Recombinant Nosema ceranae Aquaporin (AQP)
Description
Introduction to Nosema ceranae as a Pathogen
Nosema ceranae is an intracellular microsporidian parasite that primarily targets the midgut epithelial cells of adult honey bees. This pathogen has gained considerable attention in the scientific community due to its association with colony collapse disorder and its significant impact on apiculture worldwide . Unlike its close relative Nosema apis, N. ceranae demonstrates distinct infection dynamics and potentially higher virulence under certain environmental conditions .
The parasite invades host cells through a specialized apparatus known as the polar tube, which is characteristic of microsporidia . Once inside the host cell, N. ceranae exploits cellular resources to replicate and eventually produce infectious spores. Infection with this pathogen leads to numerous detrimental effects in honey bees, including impaired memory, suppressed immune responses, and potentially colony collapse under specific circumstances .
Given its capacity to compromise honey bee health at both individual and colony levels, understanding the molecular mechanisms underlying N. ceranae pathogenesis has become a priority in apicultural research. Among the various proteins involved in its life cycle and virulence, aquaporin has emerged as a protein of interest due to its potential role in parasite survival and host-pathogen interactions.
Genetic Identity and Expression
The aquaporin protein from Nosema ceranae is encoded by a gene designated as AQP, also referenced in genomic databases as NCER_102220 . This gene has been identified through comprehensive genome sequencing efforts that have mapped the approximately 8.8 Mbp genome of N. ceranae, which contains approximately 2,280 protein-coding genes .
The protein is cataloged in the UniProt database under the accession number C4VBN2, providing researchers with a standardized reference for this molecular entity . The full-length protein consists of 249 amino acids, with the expression region spanning positions 1-249, indicating that the entire protein sequence is typically expressed in recombinant systems .
Proposed Functional Roles
As a member of the aquaporin family, the N. ceranae AQP protein is primarily involved in mediating the transport of water molecules across cell membranes. This function is particularly critical for intracellular parasites, which must regulate their osmotic balance while residing within host cells. Additionally, certain aquaporins can transport small neutral solutes, including glycerol and urea, potentially enhancing the parasite's metabolic capabilities within the host environment.
For an intracellular parasite like N. ceranae, which infects the midgut epithelial cells of honey bees, aquaporin likely plays several crucial roles:
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Osmotic regulation during different life cycle stages
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Adaptation to varying environmental conditions within the host
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Potential involvement in nutrient acquisition from host cells
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Possible contribution to spore formation and germination processes
Recombinant Expression Systems
The recombinant production of Nosema ceranae Aquaporin involves expressing the protein in laboratory expression systems, typically using bacterial, yeast, or insect cell hosts. The commercially available recombinant protein is typically supplied in quantities of 50 μg, though other quantities are also available . The recombinant product is stored in a Tris-based buffer containing 50% glycerol, optimized for protein stability .
For research applications, the recombinant protein may include various tag configurations, with the specific tag type often determined during the production process to optimize expression and purification outcomes . Storage recommendations typically suggest maintaining the protein at -20°C, with extended storage at -20°C or -80°C, and avoiding repeated freeze-thaw cycles .
Research Applications
Recombinant Nosema ceranae Aquaporin serves multiple research purposes:
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Immunological Studies: The recombinant protein can be used to develop antibodies for detecting N. ceranae in infected samples, potentially enhancing diagnostic capabilities for honey bee diseases.
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Structural Analysis: Purified recombinant protein enables researchers to characterize the three-dimensional structure of N. ceranae aquaporin, providing insights into its functional mechanisms.
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Drug Development: As a potential therapeutic target, recombinant aquaporin facilitates screening of compounds that might inhibit its function, potentially leading to novel treatments for nosemosis in honey bees.
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Host-Pathogen Interaction Studies: The protein can be used to investigate how honey bee cells respond to specific N. ceranae proteins, elucidating pathogenic mechanisms.
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Evolutionary Research: Comparative studies with aquaporins from other microsporidian species can reveal evolutionary relationships and adaptive mechanisms among these parasites.
