Recombinant Plasmodium berghei ATPase ASNA1 homolog (PB000618.02.0)

What is the Plasmodium berghei ATPase ASNA1 homolog and its significance in malaria research?

The Plasmodium berghei ATPase ASNA1 homolog (PB000618.02.0) is an arsenical pump-driving ATPase that belongs to a family of proteins widely distributed across prokaryotes and eukaryotes . In Plasmodium parasites, this protein is homologous to the arsenite-stimulated ATPase found in other organisms. The protein is significant in malaria research because related homologs have been implicated in drug resistance mechanisms and parasite survival.

Comparative studies with ASNA1 homologs in other parasites, such as Eimeria tenella (EtASNA1), suggest these proteins may play critical roles in drug resistance and host cell invasion . ASNA1 proteins function as detoxification pumps that actively remove toxic compounds from cells, potentially contributing to resistance against antimalarial compounds . Understanding PB000618.02.0 provides insights into Plasmodium biology and potential drug resistance mechanisms.

How does PB000618.02.0 compare structurally and functionally to ASNA1 homologs in other Plasmodium species?

PB000618.02.0 in P. berghei shows structural and functional similarities to ASNA1 homologs in other Plasmodium species, particularly the P. falciparum homolog PFD0725c . Both proteins contain characteristic ATPase domains necessary for their function as arsenical pump-driving ATPases.

Comparative genomic analyses between human and rodent malaria parasites have revealed that while core functional domains are conserved, there may be species-specific variations that reflect adaptations to different hosts . For example, when analyzing protein sequences across six Plasmodium species (three human and three rodent malaria parasites), researchers identified conserved network modules containing these ATPases, suggesting their fundamental importance across Plasmodium species .

The functional conservation is evidenced by studies of related ASNA1 homologs, which demonstrate roles in:

• Arsenic/antimony detoxification

• Membrane protein targeting

• Response to cellular stress

• Potential involvement in drug resistance mechanisms

What are the recommended protocols for cloning and expressing recombinant PB000618.02.0?

For successful cloning and expression of recombinant PB000618.02.0, researchers should consider the following methodological approach based on successful protocols used for related ASNA1 homologs:

• Gene Amplification:

  • Design primers based on the PB000618.02.0 open reading frame, incorporating appropriate restriction sites (e.g., EcoRI and SalI)

  • Use cDNA from P. berghei sporozoites or blood stages as template

  • Perform PCR under optimized conditions (e.g., 95°C for 3 min; 32 cycles of 95°C for 45s, 60-62°C for 45s, 72°C for 2 min, followed by 10 min at 72°C)

• Cloning:

  • Purify PCR products using gel purification

  • Clone into a holding vector (e.g., pGEM-T-easy) for sequence verification

  • Subclone into an expression vector such as pGEX-6P-1 for GST-tagged protein expression

• Protein Expression:

  • Transform the recombinant plasmid into E. coli BL21(DE3)

  • Induce protein expression with 1.0 mM IPTG when bacterial culture reaches OD600 of 0.6

  • Incubate for 6 hours post-induction at an optimized temperature (typically 25-30°C for better solubility)

• Protein Purification:

  • Harvest cells by centrifugation and lyse by sonication

  • Purify the recombinant protein using affinity chromatography (GST column)

  • Confirm purity using 12% SDS-PAGE

  • Determine protein concentration using BCA protein assay

This protocol has been successfully applied to the ASNA1 homolog in E. tenella and can be adapted for P. berghei with appropriate modifications based on codon usage and expression optimization for this species .

What methods are most effective for analyzing PB000618.02.0 expression across different developmental stages of P. berghei?

