Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei

Abstract

Trypanosoma brucei, the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei. The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.

Fig. 1.

Effect of DNA polymerase RNAi on cell viability. (A to C) Clonal cell lines were grown in the absence (open circles) or presence (open diamonds) of tetracycline (1 μg/ml) to induce RNAi. Inset, Northern blot of RNA isolated from 5 × 10⁷ parasites induced for 0 (−) or 48 (+) hours of RNAi of the gene indicated. Membranes were hybridized with ³²P-labeled probes as described previously (4). Ethidium bromide (EtBr)-stained rRNA is included to show similar loading. Graph, cell density was plotted as the product of cell number and total dilution. Means and standard errors from three separate RNAi inductions are presented for clonal cell lines SMIB A24 (A), SMIC A15 (B), and SMID A13 (C). (D) Results from clonogenic assays performed with parasites that were uninduced (−) or induced (+) for 10 days of RNAi prior to plating. Viability (plating efficiency) and standard errors from two separate inductions are presented for each clonal cell line.

Fig. 2.

Kinetics of kDNA loss during DNA polymerase silencing. (A, C, E) Representative images of parasites induced for indicated day of RNAi for POLIB (A), POLIC (C), or POLID (E). (B, D, F) Kinetics of kDNA loss were determined by classifying cells as possessing normal-sized kDNA (closed circles), small kDNA (open squares), or no kDNA (closed triangles). The means and standard errors from two inductions are presented for parasites depleted of POLIB (B), POLIC (D), and POLID (F). Abbreviations: N, nucleus; K, normal-sized kDNA; sK, small kDNA; no K, no kDNA. Scale bar, 5 μM.

Fig. 3.

Loss of minicircles and maxicircles during RNAi. (A to C) Membranes from Southern blot analysis of the abundance of minicircles and maxicircles during RNAi of POLIB (A), POLIC (B), and POLID (C). (D to F). Phosphorimaging quantification of total minicircle and maxicircle abundances from the membranes presented in panels A to C. Values were normalized against the α-tubulin loading control. Open diamonds, minicircles; open circles, maxicircles.

Fig. 4.

Analysis of parasites recovered from POLIB clonogenic assays. (A, B) Parasites viable in clonogenic assays of cultures induced for POLIB RNAi were recovered and examined for the presence of kDNA and sensitivity to RNAi. (A) Differential interference contrast (DIC) and fluorescence microscopy images of DAPI-stained parasites that were grown in the presence or absence of tetracycline. (B) Growth curves of parasites recovered from clonogenic assays of POLIB RNAi-induced cultures. (C, D) Parasites viable in clonogenic assays of uninduced control cultures were recovered and assessed for RNAi sensitivity, as described in the legends to panels A and B. Scale bar, 5 μM.

Fig. 5.

Analysis of minicircle replication intermediates in parental and POLIB-depleted parasites. Neutral/alkaline two-dimensional gel electrophoresis of free minicircles. (A, top) Total DNA from parental parasites was separated in the presence of ethidium bromide and then under denaturing conditions (NaOH) prior to transfer to the membrane. Minicircle replication intermediates were detected with oligomers that specifically hybridize to leading (L)- or lagging (H)-strand intermediates. (Bottom) Higher-contrast images of membranes presented above. Contrast was adjusted equally in images of membranes to visualize the abundance of theta structures and Okazaki fragments. (B, top) Two-dimensional analysis of parasites induced for the indicated number of days of POLIB RNAi. (Bottom) Higher-contrast images of H-strand membranes presented above. (C) Phosphorimager quantification of changes in the relative abundance of unreplicated CC and newly replicated N/G intermediates in blots presented in panel B. Open squares, L-strand detection; open triangles, H-strand detection. Abbreviations: CC, covalently closed; ccD, covalently closed dimer; MG, multiply gapped; N/G, nicked/gapped; U, fraction U.

Fig. 6.

Disruption of mitochondrial membrane potential during DNA polymerase silencing. (A) Representative images of MitoTracker Red-stained parasites that were either uninduced or induced for 4 days of POLIB RNAi. (B, C) Flow cytometry analysis of MitoTracker Red-stained parasites. Unstained control, parasites treated with DMSO (used as a solvent for MitoTracker solutions); FCCP, protonophore used as a negative control; carrier, parasites treated with ethanol (used as a carrier for FCCP). (B) Histogram showing fluorescence intensities of indicated samples. (C) Relative mean fluorescence intensities (MFI) of parasites presented in panel B. The unstained background was subtracted from raw MFI values prior to graphing the adjusted MFI relative to that of uninduced cells.