Recombinant Staphylococcus saprophyticus subsp. saprophyticus Large-conductance mechanosensitive channel (mscL)
Description
Introduction to MscL Channels
Mechanosensitive channels serve as biological emergency release valves for bacteria, protecting cells from lysis during sudden osmotic downshock. The Large-conductance mechanosensitive channel (MscL) represents one of the most extensively studied bacterial mechanosensitive channels, characterized by its substantial conductance of approximately 3 nS, making it permeable to ions, water, and small proteins when opened . These channels were first discovered on the surface of giant Escherichia coli spheroplasts using patch-clamp techniques, with the E. coli MscL gene subsequently cloned in 1994 .
MscL channels function by responding to membrane tension; when bacteria experience hypoosmotic conditions, water influx creates pressure on the cell membrane, triggering the opening of these channels. This mechanism allows the rapid efflux of cytoplasmic solutes, reducing internal pressure and preventing cellular rupture. The remarkable conservation of these channels across diverse bacterial species highlights their fundamental importance to bacterial physiology and survival.
Staphylococcus saprophyticus subsp. saprophyticus MscL Protein
The MscL protein from Staphylococcus saprophyticus subsp. saprophyticus represents a specific variant of this highly conserved channel family. The native protein functions within the context of this Gram-positive coccus, which is notable as a significant cause of urinary tract infections, particularly in young, sexually active females. The complete MscL protein consists of 117 amino acids forming a pentameric complex that creates the functional channel within the bacterial membrane .
The recombinant version of this protein is produced through heterologous expression systems, typically using E. coli as the host organism. This approach allows for the production of significant quantities of the protein for research and commercial applications. The recombinant protein is often tagged, commonly with a histidine tag, to facilitate purification and detection in experimental settings .
Oligomeric Structure
The permeation pathway in MscL channels is typically funnel-shaped, with a larger opening facing the periplasmic surface of the membrane and the narrowest point located near the cytoplasm. This structural arrangement is critical for the channel's function in regulating solute passage during osmotic stress events .
Mechanosensitivity and Channel Gating
The primary function of the MscL protein is to sense mechanical tension in the cell membrane and respond by opening a large pore. This mechanosensitivity is intrinsic to the protein structure and does not require energy input or metabolic activity to function, only an appropriate stimulus . The channel can be activated by membrane tension resulting from osmotic pressure differences across the bacterial cell membrane.
When opened, the MscL channel forms one of the largest gated pores known in biological systems, allowing the passage of molecules up to 30 Å in diameter. This substantial conductance of approximately 3 nS enables the rapid efflux of cytoplasmic contents, including ions, water, and even small proteins, thereby relieving internal pressure during osmotic downshock events .
Response to Channel Agonists
Research has demonstrated that MscL channels, including those from Staphylococcus species, can be activated by small molecule compounds such as the agonist 011A. This compound has been shown to increase the sensitivity of MscL channels, slow bacterial growth, and even decrease viability in quiescent cultures . The effects of such agonists appear to be MscL-dependent, as demonstrated by comparative studies between wild-type and MscL-null bacterial strains .
Production and Purification
The recombinant Staphylococcus saprophyticus subsp. saprophyticus MscL protein is typically produced using E. coli expression systems. The gene encoding the protein is cloned into appropriate expression vectors, often with the addition of affinity tags to facilitate purification. Following expression, the protein is extracted from bacterial cells and purified using affinity chromatography and other protein purification techniques .
As a Model System for Mechanosensation
The recombinant Staphylococcus saprophyticus MscL protein serves as a valuable model system for studying bacterial mechanosensation. Its relatively simple structure compared to eukaryotic mechanosensitive channels makes it an ideal candidate for investigating the fundamental principles of mechanotransduction. Research using this and related MscL proteins has provided insights into how membrane tension is sensed and translated into protein conformational changes that result in channel opening .