Analysis of PB000618.02.0 expression across different developmental stages requires a combination of transcriptomic and proteomic approaches:

• Quantitative Real-Time PCR (qRT-PCR):

  • Isolate total RNA from different P. berghei life stages (e.g., sporozoites, liver stages, blood stages, gametocytes)

  • Treat with DNase I to remove genomic DNA contamination

  • Synthesize cDNA using reverse transcriptase and random primers

  • Design specific primers for PB000618.02.0 and a reference gene (e.g., 18S rRNA)

  • Perform qRT-PCR in triplicate with at least three biological replicates

  • Analyze using the 2^-ΔΔCt method to determine relative expression levels

• Western Blotting:

  • Prepare protein extracts from different developmental stages

  • Separate proteins by SDS-PAGE and transfer to PVDF membrane

  • Probe with specific antibodies against PB000618.02.0

  • Use appropriate secondary antibodies and detection systems

  • Quantify band intensity using image analysis software

  • Normalize to housekeeping proteins (e.g., GAPDH or actin)

• Immunofluorescence Assay (IFA):

  • Fix parasites from different developmental stages

  • Permeabilize and block non-specific binding

  • Incubate with anti-PB000618.02.0 antibodies

  • Visualize using fluorescently-labeled secondary antibodies

  • Counterstain nuclei with DAPI

  • Analyze protein localization using confocal microscopy

This multi-method approach provides comprehensive data on both transcriptional and translational regulation of PB000618.02.0 throughout the parasite life cycle, as demonstrated in studies of the E. tenella homolog .

How can comparative genomics approaches be used to investigate the function of PB000618.02.0?

Comparative genomics provides powerful tools for investigating PB000618.02.0 function through several methodological approaches:

• Sequence-Based Network Analysis:

  • Compare protein sequences from multiple Plasmodium species (human and rodent parasites)

  • Construct networks based on sequence alignment similarities

  • Partition the network into modules/clusters using methods like BGLL

  • Identify enrichment patterns across species

  • This approach can reveal whether PB000618.02.0 is conserved across all Plasmodium species or specifically enriched in rodent malaria parasites

• Ortholog Identification and Functional Inference:

  • Identify orthologs of PB000618.02.0 in other species with known functions

  • Infer potential functions based on characterized orthologs

  • For example, studies of ASNA1 in E. coli, C. elegans, and humans suggest roles in arsenite resistance, stress response, and membrane protein targeting

• Comparative Expression Analysis:

  • Compare expression patterns of PB000618.02.0 with its orthologs in other Plasmodium species

  • Identify correlation with specific phenotypes (e.g., drug resistance, virulence)

  • This can reveal potential functional conservation or divergence

• Evolutionary Analysis:

  • Perform phylogenetic analysis to understand the evolutionary history of PB000618.02.0

  • Calculate selection pressures (dN/dS ratios) to identify regions under positive selection

  • Correlate evolutionary patterns with functional domains

This approach has successfully identified genes involved in drug resistance in Plasmodium and genes potentially contributing to cerebral malaria in P. berghei , suggesting it would be valuable for understanding PB000618.02.0 function.

What is the evidence linking PB000618.02.0 to drug resistance in Plasmodium berghei?

While direct evidence specifically for PB000618.02.0 in P. berghei drug resistance is limited in the provided search results, comparative studies with ASNA1 homologs in related parasites provide important insights:

• Differential Expression in Drug-Resistant Strains:
  Studies of the E. tenella ASNA1 homolog (EtASNA1) demonstrated significantly higher expression in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains compared to drug-sensitive (DS) strains . Transcriptome sequencing showed log2 ratios of DZR/DS reaching 2.45 and MRR/DS reaching 2.27, indicating substantial upregulation .

• Correlation with Drug Concentration:
  Expression levels of EtASNA1 increased proportionally with increasing drug concentrations, suggesting a dose-dependent response mechanism . This pattern was observed in both laboratory-induced resistant strains and field isolates .

• Functional Mechanisms:
  ASNA1 proteins function as detoxification pumps that actively export toxic compounds from cells, reducing intracellular concentrations to subtoxic levels . This mechanism has been demonstrated for arsenicals and antimonials in other organisms, and may extend to antimalarial compounds in Plasmodium .

Based on this evidence from related systems, PB000618.02.0 may contribute to drug resistance in P. berghei through similar mechanisms of increased expression and active drug efflux. Further experimental validation specific to P. berghei is needed to confirm this hypothesis.

How might post-translational modifications affect PB000618.02.0 function in different parasite stages?

