Recombinant Escherichia coli Ferric enterobactin transport protein fepE (fepE)

What is the Ferric Enterobactin Transport Protein FepE and how does it function in bacterial iron acquisition?

Ferric enterobactin transport protein FepE is part of the bacterial iron acquisition system, which is crucial for bacterial survival. Bacteria produce siderophore complexes to acquire iron, an essential element for their growth and pathogenicity. These complexes are transported across the outer membrane by specialized receptor proteins . While FepA is one of the primary ferric enterobactin receptors, FepE plays a role in the regulation of this transport system. It's important to note that in some contexts, the term "fepE" may also refer to a gene involved in O-antigen biosynthesis regulation (Wzz), which affects bacterial virulence through different mechanisms .

How does FepE differ structurally and functionally from other ferric transport proteins like FepA?

FepA functions as the primary receptor protein for ferric enterobactin complexes, with a molecular weight of approximately 85 kDa . FepE, in contrast, serves as a regulatory protein in this transport system. The distinction is important when designing experiments targeting specific components of the iron acquisition pathway. When expressed as a recombinant protein, FepA has demonstrated significant immunogenicity in animal models, suggesting potential applications in preventing bacterial infections through iron-uptake inhibition .

What experimental models are most appropriate for studying FepE function?

Mouse models, particularly BALB/C mice, have been successfully used to study immune responses related to ferric enterobactin transport proteins. These models allow researchers to evaluate protein immunogenicity and potential protective effects against bacterial challenges . For the related Wzz(fepE) function, chicken embryo models have proven valuable in assessing the impact of mutations on bacterial virulence . When selecting a model, researchers should consider the specific aspect of FepE function they wish to investigate, whether it's immunological response, transport efficiency, or regulatory mechanisms.

How do mutations in fepE genes affect bacterial virulence and pathogenicity?

Mutations in fepE can significantly impact bacterial virulence. In the case of the Wzz(fepE) gene in Salmonella, a frameshift mutation caused by an adenine insertion results in an early stop codon, creating a truncated protein and rendering it a pseudogene . This mutation has been associated with reduced virulence in animal models. Specifically, the chicken embryo lethality assay demonstrated significantly lower embryo lethality rates for Salmonella strains with non-functional Wzz(fepE) compared to smooth variants with functional Wzz(fepE) . These findings suggest that functional FepE is critical for bacterial virulence, likely through its role in conferring protection against serum bactericidal activity .

What are the interactions between FepE and other components of bacterial iron acquisition systems?

While FepE functions within the broader iron acquisition system, its specific interactions with other components remain an area requiring further investigation. Research on related proteins like FepA has shown that antibodies against these proteins can recognize and bind to ferric enterobactin binding proteins, potentially inhibiting iron uptake . This suggests complex interactions within the iron acquisition pathway that could be exploited for antimicrobial strategies. When designing experiments to study these interactions, researchers should consider using co-immunoprecipitation, bacterial two-hybrid systems, or structural biology approaches to elucidate the protein-protein interaction network.

What are the recommended protocols for recombinant FepE expression and purification?

For successful recombinant FepE expression, researchers can follow protocols similar to those used for FepA. The genomic fepA gene can be amplified from E. coli, with the PCR product ligated into appropriate expression vectors such as pET28a for expression in E. coli BL21(DE3) . For protein purification, nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography has proven effective for obtaining purified recombinant protein . When expressing membrane-associated proteins like FepE, researchers should optimize conditions to ensure proper folding and functionality of the recombinant protein.

How can researchers effectively measure the immunogenicity of recombinant FepE?

To evaluate the immunogenicity of recombinant FepE, researchers can use animal models such as BALB/C mice. The purified recombinant protein can be injected to induce immunity, with antibody titers subsequently determined by ELISA . Challenge studies with various doses of pathogenic bacteria can then assess the protective efficacy of the immune response . For comprehensive immunological assessment, researchers should consider measuring both humoral and cellular immune responses, including analysis of specific antibody isotypes and T-cell responses.

What molecular techniques are most useful for studying fepE mutations?