Cross-Species Comparison Studies
The high degree of conservation among MscL proteins across bacterial species enables comparative studies that illuminate both the universal and species-specific aspects of channel function. Research has demonstrated that compounds affecting MscL channels, such as the agonist 011A, can influence multiple bacterial species, including Staphylococcus aureus and Mycobacterium smegmatis, in addition to Escherichia coli .
These cross-species studies have revealed that despite differences in amino acid sequence, the fundamental mechanosensitive properties and functional responses of MscL channels are largely conserved. This conservation underscores the essential nature of these channels in bacterial physiology and their potential as broad-spectrum targets for antimicrobial development .
As an Antibiotic Target
The essential role of MscL in bacterial survival under osmotic stress, combined with its absence in mammalian cells, positions it as a potential novel target for antibiotic development. Research has demonstrated that inappropriate activation of MscL channels can be detrimental to bacterial viability. Compounds such as 011A that increase the sensitivity of MscL channels have been shown to slow bacterial growth and decrease viability in multiple bacterial species, including Staphylococcus aureus, a significant human pathogen .
The therapeutic potential of targeting MscL is further enhanced by its expression throughout all phases of bacterial growth, including stationary phase and in biofilms. This characteristic is particularly valuable for addressing persistent infections, such as those caused by Staphylococcus aureus, which can be difficult to treat due to biofilm formation and "dormant" phenotypes .
As an Antibiotic Adjuvant
Perhaps one of the most promising applications for MscL agonists is their potential to serve as adjuvants that enhance the efficacy of existing antibiotics. Research has demonstrated that compounds like 011A can facilitate the entry of antibiotics, such as dihydrostreptomycin (DHS), into bacterial cells by essentially permeabilizing the membrane through MscL channel activation .
Molecular dynamics simulations have supported this hypothesis, showing that 011A can stabilize partially open MscL channels, potentially allowing increased antibiotic passage into the bacterial cytoplasm. This adjuvant approach could be particularly valuable for addressing antibiotic resistance by increasing the effective concentration of antibiotics within bacterial cells .
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order notes, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please communicate with us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: ssp:SSP1410
STRING: 342451.SSP1410
Q&A
What is the Staphylococcus saprophyticus mscL protein and why is it significant?
The Large-conductance mechanosensitive channel (mscL) in S. saprophyticus is a membrane protein consisting of 117 amino acids that functions as a mechanosensitive channel. This protein belongs to a class of membrane channels that respond to mechanical tension in the cell membrane, typically serving as emergency release valves during osmotic stress. The significance of studying this protein lies in understanding bacterial stress responses, particularly in S. saprophyticus, which is a leading Gram-positive cause of uncomplicated urinary tract infections (UTIs) . The protein may play crucial roles in bacterial survival under various environmental stresses, potentially contributing to pathogenicity and antibiotic resistance mechanisms.
How is recombinant S. saprophyticus mscL protein typically expressed and purified?
The recombinant S. saprophyticus mscL protein can be expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The expression typically involves cloning the mscL gene into an appropriate expression vector, transforming E. coli cells, inducing protein expression, and then extracting and purifying the protein using affinity chromatography. For optimal results, the purified protein is typically stored as lyophilized powder and should not undergo repeated freeze-thaw cycles. Working aliquots can be maintained at 4°C for up to one week . When reconstituting the protein, careful attention to buffer conditions is essential to maintain proper folding and function.
What role might mscL play in antimicrobial resistance mechanisms in S. saprophyticus?
While direct evidence linking mscL to antimicrobial resistance in S. saprophyticus is not explicitly presented in the available literature, several inferences can be made based on related research. S. saprophyticus exhibits significant increases in minimum inhibitory concentration (MIC) when transitioning from planktonic to biofilm states, with some isolates switching from susceptible to resistant categories . Mechanosensitive channels like mscL may contribute to stress responses that enable bacteria to survive antibiotic treatment.
