Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

Abstract

Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.

Keywords: Coccidium; Eimeria tenella; apicomplexa; electron transport chain; inhibitor; membrane protein; mitochondria; succinate dehydrogenase; ubiquinone.

Figure 1

(a) Electron microscopy of Eimeria tenella sporozoite; (b) high magnification of the mitochondria of the parasite; (c) staining of sporozoites by mitochondrial specific probes (MitoTracker^®, Thermo Fisher Scientific). Bars = 2 μm (a) and 5 μm (c). The positions of the mitochondria are indicated by the arrows. R: refractile bodies. N: nucleous.

Figure 2

High Resolution Clear Native Electrophoresis (HrCNE) (left and middle) and two-dimensional polyacrylamide gel electrophoresis (2D-SDS-PAGE) (right) analysis of E. tenella complex II. For HrCNE gels, 10 μg and 27 μg per lane of bovine heart mitochondria and E. tenella mitochondria-rich fraction were loaded, respectively. Left and middle panels were stained by SDH activity and CBB staining, respectively. The red arrow indicates the location of SDH stained bands of 745 kDa in the CBB stained gel, which was cut off and loaded into 2D-SDS-PAGE gel (right) and stained by CBB. A total of 15 bands, named according to their calculated molecular weight, were stoichiometrically stained by CBB (right).