Recombinant Salmonella typhimurium Quaternary ammonium compound-resistance protein sugE (sugE)
Description
Introduction to Recombinant Salmonella typhimurium Quaternary Ammonium Compound-Resistance Protein sugE (sugE)
The Recombinant Salmonella typhimurium Quaternary Ammonium Compound-Resistance Protein sugE (sugE) is a recombinant protein derived from the sugE gene, which is part of the small multidrug resistance (SMR) family. This protein is known for conferring resistance to certain quaternary ammonium compounds (QACs), which are commonly used as disinfectants. The sugE gene was initially identified in Escherichia coli as a suppressor of groEL mutations but has since been recognized for its role in drug resistance, particularly against a subset of toxic quaternary ammonium compounds .
Structure and Function of sugE
The sugE protein is a small membrane protein that functions as an efflux pump, helping bacteria to expel toxic substances, including certain quaternary ammonium compounds, from the cell. This mechanism enhances bacterial survival in environments where these compounds are present. The protein is typically expressed in bacteria like Salmonella typhimurium and Escherichia coli, where it plays a crucial role in resistance against specific antiseptics .
Table 1: Characteristics of Recombinant Salmonella typhimurium sugE Protein
| Characteristics | Description |
|---|---|
| Species | Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) |
| Gene Name | sugE |
| Protein Length | 105 amino acids |
| Tag Type | Variable (e.g., His-tag for some recombinant versions) |
| Storage Buffer | Tris-based buffer, 50% glycerol |
| Storage Conditions | -20°C or -80°C, avoid repeated freeze-thaw cycles |
Table 2: Resistance Profile of sugE Against Quaternary Ammonium Compounds
| Compound | Resistance Profile |
|---|---|
| Cetylpyridinium | Resistant |
| Cetyldimethylethyl ammonium | Resistant |
| Cetrimide | Resistant |
| Benzalkonium chloride | Not resistant |
| Benzyl-dimethyl tetradecylammonium chloride | Not resistant |
Applications and Implications
The recombinant sugE protein is used in research settings to study mechanisms of drug resistance and for the development of diagnostic tools. For instance, ELISA kits are available for detecting this protein, which can help in understanding its expression levels in different bacterial strains .
The presence of sugE in bacteria can have significant implications for public health, as it may contribute to the development of resistance against commonly used disinfectants. This highlights the need for continued surveillance and research into resistance mechanisms to ensure effective infection control strategies .
Product Specs
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Target Background
KEGG: stm:STM4338
STRING: 99287.STM4338
Q&A
What experimental strategies are used to express and purify recombinant SugE in Salmonella typhimurium?
Recombinant SugE is typically expressed in heterologous systems such as Escherichia coli due to its scalability and well-characterized protein production machinery. The full-length protein (1–105 amino acids) is often cloned with affinity tags (e.g., 6xHis-SUMO) to facilitate purification via nickel-chelate chromatography . For example, the strain LT2/SGSC1412-derived SugE is expressed in Tris-based buffers with 50% glycerol for stability, achieving >90% purity as validated by SDS-PAGE . Researchers must optimize induction conditions (e.g., IPTG concentration, temperature) to prevent aggregation, a common issue with membrane-associated proteins.
How is QAC resistance activity quantified in recombinant SugE variants?
Minimum inhibitory concentration (MIC) assays in Mueller-Hinton broth are the gold standard. For instance, Salmonella 4, ,12:i:- strains resistant to didecyldimethylammonium bromide (DDAB) exhibit MIC values of 200 μg/mL, with efflux pump activity confirmed via ethidium bromide exclusion assays . Parallel experiments using Hoechst 33342 dye accumulation in ΔsugE mutants provide direct evidence of SugE-mediated efflux . These assays require controls such as proton motive force inhibitors (e.g., carbonyl cyanide m-chlorophenyl hydrazine) to distinguish active efflux from passive diffusion.
What structural features of SugE are critical for its function?
SugE belongs to the small multidrug resistance (SMR) family, characterized by four transmembrane helices. Key residues (H24, M39, I43, A44) differentiate SugE’s substrate specificity from other SMR transporters like EmrE . Site-directed mutagenesis of Citrobacter freundii SugE revealed that single mutations (e.g., H24A) convert SugE into a QAC importer rather than exporter, as shown by hypersensitivity phenotypes and ethidium uptake assays . Structural predictions using topology reporter fusions confirm cytoplasmic N- and C-termini, consistent with SMR family architecture .
How do SugE homologs (e.g., SugE1 vs. SugE2) differ in substrate specificity and resistance profiles?
In Salmonella 4, ,12:i:-, SugE1 and SugE2 exhibit non-redundant roles. While both contribute to DDAB resistance, SugE2 deletion (ΔsugE2) significantly enhances bacterial adhesion and invasion in IPEC-J2 intestinal cells by upregulating Salmonella pathogenicity island 1 (SPI-1) genes . RNA-seq data from murine infection models show ΔsugE2 strains increase IL-17/IL-23 axis cytokines (e.g., IL-17: 28.60 pg/mL vs. 11.84 pg/mL in wild-type), linking efflux activity to immune evasion . Competitive index assays in mice reveal ΔsugE2 achieves 10-fold higher colonization in the cecum compared to wild-type, underscoring its role in virulence .
What methodologies resolve contradictions in SugE’s role as an exporter versus importer?
Contradictory findings arise from experimental systems and mutagenesis approaches. For example, E. coli SugE overexpression confers QAC resistance , whereas C. freundii SugE mutants display hypersensitivity . To reconcile this, researchers employ:
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Ethidium transport assays: Measure net flux direction (influx vs. efflux) using fluorescent quenching .
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Genetic complementation: Test cross-species functionality (e.g., expressing Salmonella SugE in E. coli ΔsugE) .
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Molecular dynamics simulations: Model residue-substrate interactions (e.g., H24’s role in cation-π bonding) .
How does SugE interact with host pathways during infection?
Intestinal extracts induce sugE2 expression 4-fold, which subsequently suppresses SPI-1 via undetermined signaling cascades . Chromatin immunoprecipitation (ChIP) assays identify SugE2-binding promoters, while transposon mutagenesis screens reveal genetic interactors (e.g., phoPQ). In vivo, SugE2-deficient strains disrupt intestinal barrier integrity, increasing serum endotoxin levels by 2.5-fold compared to wild-type .
Experimental Design Table
| Objective | Methodology | Key Parameters | Expected Outcome |
|---|---|---|---|
| SugE substrate specificity | Competitive MIC assays | QACs (e.g., DDAB, benzalkonium chloride) | IC50 values ± 5% reproducibility |
| Efflux kinetics | Real-time fluorometry (Hoechst 33342) | ΔpH, ATPase inhibitors | Efflux rate constants (k = 0.2–0.5 min⁻¹) |
| Host-pathogen interactions | Murine oral infection model | Bacterial load (CFU/g tissue), cytokine profiling | 10⁴–10⁶ CFU/g in liver/spleen |
Critical Data Contradictions and Resolutions
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Contradiction: SugE confers resistance in E. coli but hypersusceptibility in C. freundii mutants .
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Resolution: Species-specific residue conservation (e.g., C. freundii M39 vs. E. coli L39) alters substrate binding pockets.
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Contradiction: SugE2 deletion increases virulence despite reducing disinfectant resistance .
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Resolution: SPI-1 upregulation compensates for reduced QAC tolerance by enhancing invasion.
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