Post-translational modifications (PTMs) likely play critical roles in regulating PB000618.02.0 function across different parasite stages. While specific PTM data for PB000618.02.0 is not directly provided in the search results, we can extrapolate from studies of related proteins:

• Potential PTMs Affecting PB000618.02.0:

  • Phosphorylation: May regulate ATPase activity and protein-protein interactions

  • Acetylation: Could affect protein stability and subcellular localization

  • Ubiquitination: May control protein turnover and degradation

  • SUMOylation: Might influence nuclear-cytoplasmic transport and stress responses

• Stage-Specific Regulation:
  Studies of the E. tenella homolog showed significantly higher expression in second-generation merozoites and unsporulated oocysts compared to other developmental stages . This differential expression may be accompanied by stage-specific PTMs that modulate protein function according to the parasite's needs in each stage.

• Functional Implications:

  • In sporozoites: PTMs may enhance roles in host cell invasion

  • In blood stages: Modifications might optimize detoxification functions

  • During stress responses: PTMs could rapidly activate protective mechanisms without requiring new protein synthesis

Future research should employ phosphoproteomics, acetylomics, and other PTM-specific analyses to identify modifications on PB000618.02.0 and correlate them with functional changes across the parasite life cycle.

What approaches can be used to validate PB000618.02.0 as a potential drug target?

Validating PB000618.02.0 as a potential drug target requires a multi-faceted approach combining genetic, biochemical, and pharmacological methods:

• Genetic Validation:

  • Gene knockout or knockdown studies using CRISPR/Cas9 or conditional expression systems

  • Assessment of resulting phenotypes including growth, invasion capability, and drug sensitivity

  • Complementation studies to confirm phenotype specificity

• Biochemical and Structural Characterization:

  • Recombinant protein expression and purification

  • Enzymatic assays to measure ATPase activity and inhibition

  • Structural determination through X-ray crystallography or cryo-EM

  • Identification of druggable pockets through in silico analysis

• Functional Assays:

  • In vitro inhibition experiments similar to those performed with anti-EtASNA1 antibodies, which significantly inhibited sporozoite invasion of host cells

  • Drug sensitivity assays comparing wild-type and PB000618.02.0-modified parasites

  • Assessment of resistance development potential

• Specific Inhibitor Development and Testing:

  • High-throughput screening of compound libraries

  • Structure-based drug design targeting unique features of PB000618.02.0

  • Medicinal chemistry optimization of lead compounds

  • Evaluation of specificity, potency, and toxicity profiles

Studies of related proteins suggest PB000618.02.0 may be a promising target due to its potential roles in both parasite survival and drug resistance mechanisms .

How should researchers address contradictory data regarding PB000618.02.0 function and expression?

When faced with contradictory data regarding PB000618.02.0, researchers should employ a systematic approach:

Example from related research: Studies of EtASNA1 used both in vivo and in vitro experiments to analyze expression changes after infection and drug exposure, finding that while baseline expression varied between developmental stages, drug addition consistently upregulated expression .

What bioinformatic tools and databases are most valuable for analyzing PB000618.02.0?

Several specialized bioinformatic tools and databases are particularly valuable for analyzing PB000618.02.0:

• Sequence Analysis Tools:

  • BLAST (https://www.ncbi.nlm.nih.gov/BLAST/): For identifying homologs across species

  • ToxoDB (https://toxodb.org/toxo/): Plasmodium genome database for gene information

  • ProtParam (http://www.expasy.org/tools/protparam.html): For calculating molecular mass, theoretical isoelectric point, and other physicochemical properties

• Structural Prediction Tools:

  • SignalP (http://www.cbs.dtu.dk/services/SignalP/): For signal peptide prediction

  • TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/): For transmembrane region identification

  • Motifscan (http://hits.isb-sib.ch/cgi-bin/motif_scan): For protein motif prediction

  • AlphaFold: For protein structure prediction

• Network Analysis Tools:

  • BGLL algorithm: For partitioning protein sequence networks into functional modules

  • Cytoscape: For visualizing and analyzing molecular interaction networks

• Expression Analysis Resources:

  • PlasmoDB: For accessing transcriptomic and proteomic data across life cycle stages

  • Gene Expression Omnibus (GEO): For comparing expression data from different studies

• Comparative Genomics Platforms:

  • OrthoMCL: For identifying orthologous groups across Plasmodium species

  • KEGG Pathway Database: For mapping PB000618.02.0 to conserved metabolic and signaling pathways

These tools have been successfully used for analyzing related proteins and can be applied specifically to PB000618.02.0 to generate comprehensive insights into its structure, function, and evolutionary relationships .