Comparison with Nosema apis
Research comparing N. ceranae with the related species N. apis has revealed significant differences in their infection dynamics and virulence. Studies have shown that while both parasites can reach similar spore levels in the midgut after 10-12 days of infection, their growth patterns and effects on host bees differ .
Experiments investigating mixed infections of both Nosema species have shown interesting competitive dynamics. When bees were infected with different proportions of N. ceranae and N. apis spores, the relative abundance of each species changed over the course of infection. In bees infected with a low dose of N. ceranae (10%) and high dose of N. apis (90%), the proportion of N. ceranae increased slightly from 0.10 to 0.23 after 14 days, although this change was not statistically significant . Conversely, in bees infected with a high dose of N. ceranae (90%) and low dose of N. apis (10%), the proportion of N. ceranae decreased significantly from 0.90 to 0.57 over the same period .
These findings suggest complex competitive interactions between the two Nosema species within the host, with neither species consistently dominating. This dynamic relationship may have important implications for the epidemiology and management of nosemosis in honey bee populations.
Genomic Context of N. ceranae Aquaporin
The genomic analysis of N. ceranae has revealed a robust 8.8 Mbp genome assembly containing 2,280 protein-coding genes . Compared to its sister species N. apis, N. ceranae possesses a higher number of genes involved in nutrient and energy transport, as well as potential drug resistance mechanisms . This genomic profile suggests that N. ceranae may be better equipped to exploit host resources and withstand environmental stressors, potentially contributing to its observed virulence.
The presence of aquaporin within this genomic context further suggests its potential role in the adaptive capabilities of N. ceranae. As a protein involved in water and solute transport, aquaporin may contribute to the parasite's ability to survive and thrive within the host environment, potentially enhancing its virulence compared to N. apis.
Future Research Directions
Despite the information available on Nosema ceranae and its proteins, several knowledge gaps remain regarding its aquaporin specifically. Future research efforts might profitably focus on:
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Functional Characterization: Detailed studies of the transport specificities and kinetics of N. ceranae aquaporin would provide insights into its precise role in parasite physiology.
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Expression Patterns: Investigating when and where aquaporin is expressed during different stages of the N. ceranae life cycle could reveal its temporal significance in pathogenesis.
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Host-Pathogen Interactions: Studies examining how honey bee cells respond to N. ceranae aquaporin could uncover potential host defense mechanisms or susceptibility factors.
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Therapeutic Targeting: Developing compounds that specifically inhibit N. ceranae aquaporin function could lead to novel treatments for nosemosis in honey bees.
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Structural Studies: High-resolution structural analysis of N. ceranae aquaporin could reveal unique features that might be exploited for selective targeting.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them when placing your order. We will fulfill your request to the best of our ability.
Note: All protein shipments are standardly accompanied by blue ice packs. If you require dry ice shipping, please notify 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: nce:NCER_102220
Q&A
What is Nosema ceranae and why is its Aquaporin protein significant?
Nosema ceranae is a microsporidian parasite that primarily infects honey bee (Apis mellifera) gut epithelial cells. This pathogen can lead to impaired memory, suppressed host immune responses, and under certain circumstances, colony collapse . The parasite produces infectious spores that can remain viable for extended periods, allowing transmission between hosts .
Aquaporin (AQP) in N. ceranae is a transmembrane water channel protein that facilitates water movement across cell membranes, which is critical for parasite survival and proliferation . As a key component of the parasite's cellular machinery, Aquaporin represents both an important research target for understanding pathogenesis and a potential intervention point for controlling infections.
What are the optimal storage conditions for maintaining Recombinant Nosema ceranae Aquaporin activity?
For Recombinant N. ceranae Aquaporin, the recommended storage conditions are:
| Storage Parameter | Recommendation |
|---|---|
| Short-term storage | 4°C for up to one week |
| Long-term storage | -20°C or -80°C |
| Buffer composition | Tris-based buffer with 50% glycerol |
| Handling | Avoid repeated freeze-thaw cycles |
The protein is typically supplied in an optimized buffer formulation containing Tris and 50% glycerol to maintain stability . Working aliquots should be prepared to avoid repeated freezing and thawing, which can significantly reduce protein activity and stability.
How can researchers confirm Nosema ceranae infection in experimental honey bee subjects?
