, which may alter the expression or essentiality of certain genes, including fusA. This phenomenon has been observed with other targets where PNAs effective against intracellular Brucella (e.g., asd, gyrA, dnaG) showed no significant effect in pure culture .

• Nutrient availability: The intracellular environment of macrophages is not enriched with substrates that facilitate bacterial metabolism compared to rich culture media, potentially making certain genes conditionally essential in one environment but not the other .

• Host-pathogen interactions: The efficacy of EF-G inhibition may be influenced by specific host-pathogen interactions, such as the bacterial manipulation of the host UPR, which supports Brucella replication in macrophages .

• Delivery efficiency: Differences in the ability of inhibitors to penetrate bacterial membranes versus host cell and phagosomal membranes can significantly impact efficacy in different experimental settings.

• Stress responses: The intracellular environment induces specific stress responses in Brucella that may alter the vulnerability of certain targets.

These factors underscore the importance of testing potential therapeutics in both pure culture and relevant cellular infection models.

What statistical approaches are most appropriate for analyzing inhibition data from EF-G targeted experiments?

When analyzing inhibition data from EF-G targeted experiments, researchers should employ the following statistical approaches:

When conducting experiments similar to those testing PNAs against Brucella, statistical significance should be established with p-values ≤0.05, as demonstrated in previous studies (p≤0.04 in growth inhibition experiments) .

How might CRISPR-Cas systems be utilized to study fusA function in Brucella suis?

CRISPR-Cas systems offer powerful approaches to study fusA function in Brucella suis through several strategies:

• CRISPRi (CRISPR interference): Using catalytically inactive Cas9 (dCas9) fused to transcriptional repressors to achieve tunable downregulation of fusA expression without completely eliminating it, which is crucial for studying essential genes

• CRISPR-based gene editing: Creating specific point mutations in the fusA gene to study structure-function relationships and identify residues critical for EF-G activity or antibiotic interactions

• Conditional knockdown systems: Combining CRISPR with inducible promoters to achieve temporal control over fusA expression, allowing for the study of immediate effects of EF-G depletion

• Domain mapping: Using CRISPR to introduce specific domain deletions or modifications to understand the contribution of each EF-G domain to protein function and bacterial fitness

• Reporter fusions: Using CRISPR to introduce fluorescent protein fusions for tracking EF-G localization and dynamics during infection

These approaches could provide insights into the precise role of EF-G in Brucella pathogenesis and potentially identify specific vulnerabilities that could be exploited for therapeutic development.

What are the prospects for developing fusA-targeted therapeutics against intracellular Brucella infections?

The prospects for developing fusA-targeted therapeutics against intracellular Brucella infections are promising but face several challenges:

• Target validation: Studies with peptide nucleic acids targeting translation factors have demonstrated growth inhibition of Brucella in both pure culture and intracellular environments, suggesting translation machinery is a viable target .

• Delivery challenges: Effective therapeutics must cross multiple membranes (host cell, phagosomal, bacterial) to reach intracellular targets. Conjugation with cell-penetrating peptides has shown promise for PNA delivery into infected macrophages .

• Specificity considerations: While bacterial EF-G differs significantly from mammalian elongation factors, ensuring therapeutic specificity remains important to minimize host toxicity.

• Resistance development: Monitoring for potential resistance mechanisms will be essential, though targeting essential factors like EF-G may present a higher barrier to resistance.

• Combinatorial approaches: Combining fusA-targeted inhibitors with modulators of host response, such as UPR inhibitors like TUDCA, could enhance efficacy based on findings that UPR inhibition significantly reduces Brucella replication .

• Alternative delivery systems: Nanoparticle-based delivery systems could improve the intracellular delivery of fusA-targeted therapeutics.

The successful development of such therapeutics would represent a novel approach to treating intracellular bacterial infections that are often refractory to conventional antibiotics.

How does fundamental research on Brucella suis EF-G contribute to broader understanding of bacterial translation and pathogenesis?

Research on B. suis EF-G contributes significantly to our understanding of bacterial translation and pathogenesis in several ways:

• Essential gene networks: Studies targeting fusA alongside other essential genes help map the network of genes critical for Brucella survival and adaptation in different environments, revealing that genes like fusA may have variable importance depending on growth conditions .

• Host-pathogen interactions: Understanding how translation machinery components like EF-G support processes such as UPR manipulation provides insights into the molecular mechanisms of intracellular survival and replication .

• Antimicrobial development: Investigations into the inhibition of essential factors like EF-G establish proof-of-concept for novel antimicrobial strategies targeting protein synthesis in intracellular pathogens .

• Bacterial adaptation: Research on translation factors helps elucidate how bacteria adapt protein synthesis to different environments, particularly the resource-limited intracellular niche of macrophages .

• Evolutionary insights: Comparative studies of EF-G across different bacterial species can reveal evolutionary adaptations in protein synthesis machinery.