What experimental controls are essential when studying PB000618.02.0 expression in drug-resistance contexts?

When studying PB000618.02.0 expression in drug-resistance contexts, implementing rigorous controls is essential to ensure valid and reproducible results:

• Strain Controls:

  • Drug-sensitive parent strain as negative control

  • Multiple independently generated resistant strains to confirm consistency

  • Field isolates with known resistance profiles to validate laboratory findings

• Drug Exposure Controls:

  • Dose-response series to establish concentration dependence

  • Time-course experiments to capture temporal expression changes

  • Multiple drugs with different mechanisms of action to distinguish specific from general responses

• Developmental Stage Controls:

  • Stage-matched parasites for all comparisons

  • Analysis across multiple life cycle stages to identify stage-specific effects

  • Synchronization protocols to minimize stage heterogeneity

• Molecular Controls:

  • Multiple reference genes for qRT-PCR normalization (e.g., 18S rRNA)

  • Housekeeping proteins as loading controls for western blots

  • Antibody specificity controls (pre-immune serum, peptide competition)

• Methodological Controls:

  • Technical replicates (minimum triplicate) for all assays

  • Biological replicates (minimum three independent experiments)

  • No-template and no-reverse transcriptase controls for qRT-PCR

In studies of the E. tenella ASNA1 homolog, researchers implemented these controls by comparing expression across different resistant strains (DZR, MRR, SMR) to sensitive strains, examining multiple developmental stages, and performing both in vivo and in vitro experiments to validate findings .

How might CRISPR-Cas9 gene editing be optimized for studying PB000618.02.0 function in P. berghei?

Optimizing CRISPR-Cas9 gene editing for studying PB000618.02.0 in P. berghei requires careful consideration of several methodological aspects:

• Guide RNA Design:

  • Select target sites with minimal off-target effects using algorithms specific for P. berghei genome

  • Design multiple gRNAs targeting different regions of PB000618.02.0

  • Consider the GC content and secondary structure of gRNAs for optimal efficiency

  • Test gRNA efficiency using in vitro cleavage assays before transfection

• Donor Template Strategy:

  • For gene knockout: Design homology arms of 500-1000 bp flanking a selection marker

  • For point mutations: Include 1-2 kb homology arms and silent mutations to prevent re-cutting

  • For tagging: Ensure the tag does not interfere with protein function by placing it at the C-terminus

  • Consider codon optimization for P. berghei

• Delivery Methods:

  • Optimize electroporation parameters for maximum transfection efficiency

  • Consider ribonucleoprotein (RNP) complex delivery to reduce off-target effects

  • Use selection markers appropriate for P. berghei (e.g., pyrimethamine resistance)

• Phenotypic Validation:

  • Design comprehensive phenotypic assays to assess:

    • Growth rates in vivo and in vitro

    • Drug susceptibility profiles

    • Host cell invasion efficiency

    • Developmental progression

  • Compare results to those observed with the E. tenella homolog, where anti-EtASNA1 antibody incubation significantly inhibited sporozoite invasion

• Conditional Systems:

  • Implement conditional knockdown systems (e.g., auxin-inducible degron) if PB000618.02.0 proves essential

  • Consider stage-specific promoters to restrict modifications to specific life cycle stages

  • Develop complementation systems to validate phenotypes and perform structure-function studies

This optimized approach will enable precise genetic manipulation of PB000618.02.0 to determine its functions in parasite biology and drug resistance mechanisms.

What comparative proteomic approaches would best identify interaction partners of PB000618.02.0?

