Frank Seeber

Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein‐attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii‐infected cells in lipoate‐deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate‐deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

Metabolic pathways in the apicoplast of apicomplexa

Intracellular parasites of the phylum Apicomplexa harbor a plastid-like organelle called apicoplast that is the most reduced organelle of this type known. Due to the medical importance of some members of Apicomplexa, a number of fully sequenced genomes are available that have allowed to assemble metabolic pathways also from the apicoplast and have revealed initial clues to its essential nature for parasite survival in the host. We provide a compilation of Internet resources useful to access, reconstruct, verify, or annotate metabolic pathways. Then we show detailed and updated metabolic maps and discuss the three major biosynthetic pathways leading to the generation of isoprenoids, fatty acids, and heme, and compare these routes in the different species. Moreover, several auxiliary pathways, like iron-sulfur cluster assembly, are covered and put into context with the major metabolic routes. Finally, we highlight some aspects that emerged from recent publications and were not discussed previously with regard to Apicomplexa.

Consensus sequence of translational initiation sites from Toxoplasma gondii genes

Translation initiation of mRNA in eukaryotes usually occurs at the first AUG codon nearest to its 5′ end. In this so-called scanning model for initiation the 40S ribosomal subunit is thought to bind at the 5′ cap structure of the mRNA and, in moving toward the 3′ direction, to scan the sequence for the first AUG codon in a favorable context, which would then lead the ribosome to start translation (for a recent review see Pain 1996). A consensus sequence for this favorable context (often termed the Kozak sequence) has been compiled for several groups of organisms, including protozoa (Kozak 1989; Yamauchi 1991). As more and more genes from Toxoplasma gondii have been sequenced, it has become apparent that a reevaluation of this early protozoan consensus sequence is necessary. The sequences for which sufficient 5′ UTR sequence information is available (26 in total, September 1996), together with their Genbank accession numbers, are shown in Fig. 1A. Sequences were compared from position )20 relative to the ATG start codon (as stated by the respective authors of the entry) up to position +4. For prevention of confusion the DNA code instead of the RNA code was used throughout. It is apparent that although only a limited number of sequences were compared, a clear consensus sequence emerges (see Table 1), with A occurring almost exclusively at position )3 and a strong bias being apparent toward A at position )2 and toward C at position )4. Positions )1 and )6 show a trend toward A and G, respectively, whereas at position )5, no clear nucleotide bias can be observed. In over half of the coding sequences, G is found at position +4, whereas in the rest, A, T, and C are evenly distributed. Thus, the consensus translational initiation sequence ‘‘gNCAAa ATG g’’ can be assigned for T. gondii genes, which is similar but not identical to the Kozak sequence of higher eukaryotes (see Table 1). The T. gondii sequence shows an almost perfect conservation of the universally found A at )3, which is assumed to be of major importance in the recognition of the following ATG. With one exception, the others possess a G at this position. Thus, almost all compared sequences have a purine at this site. This new consensus sequence differs considerably from the one previously published for sporozoan parasites, namely, /ANAAAA ATG A (Yamauchi 1991), due to the fact that this early compilation consisted mainly of Plasmodium sequences (27) and only 3 T. gondii genes and did not take into account the high AT-content of the Plasmodium genome (82%; Weber 1987) as compared with the more GC-rich T. gondii nuclear genome (52%, calculated from 69 kb of reported T. gondii nuclear encoded DNA in the GenBank data base; data not shown). With this consensus sequence at hand, it should now be possible to screen T. gondii gene data bases (especially the fast-growing EST data base) using pattern search programs (e.g., PatScan) to help identify either potential start codons or correct reading frames. For instance, we have assembled the coding sequence and its 5′ UTR of T. gondii calmodulin by including or eliminating several ESTs with homology to calmodulin or related proteins and then comparing these with our own polymerase chain reaction (PCR)-derived coding sequence for that gene (Seeber et al., in preparation). The translation initiation site for the T. gondii calmodulin sequence thus derived from two independent ESTs is AAACAA ATG G, further strengthening the assigned consensus sequence. We have also used the above consensus sequence to try to explain why the second in-frame ATG (relative to Parasitol Res (1997) 83: 309–311  Springer-Verlag 1997

Apicomplexan mitochondrial metabolism: a story of gains, losses and retentions.