Recent comparative genomics studies have revealed correlations between stress response genes and antimicrobial resistance in S. saprophyticus . Computational modeling has identified several genes associated with cefoxitin and oxacillin resistance in mecA-negative isolates, some with predicted functions in stress response and cell wall synthesis . Given that mscL functions in bacterial stress responses, it may interact with these resistance mechanisms, potentially modulating membrane permeability or integrity during antibiotic exposure.
How might mscL function differ between planktonic and biofilm forms of S. saprophyticus?
S. saprophyticus biofilms demonstrate significantly increased resistance to antimicrobial agents compared to their planktonic counterparts . The minimum inhibitory concentration in biofilm (MICB) can increase more than 32-fold compared to planktonic MIC values for various antibiotics, including vancomycin, oxacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and norfloxacin . This suggests substantial physiological differences between these growth states.
The mscL protein, being a mechanosensitive channel involved in osmotic regulation, may exhibit altered expression or function in biofilm environments. The dense extracellular matrix of biofilms creates a different physical environment that could affect membrane tension sensing. Additionally, the microenvironments within biofilms, which often include gradients of nutrients, oxygen, and pH, might induce differential regulation of stress response proteins like mscL. Experimental approaches to investigate these differences would include comparing mscL expression levels between planktonic and biofilm states using RT-qPCR or proteomics, and analyzing the protein's functional properties in membrane vesicles derived from both growth conditions.
What potential interactions exist between mscL and mobile genetic elements in S. saprophyticus?
Genomic analyses of S. saprophyticus have revealed that 29.8% (82/275) of isolates carry staphylococcal cassette chromosome mobile elements, with 25.6% (21/82) of these being mecA-positive . Additionally, antibiotic resistance genes such as erm and erm(44)v carried by bacteriophages have been correlated with high phenotypic non-susceptibility to erythromycin and clindamycin .
While direct evidence for interactions between mscL and mobile genetic elements is not explicitly presented, the presence of these elements in the S. saprophyticus genome suggests potential for horizontal gene transfer that could affect mscL function or regulation. Research questions could include: (1) whether variants of mscL exist due to recombination events; (2) if mobile elements influence mscL expression; and (3) whether physiological stress that activates mscL also induces mobility of genetic elements. Investigating these questions would require whole genome sequencing, transcriptomic analysis, and genetic manipulation studies.
What are the recommended protocols for studying mscL function in S. saprophyticus?
To investigate mscL function in S. saprophyticus, several methodological approaches can be employed:
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Electrophysiological studies: The recombinant His-tagged mscL protein can be reconstituted into artificial lipid bilayers for patch-clamp experiments to characterize channel conductance, gating tension, and ion selectivity.
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Fluorescence-based assays: Utilizing fluorescent probes that are released upon channel opening provides a high-throughput approach to study channel activation under various conditions.
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Genetic manipulation: Creating knockout or knockdown strains of mscL in S. saprophyticus would allow for phenotypic characterization. This can be challenging in S. saprophyticus but could be approached using techniques such as CRISPR-Cas9 or antisense RNA.
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Osmotic shock survival assays: Comparing wild-type and mscL-mutant strains in their ability to survive hypoosmotic shock provides functional insights into the protein's role in osmotic regulation.
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Protein-protein interaction studies: Co-immunoprecipitation or bacterial two-hybrid systems can identify potential interaction partners of mscL, providing insights into its broader functional network.
How can researchers effectively express and purify functional recombinant S. saprophyticus mscL?
The expression and purification of functional recombinant S. saprophyticus mscL requires careful optimization due to its membrane protein nature. The recommended methodology includes:
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Expression system selection: While E. coli is commonly used , expression levels and proper folding may vary depending on the specific strain and expression conditions. BL21(DE3) or C41/C43(DE3) strains are often effective for membrane protein expression.
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Vector design: Including an N-terminal His-tag facilitates purification, but the tag position (N- or C-terminal) should be optimized to avoid interfering with function.
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Induction conditions: Lower temperatures (16-25°C) and reduced inducer concentrations often improve the yield of properly folded membrane proteins.
