| The analysis of genomic data found that the coding gene of 2-Methylcitrate cycle(2-MCC) pathway enzymes exist in Eimeria tenella. In this study, through cloning and prokaryotic expression of E. tenella 2-MCC pathway related genes, and analysis the enzyme kinetic and enzyme inhibition kinetic to found effective lead compounds. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs.The main works of this studies include:The ORF sequences of EtMS, EtACN, EtCS, EtACS, and the conserved domain sequences of EtMD and EtML have cloned from E. tenella total RNA by RT-PCR, the results confirmed the presence of 2-MCC pathway in chicken coccidiosis.The bioinformatics analysis results found the predicted sequence of EtprpE was identical to the EtACS gene sequence, indicating the ACS may have prpE function in E. tenella.EtMS?EtACN?EtCS ? EtACS have successfully expressed in Escherichia coli expression system.Obtained active recombinant expression protein MBP-EtMS, and have carried on the enzymatic kinetic analysis. Confirmed that EtMS can not only use the propionyl coenzyme A as a substrate but also use acetyl coenzyme A as substrate.Genomic DNA methylation is part of a number of key biological processes in organisms, such as gene expression regulation, genomic imprinting, etc. and is essential for cell cycles. To date, little was known in apicomplexan parasites about cytosine methylation in genomic DNA, although it is confirmed that this important epigenetic modification exists in lots of lower eukaryotes, plants, and animals. We examined the levels of cytosine DNA methylation in avian coccidia, and in other important apicomplexan parasite genomes with an ELISA-like detection kit (Abcam). The results demonstrated that low levels of 5-methylcytosine (5-mC) are widespread in Eimeria spp., Toxoplasma gondii, Cryptosporidium spp., and Neospora caninum. The contents of 5-mC were 0.20 ± 0.02% in E. tenella sporulated oocysts,0.18±0.02% in E. tenella merozoites,0.22±0.04% in T. gondii tachyzoites,0.28±0.03% in N. caninum tachyzoites, and 0.06±0.01%, 0.11±0.01%, and 0.09±0.01% in C. andersoni, C. baileyi, and C. parvum sporulated oocysts, respectively. In addition, we found that 5-mC contents in E. tenella varied considerably at different life stages, with sporozoites having the highest percentage of 5-mC (0.78 ± 0.17%). Similar stage differences in 5-mC were also found in E. maxima, E. necatrix, and E. acervulina, the levels of 5-mC in their sporozoites being 3.9-,2.0-,2.4-, and 2.0-fold higher than that in sporulated oocysts, respectively (p< 0.05).These results suggest that DNA methylation may play roles in the stage conversion of E. tenella. Furthermore, DNA methylation levels of E. tenella sporozoites can be inhibited in vitro by 5-azacytidine added to culture medium, an total DNA methyltransferase-like activity can be detected in sporozoites. The DNA methyltransferase-like activity levels are 3.3±0.28×103,8.4±1.3×103, and 9.3±0.4×103 RFU/h/mg at 4 ?g, 8 ?g, and 12 ?g of whole cell extracts, respectively, suggesting the potential for a DNA methyltransferase-like mechanism in E. tenella. In conclusion, genomic DNA methylation may be widespread in these common apicomplexan parasites, and represent a primary and significant step in our understanding of DNA methylation and its regulations on gene expression in E. tenella. |
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