Apicomplexans form a large group of obligate intracellular parasites that occupy diverse environmental niches. To adapt to their hosts, these parasites have evolved sophisticated strategies to access host-cell nutrients and minimize exposure to the hosts defence mechanisms. Concomitantly, they have drastically reshaped their own metabolic functions by retaining, losing or gaining genes for metabolic enzymes. Although several Apicomplexans remain experimentally intractable, bioinformatic analyses of their genomes have generated preliminary metabolic maps. Here, we compare the metabolic pathways of five Apicomplexans, focusing on their different mitochondrial functions, which highlight their adaptation to their individual intracellular habitats.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid‐like organelle (apicoplast) by the mevalonate independent 1‐deoxy‐d‐xylulose‐5‐phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe–4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin‐NADP+ reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin‐NADP+ reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

Biogenesis of iron–sulphur clusters in amitochondriate and apicomplexan protists

During the last 4 years there has been an enormous interest in the question how iron-sulphur ([Fe-S]) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes. The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution. In yeast and probably all other eukaryotes the mitochondrion is the place where [Fe-S] clusters are synthesised, even for extramitochondrial [Fe-S] cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway. However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this. In this article I will summarise the current view of [Fe-S] cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.

Apicomplexan parasites contain a single lipoic acid synthase located in the plastid

Apicomplexan parasites contain a vestigial plastid called apicoplast which has been suggested to be a site of [Fe–S] cluster biogenesis. Here we report the cloning of lipoic acid synthase (LipA) from Toxoplasma gondii, a well known [Fe–S] protein. It is able to complement a LipA‐deficient Escherichia coli strain, clearly demonstrating that the parasite protein is a functional LipA. The N‐terminus of T. gondii LipA is unusual with respect to an internal signal peptide preceding an apicoplast targeting domain. Nevertheless, it efficiently targets a reporter protein to the apicoplast of T. gondii whereas co‐localization with the fluorescently labeled mitochondrion was not detected. In silico analysis of several apicomplexan genomes indicates that the parasites, in addition to the presumably apicoplast‐resident pyruvate dehydrogenase complex, contain three other mitochondrion‐localized target proteins for lipoic acid attachment. We also identified single genes for lipoyl (octanoyl)‐acyl carrier protein:protein transferase (LipB) and lipoate protein ligase (LplA) in these genomes. It thus appears that unlike plants, which have only two LipA and LipB isoenzymes in both the chloroplasts and the mitochondria, Apicomplexa seem to use the second known lipoylating activity, LplA, for lipoylation in their mitochondrion.

The plastid-derived organelle of protozoan human parasites as a target of established and emerging drugs.

Human diseases like malaria, toxoplasmosis or cryptosporidiosis are caused by intracellular protozoan parasites of the phylum Apicomplexa and are still a major health problem worldwide. In the case of Plasmodium falciparum, the causative agent of tropical malaria, resistance against previously highly effective drugs is widespread and requires the continued development of new and affordable drugs. Most apicomplexan parasites possess a single plastid-derived organelle called apicoplast, which offers the great opportunity to tailor highly specific inhibitors against vital metabolic pathways resident in this compartment. This is due to the fact that several of these pathways, being of bacterial or algal origin, are absent in the mammalian host. In fact, the targets of several antibiotics already in use for years against some of these diseases can now be traced to the apicoplast and by knowing the molecular entities which are affected by these substances, improved drugs or drug combinations can be envisaged to emerge from this knowledge. Likewise, apicoplast-resident pathways like fatty acid or isoprenoid biosynthesis have already been proven to be the likely targets of the next drug generation. In this review the current knowledge on the different targets and available inhibitors (both established and experimental) will be summarised and an overview of the clinical efficacy of drugs that inhibit functions in the apicoplast and which have been tested in humans so far will be given.

Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways

Background Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. Methodology/Principal Findings Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. Conclusions/Significance Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens.