A passion for parasites.

I knew nothing and had thought nothing about parasites until 1971. In fact, if you had asked me before then, I might have commented that parasites were rather disgusting. I had been at the Johns Hopkins School of Medicine for three years, and I was on the lookout for a new project. In 1971, I came across a paper in the Journal of Molecular Biology by Larry Simpson, a classmate of mine in graduate school. Larry's paper described a remarkable DNA structure known as kinetoplast DNA (kDNA), isolated from a parasite. kDNA, the mitochondrial genome of trypanosomatids, is a DNA network composed of several thousand interlocked DNA rings. Almost nothing was known about it. I was looking for a project on DNA replication, and I wanted it to be both challenging and important. I had no doubt that working with kDNA would be a challenge, as I would be exploring uncharted territory. I was also sure that the project would be important when I learned that parasites with kDNA threaten huge populations in underdeveloped tropical countries. Looking again at Larry's paper, I found the electron micrographs of the kDNA networks to be rather beautiful. I decided to take a chance on kDNA. Little did I know then that I would devote the next forty years of my life to studying kDNA replication.

TbKAP6, a mitochondrial HMG box-containing protein in Trypanosoma brucei, is the first trypanosomatid kinetoplast-associated protein essential for kinetoplast DNA replication and maintenance.

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei, proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata, some small basic histone H1-like kinetoplast-associated proteins (CfKAP) have been shown to condense isolated kDNA networks in vitro. High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro. These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.

Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1.

In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock-down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.

Network news: the replication of kinetoplast DNA.

One of the most fascinating and unusual features of trypanosomatids, parasites that cause disease in many tropical countries, is their mitochondrial DNA. This genome, known as kinetoplast DNA (kDNA), is organized as a single, massive DNA network formed of interlocked DNA rings. In this review, we discuss recent studies on kDNA structure and replication, emphasizing recent developments on replication enzymes, how the timing of kDNA synthesis is controlled during the cell cycle, and the machinery for segregating daughter networks after replication.

TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance.

The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8 is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.

Depletion of mitochondrial acyl carrier protein in bloodstream-form Trypanosoma brucei causes a kinetoplast segregation defect.

Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis.

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.

The killing of African trypanosomes by ethidium bromide.

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.

TbPIF1, a Trypanosoma brucei mitochondrial DNA helicase, is essential for kinetoplast minicircle replication.

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.

TbPIF5 is a Trypanosoma brucei mitochondrial DNA helicase involved in processing of minicircle Okazaki fragments.

Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5' to 3' DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.

Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication.

Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.

Silencing of genes required for glycosylphosphatidylinositol anchor biosynthesis in Burkitt lymphoma.

OBJECTIVE: To investigate the mechanism of glycosylphosphatidylinositol (GPI) anchor deficiency in Burkitt lymphoma cell lines. METHODS: We identified a large GPI anchor protein deficient population in three different Burkitt lymphoma cell lines through proaerolysin treatment of the cells and flow cytometry analysis using a proaerolysin variant (FLAER). The mechanism of GPI anchor protein deficiency was studied by DNA gene sequencing, a cell-free assay to investigate the GPI anchor biosynthetic pathway, microarray analysis, and quantitative real-time polymerase chain reaction. RESULTS: Burkitt lymphoma cell lines harbor large populations of FLAER(neg) cells, which are resistant to proaerolysin. In all three cell lines, silencing of a gene involved in an early step in GPI-anchor biosynthesis was responsible for the lack of GPI-anchored proteins on the cell surface. Quantitative polymerase chain reaction and microarray analysis demonstrate that the level of mRNA for PIGL and PIGY is lower in the FLAER(neg) Ramos cells and that mRNA levels of PIGY are reduced in the Akata and Daudi cells. Hypermethylation of these genes was associated with the low levels of mRNA and treatment of the cells with 5-aza-2' deoxycytidine restored cell surface GPI-anchored proteins to the FLAER(neg) cells. CONCLUSION: GPI-anchored protein deficiency in Burkitt lymphoma cells is not due to a genetic mutation (e.g., PIGA); rather, the lack of GPI-anchored proteins results from transcriptional silencing of PIGL and PIGY.

A new function of Trypanosoma brucei mitochondrial topoisomerase II is to maintain kinetoplast DNA network topology.

The mitochondrial genome of Trypanosoma brucei, called kinetoplast DNA, is a network of topologically interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles and reattachment of the progeny. Here we report a new function of the mitochondrial topoisomerase II (TbTOP2mt). Although traditionally thought to reattach minicircle progeny to the network, here we show that it also mends holes in the network created by minicircle release. Network holes are not observed in wild-type cells, implying that this mending reaction is normally efficient. However, RNAi of TbTOP2mt causes holes to persist and enlarge, leading to network fragmentation. Remarkably, these network fragments remain associated within the mitochondrion, and many appear to be appropriately packed at the local level, even as the overall kinetoplast organization is dramatically altered. The deficiency in mending holes is temporally the earliest observable defect in the complex TbTOP2mt RNAi phenotype.

What happens when Trypanosoma brucei leaves Africa.

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.

Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

The 3-hydroxyacyl-ACP dehydratase of mitochondrial fatty acid synthesis in Trypanosoma brucei.

The trypanosomatid parasite Trypanosoma brucei synthesizes fatty acids in the mitochondrion using the type II fatty acid synthesis (FAS) machinery. When mitochondrial FAS was characterized in T. brucei, all of the enzymatic components were identified based on their homology to yeast mitochondrial FAS enzymes, except for 3-hydroxyacyl-ACP dehydratase. Here we describe the characterization of T. brucei mitochondrial 3-hydroxyacyl-ACP dehydratase (TbHTD2), which was identified by its similarity to the human mitochondrial dehydratase. TbHTD2 can rescue the respiratory deficient phenotype of the yeast knock-out strain and restore the lipoic acid content, is localized in the mitochondrion and exhibits hydratase 2 activity.

Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei.

Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.

Effect of hydroxyurea on procyclic Trypanosoma brucei: an unconventional mechanism for achieving synchronous growth.

Procyclic Trypanosoma brucei cells were synchronized with 0.2 mM hydroxyurea. The cells did not arrest at the G(1)/S boundary but proceeded through one round of replication and arrested near the end of S phase. The mitochondrial genome (kinetoplast DNA network) replicated, forming two progeny networks, but the repair of minicircle gaps was inhibited.

p166, a link between the trypanosome mitochondrial DNA and flagellum, mediates genome segregation.

Kinetoplast DNA (kDNA), the trypanosome mitochondrial genome, is a giant network containing several thousand interlocked DNA rings. Within the mitochondrion, kDNA is condensed into a disk-shaped structure positioned near the flagellar basal body. The disk is linked to the basal body by a remarkable transmembrane filament system named the tripartite attachment complex (TAC). Following kDNA replication, the TAC mediates network segregation, pulling the progeny networks into the daughter cells by their linkage to the basal bodies. So far TAC has been characterized only morphologically with no known protein components. By screening an RNAi library, we discovered p166, a protein localizing between the kDNA and basal body in intact cells and in isolated flagellum-kDNA complexes. RNAi of p166 has only small effects on kDNA replication, but it causes profound defects in network segregation. For example, kDNA replication without segregation causes the networks to grow to enormous size. Thus, p166 is the first reported molecular component of the TAC, and its discovery will facilitate study of kDNA segregation machinery at the molecular level.

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.

Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.