Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella

Abstract

The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.

Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella

“…We also demonstrated the importance of EteIF3s7 in host cell invasion in invasion inhibition experiments. In our previous study, we showed that some anti-protein specific polyclonal antibodies reduced E. tenella sporozoites invasion in vitro (Han et al, 2013;Jiang et al, 2012b). In our current study, the reduced capacity of sporozoites preincubated with an anti-rEteIF3s7 polyclonal antibody to invade DF-1 cells demonstrated the importance of EteIF3s7 in host cell invasion.…”

“…The gene expression profiles of the various developmental stages of Eimeria likely differ due to the different morphological characteristics and living environments required for the different stages of the life cycle (Han et al, 2010;Miska et al, 2004;Ng et al, 2002). Because sporozoites invade the epithelial cells lining the intestine, genes differentially expressed in sporozoites might be involved in host cell invasion (Han et al, 2013;Kinnaird et al, 2004;Wiersma et al, 2004).…”

Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

“…Our previous studies showed that in vitro invasion inhibition assays reduced E. tenella sporozoite invasion with specific polyclonal antibodies [30], [31], [55], [56]. Western blots showed that rabbit anti-rEtPDIL antibody recognized a single band of the expected 24 kDa in E. tenella sporozoites, merozoites and sporulated oocysts, but not unsporulated oocysts.…”

“…The chicken embryo fibroblast cell line DF-1, derived from East Lansing Line (ELL-0) chicken embryos, was used for infection, inhibition assays and immunofluorescence experiments [30], [31]. Cells were infected at a ratio of one sporozoite per cell in complete medium (DMEM, Invitrogen, USA) containing 10% fetal calf serum (FCS) at 41°C.…”

Two Calcium-Dependent Protein Kinases fromChlamydomonas reinhardtiiare transcriptionally regulated by nutrient starvation

“…[56][57][58][59][60][61][62] Studies on protozoans have shown that CDPKs are localized in the schizonts and pellicle in Plasmodium, in sporozoites in Eimeria, and also in the cilia and body of Paramecium. [63][64][65][66][67] CDPK3 is localized in the C. reinhardtii membrane-matrix fraction of the flagella. 19 Another 65 kDa CDPK has been shown to be localized in the C. moewusii flagella.…”