Proteomic comparison of four Eimeria tenella life-cycle stages: unsporulated oocyst, sporulated oocyst, sporozoite and second-generation merozoite

Abstract

We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage-specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most "glideosome" proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle "off shoot" of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.

Figure 1. pH 3–10 NL proteome map of E. tenella merozoites

Soluble proteins from 10⁸ merozoites were resolved by IEF over a broad, nonlinear pH 3–10 range followed by molecular mass on a 12.5% w/v acrylamide gel under denaturing conditions. Protein spots are visualised using silver stain. All protein identifications are labelled on the gel, the spots are numbered and the list of protein identifications is given in Table S3.

Figure 2. Gel pH 3–10 NL (A) resolving E. tenella sporozoite proteins highlighting proteins identified from multiple spots

Soluble proteins from 10⁸ sporozoites were resolved by IEF over a broad, nonlinear pH 3–10 range followed by molecular mass on a 12.5% w/v acrylamide gel under denaturing conditions. Protein spots are visualised using Coomassie Colloidal stain. Representative proteins identified in multiple spots are labelled on the gel, SO7 antigen was identified in all spots ringed blue. The spots are numbered and the complete list of protein identifications are given in Table S5.

Figure 3. Protein numbers detected in the merozoite, sporozoite, early and late oocyst proteomes

Venn diagram to show the numbers of proteins identified in merozoite, sporozoite, and early and late oocyst stages by either MudPIT or 2D LC-MS/MS. Proteins shared by the different life cycle stages are shown.

Figure 4. Numbers of proteins identified from the functional categories in the merozoite, sporozoite, early and late oocyst proteomes

Homology to better characterised proteins allowed MIPs functional categorisation of the proteins detected by either MudPIT or 2D gel LC-MS/MS (Table S1). Protein diversity is represented as the number of proteins found in each category, expressed as a percentage of the total detected in each stage.

Figure 5. Protein abundances associated with the functional categories in the merozoite, sporozoite, early and late oocyst proteomes

The abundance of proteins detected by MudPIT associated with each functional category is represented by A) the total number of peptides detected B) the total spectrum count and C) the sum of the X_corr scores. Each category is expressed as a percentage of the total detected in each stage.

Figure 6. Protein abundances involved in metabolic pathways in merozoites, sporozoites, early and late oocysts

Metabolism associated proteins detected by MudPIT were further categorised into metabolic pathways and the relative protein investment in each pathway by each life cycle stage is shown. The total numbers of peptides (A & B), spectrum (C & D) and sum of X_corr scores (E & F), are represented as a proportion of the total involved in metabolism in each life cycle stage in A, C and E, or as the ratio of merozoite:sporozoite protein proportions (B, D and F).