Recombinant Eimeria tenella 50S ribosomal protein L14, apicoplast (rpl14)

What is the apicoplast in Eimeria tenella and why is rpl14 significant?

The apicoplast is an algal-originated plastid present in apicomplexan parasites, including Eimeria tenella. The E. tenella apicoplast genome is a 35-kb circular AT-rich element whose gene organization resembles more closely that of Toxoplasma gondii than Plasmodium falciparum . The 50S ribosomal protein L14 (rpl14) is encoded by this apicoplast genome and plays a crucial role in the parasite's protein synthesis machinery. Its significance lies in its essential function for parasite survival and its divergence from host proteins, making it an attractive target for anti-parasitic interventions. The apicoplast and its constituent proteins like rpl14 are being studied as potential drug targets against coccidiosis in livestock .

How does Eimeria tenella rpl14 compare structurally to homologous proteins in other apicomplexan parasites?

While the E. tenella apicoplast genome contains an almost identical set of genes to those found in P. falciparum and T. gondii, its encoded genes (including rpl14) share low or moderate homologies with their counterparts in these other apicomplexans . This evolutionary divergence provides unique research opportunities. Phylogenetic reconstructions using maximum likelihood and Bayesian inference methods based on plastid-encoded rpo proteins have placed the apicoplast as a sister to Euglena within the green lineage, though other studies suggest a common red algal ancestry for apicomplexan and dinoflagellate plastids . These conflicting findings suggest that the ancestral host that gave rise to the apicoplast might have already contained some primary green plastid genes, adding complexity to our understanding of rpl14 evolution.

What expression systems are commonly used for producing recombinant Eimeria tenella rpl14?

Recombinant E. tenella 50S ribosomal protein L14 can be expressed in multiple host systems including E. coli, yeast, baculovirus, or mammalian cells . Each expression system offers different advantages:

• E. coli provides high yield and cost-effectiveness but may lack appropriate post-translational modifications

• Yeast systems offer eukaryotic processing capabilities with moderate yield

• Baculovirus systems provide high-level expression of complex proteins

• Mammalian cell systems ensure proper folding and post-translational modifications most similar to the native protein

The product typically achieves ≥85% purity as determined by SDS-PAGE analysis . When selecting an expression system, researchers should consider the intended application, required protein folding, and post-translational modifications needed for functional studies.

How can the apicoplast rpl14 be targeted for developing novel anticoccidial drugs?

The unique nature of the apicoplast and its prokaryotic-type ribosomal proteins like rpl14 make them excellent targets for selective inhibition in drug development strategies. To target rpl14 for anticoccidial drug development, researchers can:

• Perform high-throughput screening of compound libraries against purified recombinant rpl14 to identify molecules that bind specifically to the parasite protein

• Develop in vitro translation assays using E. tenella apicoplast ribosomes to assess compounds that inhibit protein synthesis

• Test structural analogs of antibiotics known to target bacterial 50S ribosomal proteins (like macrolides or lincosamides)

• Design rational inhibitors based on crystal structure analysis of E. tenella rpl14

The USDA Agricultural Research Service has prioritized the development of control and intervention strategies for avian coccidiosis, including potential drug targets like apicoplast proteins . Researchers should establish reliable in vitro assays to assess the efficacy of potential inhibitors and document the level of resistance by various Eimeria species to existing coccidiostats before developing new therapeutic approaches .

What methodologies are effective for studying rpl14 function in Eimeria tenella?

To effectively study rpl14 function, researchers can employ several approaches:

• Gene Knockout/Knockdown Studies: Transfection systems for Eimeria allow for genetic manipulation to assess gene function. Transgenic Eimeria can stably express exogenous genes when the plasmid is successfully integrated into the genome .

• Transcriptional Analysis: Gene expression patterns can be monitored throughout the parasite life cycle. Some genes involved in metabolism vary in their expression as parasites mature, which could provide insights into rpl14 regulation .

• Protein-Protein Interaction Studies: Identify binding partners of rpl14 within the ribosomal complex and potentially with other cellular components.

• Structural Biology Approaches: X-ray crystallography or cryo-EM can reveal the three-dimensional structure of rpl14 alone or within the ribosomal complex.

• Comparative Genomics: Analysis across different Eimeria species and other apicomplexans can provide insights into conserved functional regions.

When developing recombinant Eimeria for such studies, researchers should note that the integration sites of exogenous plasmids in the genome can vary in one transgenic population, potentially affecting expression levels and phenotype .

How can Eimeria tenella be engineered as a vaccine vector expressing modified rpl14?

E. tenella has significant potential as a vaccine delivery vehicle due to its ability to trigger immune responses. Engineering E. tenella to express modified rpl14 or using rpl14 as a carrier for heterologous antigens involves several critical steps:

• Vector Construction: Design expression constructs with appropriate promoters. The His4 or Actin promoter allows constitutive expression throughout the life cycle, while Mic2 or SAG13 promoters provide stage-specific expression .

