Non‐cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

Abstract

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferinlluciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.Trypanosoma brucei brucei is of medical and economic importance as the agent of human African sleeping sickness and nagana in cattle. The life cycle of the parasite in the mammalian host begins with metacyclic forms injected with the bite of the tsetse fly vector. These metacyclics subsequently transform into the rapidly dividing long slender forms which in turn give rise to intermediate and short stumpy forms, stages suggested to be pre-adapted to survive in the vector if ingested with a blood meal. In this report we refer to the intermediate and short stumpy bloodstream forms as transitional bloodstream forms. Several aspects of the metabolism of bloodstream 7: brucei brucei are known to be unique and have been identified as targets for chemotherapy (Fairlamb, 1989;Clarkson and Brohn, 1976). Of special interest is the mitochondrion which undergoes marked differentiation and development as the organism progresses through its life cycle (Vickerman, 1965). The procyclic or insect midgut form has cytochromes and Krebs cycle function whereas these are both reported to be absent in bloodstream form mitochondria which have high respiratory activity but a limited range of function (for review see Fairlamb and Opperdoes, 1976).Glycolysis is generally considered the sole source of ATP in all bloodstream forms. However, earlier work by Vickerman (1965) and Flynn and Bowman (1973) showed that populations with significant numbers of transitional bloodstream forms were able to maintain cell motility and oxygen utilization in the presence of 2-oxoglutarate, a Krebs cycle intermediate. In contrast, long slender fosms lysed in the absence of Correspo...

Adaptations in the Glucose Metabolism of Procyclic Trypanosoma brucei Isolates from Tsetse Flies and during Differentiation of Bloodstream Forms

“…Therefore, the mitochondria of short stumpy trypanosomes are metabolically divergent from the mitochondria in long slender BSF T. brucei cells. These results are consistent with prior work (4,5,8,11). The functional changes might be a preadaptation that allows short stumpy BSF T. brucei to function in the intestines of infected tsetse flies and enables them to differentiate further into PCF trypanosomes.…”

“…Differential gene expression and the formation of cristea in the inner mitochondrial membrane have been shown to occur during this transition (8,11,19). Furthermore, the trypanosomal homologue of complex I of the respiratory chain is expressed in short stumpy BSF trypanosomes (4,5,18). Our experiments show that this more elaborate composition of the electron transport chain is also used by this stage, as the production of acetate implies that acetyl-CoA is formed, which is catalyzed by the pyruvate dehydrogenase complex and results in the production of NADH inside the mitochondrion.…”

Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

“…As cell numbers increase during a parasitaemia, bloodstream parasites transform from proliferative "slender" to nonproliferative "stumpy" forms (Brown et al, 1973;Matthews, 1999). These activate the expression of some mitochondrial components (Bienen et al, 1991(Bienen et al, , 1993, although full development of cytochrome-dependent respiration does not occur until transmission to the tsetse. This metabolic adaptation is accompanied by changes in cell morphology, patterns of gene expression and surface antigen presentation, culminating in a fully differentiated procyclic form that inhabits the tsetse midgut.…”

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

“…The cells were then resuspended, and the oxygen consumed by 2.0 ϫ 10 6 cells per ml was recorded in the presence and the absence of the test compounds. The respiration of T. b. brucei mitochondrial preparations was measured by following a protocol adapted from that described in previous publications (4,30). Briefly, the mitochondria were suspended to 0.8 mg/ml in mannitol-based assay buffer (pH 7.4) lacking glucose, and a final concentration of 20 mM L-␣-glycerol phosphate was then added as the respiratory substrate.…”