To identify interaction partners of PB000618.02.0, several comparative proteomic approaches can be employed:

• Co-Immunoprecipitation (Co-IP) Combined with Mass Spectrometry:

  • Generate specific antibodies against PB000618.02.0 or use tagged recombinant protein

  • Perform Co-IP from different developmental stages and under various conditions (e.g., drug exposure)

  • Analyze precipitated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Compare to appropriate controls (pre-immune serum, unrelated protein)

  • Validate interactions using reciprocal Co-IP or other methods

• Proximity-Based Labeling Techniques:

  • Generate fusion proteins of PB000618.02.0 with BioID or APEX2

  • Express in parasites and activate labeling at specific time points or conditions

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures transient interactions and works in native cellular environments

• Yeast Two-Hybrid Screening:

  • Use PB000618.02.0 as bait against a P. berghei cDNA library

  • Screen for positive interactions and verify using secondary assays

  • Perform domain mapping to identify specific interaction regions

• Quantitative Interaction Proteomics:

  • Compare the interactome of PB000618.02.0 between drug-sensitive and resistant parasites

  • Identify differentially interacting proteins using SILAC or TMT labeling

  • Correlate changes with functional properties and drug resistance phenotypes

• Cross-Species Comparative Analysis:

  • Compare PB000618.02.0 interaction networks with those of homologs in other species

  • Identify conserved interaction partners that may represent core functional complexes

  • This approach leverages known interactions of ASNA1 homologs in other organisms, such as E. tenella

These complementary approaches would generate a comprehensive interaction network for PB000618.02.0, providing insights into its functional roles in different cellular processes and potential involvement in drug resistance mechanisms.

What strategies can overcome difficulties in expressing soluble recombinant PB000618.02.0?

Researchers often encounter challenges when expressing Plasmodium proteins in heterologous systems. Here are effective strategies to overcome solubility issues with recombinant PB000618.02.0:

• Expression System Optimization:

  • Try multiple expression systems beyond E. coli (e.g., yeast, insect cells)

  • Test different E. coli strains optimized for difficult proteins (Rosetta, Arctic Express, SHuffle)

  • Compare results with successful expression of the E. tenella homolog in E. coli BL21(DE3)

• Fusion Tag Selection:

  • Test multiple solubility-enhancing tags:

    • MBP (maltose-binding protein) for improved solubility

    • SUMO tag for enhanced folding

    • Thioredoxin (Trx) for disulfide bond formation

    • Compare with GST-tag approach used successfully for EtASNA1

• Expression Condition Optimization:

  • Reduce induction temperature (16-25°C) to slow protein folding

  • Decrease IPTG concentration (0.1-0.5 mM) to reduce expression rate

  • Supplement media with osmolytes (sorbitol, glycine betaine)

  • Add specific metal ions required for proper folding

• Domain-Based Approach:

  • Express individual domains rather than full-length protein

  • Design constructs based on bioinformatic predictions of domain boundaries

  • Create a panel of truncated constructs to identify soluble fragments

• Co-expression Strategies:

  • Co-express with chaperone proteins (GroEL/GroES, DnaK/DnaJ)

  • Co-express with known interaction partners to stabilize the protein

  • Include rare tRNA codons to overcome codon bias issues

These strategies have proven effective for expressing difficult parasite proteins and can be applied systematically to overcome challenges with PB000618.02.0 expression, building upon the successful methodology used for the E. tenella homolog .

How can researchers troubleshoot inconsistent results in drug resistance assays involving PB000618.02.0?

Inconsistent results in drug resistance assays involving PB000618.02.0 can be addressed through systematic troubleshooting:

• Strain Verification and Maintenance:

  • Confirm resistance phenotype regularly through dose-response assays

  • Maintain drug pressure during culturing to prevent resistance loss

  • Use early passage parasites to minimize genetic drift

  • Sequence PB000618.02.0 to verify absence of mutations or polymorphisms

  • Apply lessons from studies of drug-resistant E. tenella strains, which maintained stable resistance phenotypes

• Standardization of Experimental Conditions:

  • Control for parasite life cycle stage and synchronization

  • Standardize inoculum size and parasitemia

  • Maintain consistent drug preparation and storage conditions

  • Minimize variation in host factors (e.g., use mice of same age, sex, and strain)

  • Implement precise timing for all experimental steps

• Technical Optimization:

  • Validate antibody specificity for PB000618.02.0 detection

  • Optimize primer design and qRT-PCR conditions for expression analysis

  • Include multiple housekeeping genes for normalization

  • Use quantitative methods for protein detection (e.g., western blot with standard curves)