• Transfection Methods: Transfect E. tenella sporozoites with the constructed plasmid using established methods such as electroporation or nucleofection.

• Selection Strategy: Apply appropriate selection pressures to obtain stable transfectants. Both drug selection and fluorescence-activated cell sorting (FACS) can be employed, with dual selection leading to more stable transgenic populations .

• Clonal Selection: Isolate single oocyst or sporocyst clones to establish homogeneous transgenic lines. Single sporocyst clone progeny have been shown to contain one copy of exogenous DNA as determined by Southern blotting .

• Expression Verification: Confirm expression of the modified rpl14 through fluorescence microscopy, immunofluorescence assays, or Western blotting.

The capacity of heterologous antigen expression in recombinant Eimeria is the critical criterion for developing it as a novel vaccine delivery vehicle . The expression pattern will depend on the chosen promoter and can be targeted to specific developmental stages.

What immune responses can be elicited by recombinant Eimeria tenella expressing modified antigens?

Recombinant E. tenella can trigger both humoral and cell-mediated immune responses against expressed heterologous antigens. Previous studies have demonstrated that:

• Immunization with recombinant Eimeria expressing viral proteins induced low-titer, transgene-specific antibodies in chickens .

• Recombinant E. tenella expressing Toxoplasma gondii surface antigen 1 (Et-TgSAG1) elicited TgSAG1-specific IgY antibody production and IFN-γ-secreting peripheral blood mononuclear cell proliferation after a single oral immunization in chickens .

• Intraperitoneal immunization with Et-TgSAG1 sporozoites efficiently triggered humoral immune responses with predominant IgG2a production in mice, with the antisera recognizing the native SAG1 in T. gondii tachyzoites .

• Recombinant E. tenella expressing immunoprotective antigens like E. maxima immune mapped protein 1 (EmIMP1) elicited antigen-specific antibody production, with increased titers after booster immunization .

• Protection levels can be substantial - recombinant E. tenella expressing Campylobacter jejuni vaccine candidate CjaA induced 91% and 86% immune protection against subsequent C. jejuni challenge after single or serial oral vaccination, respectively .

When designing rpl14-based vaccines, researchers should consider that some recombinant Eimeria lines may show variable antibody reactivity against certain antigens, as seen with E. maxima apical membrane antigen (EmAMA1) .

What methodological approaches are recommended for evaluating the efficacy of rpl14-based vaccine candidates?

To effectively evaluate rpl14-based vaccine candidates, researchers should implement a comprehensive assessment protocol:

• Antibody Response Measurement:

  • Quantify antigen-specific antibodies using ELISA

  • Determine antibody isotype distribution (IgY, IgA, IgM in chickens)

  • Assess functional antibody activity through neutralization assays

• Cell-Mediated Immunity Assessment:

  • Measure antigen-specific T-cell proliferation

  • Quantify IFN-γ and other cytokine production by ELISPOT or intracellular cytokine staining

  • Analyze T-cell subset activation (CD4+, CD8+)

• Challenge Studies:

  • Conduct controlled infection with virulent Eimeria or heterologous pathogens

  • Monitor oocyst shedding as a quantitative measure of protection

  • Assess intestinal lesion scores and histopathological changes

  • Evaluate weight gain and feed conversion ratio in poultry

• Alternative Delivery Assessment:

  • Compare different delivery systems, such as gel bead application tested for live Eimeria oocyst vaccines

  • Measure uptake efficiency and effects on chick performance after placement in poultry houses

• Population Dynamics Analysis:

  • Study the epidemiology of Eimeria during different coccidiosis control programs

  • Assess the impact of vaccination on parasite population dynamics in poultry houses

• Cross-Protection Evaluation:

  • Test protection against various Eimeria species and strains

  • Assess protection against heterologous pathogens when using rpl14 as a carrier for other antigens

The USDA Agricultural Research Service recommends developing and evaluating the efficacy of new vaccine regimens using novel vaccine vector systems as part of their strategic approach to controlling avian coccidiosis .

What purification protocols yield high-quality recombinant Eimeria tenella rpl14?

To obtain high-quality recombinant E. tenella rpl14 for research applications, a multi-step purification process is recommended:

• Initial Capture:

  • When expressed with a His-tag, use one of three His-tag capture materials: high yield, EDTA resistant, or DTT resistant, depending on downstream applications

  • Analyze eluate fractions by SDS-PAGE to identify protein-containing fractions

• Secondary Purification:

  • Subject the best fractions from initial purification to size exclusion chromatography

  • Analyze eluate fractions by SDS-PAGE and Western blot to confirm identity and purity

• Quality Control:

  • Verify purity (target: >95% as determined by SDS-PAGE)

  • Confirm protein concentration using absorbance at 280nm, measured against a specific reference buffer

  • Calculate concentration using the specific absorption coefficient determined by tools such as Expasy's protparam

• Sterilization and Endotoxin Management:

  • Filter the final product through a 0.22 μm filter to ensure sterility

  • Consider endotoxin removal for applications sensitive to bacterial endotoxins

This approach typically yields protein with ≥85% purity as determined by SDS-PAGE , suitable for most research applications.