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Membrane extraction: Efficient extraction requires appropriate detergents; n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucoside (OG) are commonly used for mechanosensitive channels.
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Purification protocol: Immobilized metal affinity chromatography (IMAC) using the His-tag , followed by size exclusion chromatography, typically yields high-purity protein.
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Quality control: Circular dichroism spectroscopy and thermal stability assays should be employed to verify proper folding.
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Storage: The purified protein should be stored as a lyophilized powder to maintain stability, avoiding repeated freeze-thaw cycles .
What biofilm models are appropriate for studying mscL in S. saprophyticus?
Given the clinical relevance of S. saprophyticus biofilms in urinary tract infections , appropriate biofilm models are crucial for studying mscL function in this context. Recommended approaches include:
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Static biofilm models: The polystyrene plate adherence method has been successfully used to identify biofilm-producing S. saprophyticus isolates . This approach is suitable for initial screening and basic functional studies.
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Catheter-based models: Utilizing catheter segments in broth culture with constant stirring can effectively mimic clinically relevant biofilms, as demonstrated in scanning electron microscopy studies of S. saprophyticus .
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Flow systems: Continuous flow chambers better represent the dynamic environment of the urinary tract and allow for real-time observation of biofilm formation and response to perturbations.
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Antimicrobial susceptibility testing in biofilms: The determination of minimum inhibitory concentration in biofilm (MICB) and minimum bactericidal concentration in biofilm (MBCB) provides functional readouts for assessing the role of mscL in antibiotic resistance within biofilms .
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Co-culture models: Incorporating host cells (bladder epithelial cells) or other microbiome members can provide more physiologically relevant conditions for studying mscL function in UTI scenarios.
What are the current limitations in studying S. saprophyticus mscL and how might they be addressed?
Current research on S. saprophyticus mscL faces several methodological and conceptual challenges:
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Limited genetic tools: Unlike model organisms such as E. coli or S. aureus, genetic manipulation of S. saprophyticus remains challenging. Developing optimized transformation protocols and genetic tools specific for S. saprophyticus would significantly advance the field.
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Biofilm heterogeneity: The heterogeneous nature of biofilms complicates the study of protein function within these structures. Single-cell approaches and spatial transcriptomics may help address this challenge.
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Physiological relevance: In vitro studies may not fully recapitulate the in vivo environment of the urinary tract. Developing improved animal models or organoid-based systems could provide more relevant insights.
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Structural data: The lack of high-resolution structural data for S. saprophyticus mscL limits understanding of its functional mechanisms. Cryo-electron microscopy or X-ray crystallography studies are needed.
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Integration with systems biology: Connecting mscL function to broader cellular networks remains challenging. Multi-omics approaches integrating transcriptomics, proteomics, and metabolomics would provide a more comprehensive view.
How can researchers correlate mscL function with antibiotic resistance phenotypes in S. saprophyticus?
To establish meaningful correlations between mscL function and antibiotic resistance in S. saprophyticus, researchers should consider the following approaches:
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Genomic-phenotypic correlations: Building on existing comparative genomics approaches , researchers can analyze correlations between mscL sequence variants or expression levels and antibiotic resistance profiles across diverse S. saprophyticus isolates.
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Controlled expression studies: Creating strains with variable mscL expression levels and assessing their resistance to different antibiotics can establish causal relationships.
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Membrane permeability assays: Since mscL affects membrane integrity, measuring changes in membrane permeability in response to antibiotics in wild-type versus mscL-mutant strains would provide functional insights.
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Real-time monitoring: Developing reporter systems that simultaneously track mscL activity and antibiotic-induced stress responses would help establish temporal relationships between these processes.
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Combinatorial approaches: Testing how mscL mutations interact with known resistance determinants (such as mecA or blaZ ) would reveal potential synergistic or antagonistic relationships in resistance mechanisms.
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Biofilm-specific analyses: Given the dramatic differences in antibiotic susceptibility between planktonic and biofilm states , specialized approaches to measure mscL function specifically within biofilm structures are needed.
