Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella.

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

Cloning, expression, and functional identification of aquaporin genes from Eimeria tenella.

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in chicken. Aquaporins (AQP) are essential mediators of water regulation and nutritional intake in parasites, and it may be a suitable molecule for chemotherapeutic target and vaccine candidate. We identified two aquaporin genes in Eimeria tenella (EtAQP1 and EtAQP2) with their full sequence, and the expression profiles were analyzed across different stages of E. tenella life cycle. The expression of EtAQP1 and EtAQP2 in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. The immunohistochemical analysis confirms the restriction of antiserum staining to the surface of transfected Xenopus oocytes. Like other AQP family, EtAQPs are transmembrane proteins that are likely important molecules that facilitate solute uptake for parasite intracellular growth and therapeutic targets.