Confirming N. ceranae infection in honey bees requires specific methodological approaches:
How does Nosema ceranae Aquaporin function compare to aquaporins from other microsporidian species?
Comparative analysis of microsporidian aquaporins reveals important functional and evolutionary insights:
| Microsporidian Species | Aquaporin Characteristics | Notable Differences |
|---|---|---|
| Nosema ceranae | 249 amino acids, contains NPA motifs | Functions at higher temperatures compared to N. apis |
| Nosema apis | Similar structure to N. ceranae AQP | Less temperature tolerant; spores lose infectivity at higher temperatures |
| Encephalitozoon intestinalis | 251 amino acids | Slightly longer sequence with different C-terminal region |
| Encephalitozoon cuniculi | 250 amino acids | Different transmembrane organization |
| Enterocytozoon bieneusi | 242 amino acids | Shorter sequence with altered channel selectivity |
N. ceranae Aquaporin shows distinct functional adaptations that may contribute to its broader temperature tolerance compared to other microsporidian aquaporins . While sharing the core structural features of the MIP family, variations in specific amino acid residues within the transmembrane domains and selectivity filters likely account for functional differences that adapt these proteins to their respective host environments.
What methodologies are most effective for studying Nosema ceranae Aquaporin function?
Effective methodologies for studying N. ceranae Aquaporin function include:
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Liposome and proteoliposome reconstitution: The protein can be incorporated into artificial lipid bilayers to measure water transport activity. Dynamic light scattering can then be used to determine particle size and assess functional integration .
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Detergent solubilization optimization: The zwitterionic mild detergent (3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) has been identified as a suitable surfactant for aquaporin solubilization .
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Expression optimization: For N. ceranae Aquaporin, optimal expression conditions in E. coli BL21 (DE3) include induction with 0.5 mM IPTG at 25°C for 20 hours, as demonstrated with similar aquaporins .
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Functional water transport assays: Stopped-flow light scattering to measure the rate of water movement across reconstituted proteoliposomes in response to osmotic gradients.
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Affinity chromatography: Utilizing histidine tags for purification via nickel-NTA or similar affinity resins .
How might targeting Aquaporin affect Nosema ceranae pathogenicity and potential treatment strategies?
Targeting Aquaporin represents a promising approach for controlling N. ceranae infections:
As a water channel protein critical for parasite survival, Aquaporin inhibition could potentially disrupt N. ceranae's ability to maintain osmotic balance during infection and proliferation. This approach differs from current treatment strategies that rely primarily on fumagillin-based compounds, which require at least four weeks of application to break the infection cycle .
Current evidence suggests that effective N. ceranae control strategies might combine:
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Aquaporin inhibitors: Compounds that specifically block water transport through N. ceranae Aquaporin channels
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Nutritional supplements: Pollen supplementation has shown effectiveness comparable to fumagillin for moderate infections (<3 million spores/bee)
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Targeted delivery methods: The "drench method" commonly used by commercial beekeepers for administering treatments could be adapted for Aquaporin-targeting compounds
A multi-modal approach targeting both Aquaporin function and providing nutritional support may offer synergistic benefits in managing N. ceranae infections while reducing reliance on traditional antimicrosporidian compounds.
What is the relationship between environmental factors and Nosema ceranae Aquaporin function?
Temperature significantly impacts N. ceranae infectivity and potential Aquaporin function:
N. ceranae spores quickly lose their infectivity when exposed to low temperatures , suggesting temperature-dependent aspects of parasite biology, potentially including Aquaporin function. This temperature sensitivity differs from N. apis, indicating species-specific adaptations in membrane proteins like Aquaporin.
Research on psychrophilic (cold-adapted) aquaporins from other organisms indicates that structural adaptations enable function at low temperatures . Comparative analysis of N. ceranae Aquaporin with psychrophilic aquaporins might reveal key structural differences that explain temperature-dependent functionality.
The seasonal pattern of N. ceranae infections, with regional variations in prevalence , suggests that environmental factors including temperature may regulate Aquaporin expression or function, potentially offering insights for timing intervention strategies.
What are key considerations when designing experiments to study Nosema ceranae Aquaporin in the context of honey bee infections?