Several molecular techniques can be employed to study fepE mutations. PCR and Sanger sequencing provide reliable methods for confirming mutations, as demonstrated in the analysis of the Wzz(fepE) frameshift mutation . For genome-wide analyses, next-generation sequencing followed by bioinformatic analysis using tools like BWA/MEM for reference mapping and snippy for variant calling can identify mutations . Visualization tools such as Integrated Genome Viewer (IGV) facilitate the interpretation of these variants . When designing primers for mutation analysis, researchers should consider the flanking regions of the target sequence to ensure specific amplification.

How should researchers analyze the effects of FepE on bacterial phenotypes?

Analysis of FepE effects on bacterial phenotypes requires multiple approaches. For O-antigen related functions of Wzz(fepE), comparative analysis of smooth and rough bacterial variants can reveal phenotypic differences . Virulence assays, such as the chicken embryo lethality assay, provide quantitative measures of pathogenicity differences . When analyzing the effects on iron acquisition, researchers should consider growth assays under iron-limited conditions and measurements of siderophore production and utilization. Statistical analysis should employ appropriate tests for comparing experimental groups, with significance thresholds clearly defined (e.g., p < 0.05, ** p < 0.01, and *** p < 0.001) .

What bioinformatic approaches are most valuable for comparative analysis of FepE across bacterial species?

For comparative analysis of FepE across bacterial species, several bioinformatic approaches are recommended. Nucleotide BLAST can identify homologous genes in different bacterial genomes . For analyzing microbiome data related to FepE function, techniques such as principal coordinate analysis (PCoA) and clustering heatmaps can visualize variations across different groups . Linear discriminant analysis of effect size (LEfSe), non-parametric paired t-tests (p < 0.05), and fold-change (FC > 1.5) methods can identify significant changes in microbial abundance related to FepE function . When performing these analyses, researchers should ensure appropriate data normalization and consider potential batch effects.

What control groups are essential for FepE functional studies?

When designing experiments to study FepE function, several control groups are essential. For immunogenicity studies, both negative controls (unimmunized animals) and positive controls (animals immunized with known immunogenic proteins) should be included . For mutation studies, comparison between wild-type strains and those carrying specific fepE mutations provides valuable insights . In the case of Wzz(fepE), comparing smooth variants (with functional Wzz(fepE)) to rough variants (with mutated Wzz(fepE)) has revealed significant functional differences . Additionally, complementation studies, where the mutated gene is restored, can confirm that observed phenotypes are specifically due to the fepE mutation.

How should researchers address potential confounding factors in FepE studies?

Several confounding factors may affect FepE studies. Iron availability in experimental media can significantly impact the expression and function of iron acquisition systems. Researchers should standardize iron concentrations and consider using iron chelators to create defined conditions . When studying bacterial virulence, variations in bacterial growth rates may confound the interpretation of virulence assays. Growth curve analysis should be performed to account for these differences. For immunological studies, factors such as animal age, sex, and genetic background can influence immune responses and should be controlled or analyzed as potential variables .

What are the promising applications of FepE research in addressing bacterial infections?

Research on FepE and related proteins shows promise for developing novel strategies against bacterial infections. The immunogenicity of recombinant ferric enterobactin binding proteins suggests potential vaccine applications . By targeting iron acquisition systems, which are essential for bacterial survival, researchers may develop approaches to restrict bacterial propagation . The observation that immunized mice challenged with higher doses of selected bacteria survived indicates the protective potential of this approach . Future research should focus on optimizing antigen formulations, delivery systems, and combining FepE-based strategies with other antimicrobial approaches.

What emerging technologies could enhance our understanding of FepE structure and function?

Several emerging technologies could advance FepE research. Cryo-electron microscopy could provide detailed structural insights into FepE and its interactions with other components of the iron acquisition system. CRISPR-Cas9 genome editing allows for precise manipulation of fepE genes to study specific domains and functional residues. Single-cell technologies can reveal heterogeneity in bacterial populations regarding FepE expression and function. Metabolomic approaches, similar to those used in FEPE studies , could identify metabolic changes associated with FepE function or mutation, providing a more comprehensive understanding of its role in bacterial physiology.