How can researchers detect and quantify the expression of rpl14 in different life stages of Eimeria tenella?

To effectively detect and quantify rpl14 expression across different life stages of E. tenella, researchers can employ several complementary techniques:

• RNA-based Methods:

  • Quantitative RT-PCR: Design primers specific to rpl14 mRNA for sensitive detection

  • RNA-Seq: Analyze the transcriptome at various developmental stages to compare rpl14 expression levels, similar to approaches used for studying gene regulation during E. acervulina sporulation

  • Northern blotting: Visualize rpl14 transcript size and abundance

• Protein-based Methods:

  • Western blotting: Use antibodies against rpl14 or epitope tags in recombinant parasites

  • Immunofluorescence assays: Visualize the localization and expression of rpl14 in different parasite stages

  • Mass spectrometry: Perform quantitative proteomics across life cycle stages

• Reporter Systems:

  • For recombinant parasites, fluorescent reporter genes can be fused to rpl14 or placed under the control of the rpl14 promoter

  • The reporter gene expression can be constitutively expressed throughout the life cycle when driven by promoters like His4 or Actin

• Single-Cell Approaches:

  • Single-cell RNA-Seq to detect cell-to-cell variation in expression

  • Flow cytometry for reporter-expressing recombinant parasites

When studying expression patterns, it's important to note that genes related to metabolism often vary in their expression as parasites mature , and the regulation of apicoplast genes like rpl14 may follow similar patterns.

What genetic manipulation strategies are most effective for studying rpl14 function in Eimeria tenella?

Several genetic manipulation strategies can be employed to study rpl14 function in E. tenella:

• Stable Transfection:

  • Exogenous plasmids can be successfully integrated into the genome of E. tenella, though integration sites may vary within a transgenic population

  • After continuous selection using both drug pressure and FACS, transgenic populations tend to stabilize

  • Single oocyst progeny of transgenic Eimeria can stably express the reporter gene without selection pressures

• Promoter Selection:

  • The His4 or Actin promoter drives constitutive expression throughout the parasite life cycle

  • The Mic2 or SAG13 promoter restricts expression, excluding the unsporulated oocyst stage

  • Choosing the appropriate promoter is critical for studying stage-specific functions of rpl14

• Integration Analysis:

  • Genome walking or plasmid rescue methods can reveal integration sites in the parasite genome

  • Southern blotting can confirm the copy number of exogenous DNA in the genome

• Phenotypic Assessment:

  • Various integration sites may affect parasite phenotype, including fecundity

  • Some transgenic lines show reduced fecundity (e.g., TE1), while others show increased fecundity (e.g., EtM2e showed 2-fold increase compared to parental strain)

• CRISPR-Cas9 Approaches:

  • While not explicitly mentioned in the search results, CRISPR-Cas9 technology has been adapted for use in other apicomplexan parasites and could potentially be applied to E. tenella for targeted gene editing of rpl14

When designing genetic manipulation experiments, researchers should be aware that different recombinant Eimeria lines may exhibit variable phenotypes, likely due to positional effects of transgene integration .

What are the emerging approaches for targeting apicoplast proteins like rpl14 for novel anticoccidial interventions?

Emerging approaches for targeting apicoplast proteins like rpl14 for anticoccidial interventions include:

How might advances in apicoplast biology inform evolutionary relationships among apicomplexan parasites?

The study of apicoplast biology, including proteins like rpl14, provides valuable insights into evolutionary relationships among apicomplexans:

• Phylogenetic Analysis: The E. tenella apicoplast genome sequence has contributed to reexamining apicoplast genome evolution. Phylogenetic reconstructions using plastid-encoded rpoB, rpoC1, and rpoC2 proteins placed the apicoplast as a sister to Euglena within the green lineage .

• Conflicting Evolutionary Signals: While some studies place apicoplasts within the green lineage, others based on plastid gene organization and nuclear-encoded plastid proteins support a common red algal ancestry of apicomplexan and dinoflagellate plastids .

• Endosymbiotic Gene Transfer: If apicoplasts indeed originated from a red algal ancestor, the green relationship of apicomplexan genes might suggest that the ancestral host already contained primary green plastid genes before acquiring the red plastid .

• Genomic Comparisons: The E. tenella plastid genome organization resembles that of T. gondii more closely than P. falciparum, suggesting closer evolutionary relationships between certain apicomplexan groups .

• Protein Divergence Analysis: The varying degrees of homology between E. tenella apicoplast proteins and their counterparts in other apicomplexans provide molecular clocks for estimating divergence times .

Further research on apicoplast proteins like rpl14 across different apicomplexan species will continue to refine our understanding of their evolutionary history and relationships, potentially resolving current conflicts in phylogenetic signals.