• Comprehensive Data Analysis:

  • Apply appropriate statistical methods for small sample sizes

  • Conduct power analyses to determine required replicate numbers

  • Use statistical tests that account for biological variability

  • Consider hierarchical or mixed-effects models for complex datasets

• Multi-method Validation:

  • Confirm key findings using complementary techniques

  • Compare in vitro and in vivo results to identify context-dependent effects

  • Use both transcriptional (qRT-PCR) and translational (western blot) analyses

By implementing these troubleshooting strategies, researchers can minimize inconsistency and generate reliable data regarding the role of PB000618.02.0 in drug resistance mechanisms.

How might the study of PB000618.02.0 contribute to next-generation antimalarial strategies?

Understanding PB000618.02.0 could significantly impact the development of novel antimalarial strategies through several pathways:

• Target-Based Drug Development:

  • If validated as essential, PB000618.02.0 could become a direct drug target

  • Structural studies could enable rational design of specific inhibitors

  • The apparently critical role of ASNA1 homologs in parasite survival and invasion, as demonstrated for EtASNA1 , suggests PB000618.02.0 may be essential for P. berghei

• Resistance Mechanism Circumvention:

  • Understanding PB000618.02.0's role in drug resistance could inform combination therapies

  • Inhibitors of PB000618.02.0 might restore sensitivity to existing antimalarials

  • Similar to how the upregulation of EtASNA1 in drug-resistant E. tenella strains suggests its involvement in resistance mechanisms

• Cross-Species Therapeutic Strategies:

  • Comparative analysis of ASNA1 homologs across Plasmodium species could reveal conserved vulnerabilities

  • Targeting these conserved elements might produce broad-spectrum antimalarials

  • Network analysis approaches have successfully identified conserved modules across Plasmodium species that could be targeted

• Host-Parasite Interaction Disruption:

  • If PB000618.02.0 is involved in host cell invasion, as suggested by studies of EtASNA1 , blocking this function could prevent infection establishment

  • Anti-PB000618.02.0 antibodies or peptide mimetics could potentially neutralize this activity

  • In vitro inhibition experiments with anti-EtASNA1 antibodies significantly inhibited host cell invasion

• Biomarker Development:

  • Expression levels of PB000618.02.0 might serve as biomarkers for predicting drug resistance

  • Similar to how EtASNA1 expression levels correlated with resistance to multiple drugs

By pursuing these research directions, studies of PB000618.02.0 could contribute significantly to addressing the ongoing challenge of antimalarial drug resistance and the need for new therapeutic approaches.

What interdisciplinary approaches might accelerate understanding of PB000618.02.0 function?

Accelerating our understanding of PB000618.02.0 requires innovative interdisciplinary approaches that integrate multiple scientific disciplines:

• Systems Biology and Network Analysis:

  • Integrate transcriptomics, proteomics, and metabolomics data to place PB000618.02.0 in broader biological networks

  • Apply network module analysis methods like BGLL to identify functional associations

  • Model the effects of PB000618.02.0 perturbation on parasite biology using computational simulations

• Structural Biology and Biophysics:

  • Determine high-resolution structures using cryo-EM or X-ray crystallography

  • Analyze protein dynamics through molecular dynamics simulations

  • Characterize protein-protein and protein-drug interactions using biophysical methods (ITC, SPR)

• Chemical Biology and Drug Discovery:

  • Develop chemical probes that specifically target PB000618.02.0

  • Perform chemogenomic profiling to understand the relationship between chemical structure and biological activity

  • Screen for small molecules that modulate PB000618.02.0 function

• Advanced Imaging Techniques:

  • Apply super-resolution microscopy to visualize PB000618.02.0 localization and dynamics

  • Use live-cell imaging to track protein movement during host cell invasion and parasite development

  • Employ correlative light and electron microscopy to connect function with ultrastructure

• Evolutionary Biology and Comparative Genomics:

  • Analyze PB000618.02.0 conservation and divergence across Apicomplexa

  • Identify selection pressures that have shaped ASNA1 evolution

  • Apply comparative methods that have successfully identified disease-related genes in P. berghei through comparison with other Plasmodium species