When designing experiments investigating N. ceranae Aquaporin in honey bee infections, researchers should consider:
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Infection protocol standardization:
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Environmental variables control:
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Experimental timeline considerations:
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Appropriate controls:
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Multiple assessment metrics:
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Survival time measurements
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Food consumption rates
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Molecular markers of infection
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Physiological indicators of bee health
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How can researchers effectively measure water transport activity of recombinant Nosema ceranae Aquaporin?
Effective methodological approaches for measuring water transport activity include:
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Proteoliposome swelling assays:
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Reconstitute purified Aquaporin into liposomes containing a self-quenching fluorescent dye
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Subject proteoliposomes to osmotic gradients and measure fluorescence changes as water flux occurs
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Compare with control liposomes lacking Aquaporin to determine specific activity
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Stopped-flow spectroscopy:
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Mix proteoliposomes rapidly with solutions of different osmolarity
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Measure light scattering changes as vesicles shrink or swell
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Calculate water permeability coefficients from the rate of volume change
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Yeast functional complementation:
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Express N. ceranae Aquaporin in aquaporin-deficient yeast strains
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Subject yeast to osmotic shock and measure survival or recovery rates
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Compare with control strains expressing known functional aquaporins
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Oocyte swelling assays:
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Express Aquaporin in Xenopus oocytes through mRNA injection
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Place oocytes in hypotonic solution and measure volume changes
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Calculate water permeability from volumetric changes over time
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Each method requires specific controls to distinguish between Aquaporin-mediated water transport and passive diffusion across membranes. Experiments should include inhibitor studies using known aquaporin blockers (like mercury compounds) to confirm channel-specific activity.
How should researchers interpret contradictory findings regarding Nosema ceranae virulence and potential Aquaporin contributions?
The literature contains contradictory findings about N. ceranae virulence, presenting challenges for researchers studying Aquaporin's role:
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Contextualizing virulence findings:
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In Spain, N. ceranae was found to be devastating to colonies and individual bees
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Studies in other regions showed less dramatic effects or no significant association with colony losses
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A 15-year longitudinal study found statistically significant but biologically insignificant effects of N. ceranae on colony losses when compared to Varroa destructor
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Methodological approach to resolving contradictions:
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Implement multivariate analyses to assess the relative influence of multiple factors simultaneously
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Use classification tree analysis to determine hierarchical relationships between pathogens
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Calculate effect sizes in addition to statistical significance to determine biological relevance
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Account for geographical variables, as N. ceranae effects appear to be regional phenomena
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Interpreting Aquaporin's contribution:
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Consider pathogen-specific adaptations of Aquaporin that may explain regional virulence differences
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Analyze Aquaporin expression levels in relation to virulence markers
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Evaluate temperature-dependent functionality that may explain geographical variation in pathogenicity
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Examine potential interactions between Aquaporin function and co-occurring stressors
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The most robust approach involves comprehensive experimental designs that simultaneously assess multiple variables, including Aquaporin expression and function, regional factors, temperature effects, co-infections (particularly Varroa destructor), and nutritional status of colonies.
What structural information about Nosema ceranae Aquaporin can inform functional studies?
Structural analysis of N. ceranae Aquaporin provides crucial insights for functional investigations:
The amino acid sequence of N. ceranae Aquaporin reveals key structural features common to the aquaporin family but with parasite-specific adaptations. Based on analyses of related aquaporins, researchers should focus on:
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Transmembrane domain organization:
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Six transmembrane α-helices forming the water-selective channel
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Loop regions that contribute to channel selectivity and regulation
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N- and C-terminal domains potentially involved in localization or regulation
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Functional motifs:
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NPA (Asparagine-Proline-Alanine) motifs forming the selectivity filter
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Residues lining the pore that determine water specificity versus other small molecules
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Regions contributing to temperature sensitivity and adaptation
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Potential regulatory sites:
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Phosphorylation sites that might regulate channel activity
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Protein-protein interaction domains
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Membrane targeting sequences
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Comparative modeling using known aquaporin structures can help predict N. ceranae Aquaporin's three-dimensional conformation and identify unique features that might explain its specific functional properties in the parasite life cycle, particularly in relation to temperature adaptation and host interaction.
