The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei.

Messenger RNA is modified by the addition of a 5' methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo.

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania.

Drug discovery pipelines for the "neglected diseases" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.

The genomic basis of parasitism in the Strongyloides clade of nematodes.

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei.

OBJECTIVES: The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. METHODS: Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. RESULTS: Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. CONCLUSIONS: NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype.

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome.

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class.

Global Gene Expression Profiling through the Complete Life Cycle of Trypanosoma vivax.

The parasitic flagellate Trypanosoma vivax is a cause of animal trypanosomiasis across Africa and South America. The parasite has a digenetic life cycle, passing between mammalian hosts and insect vectors, and a series of developmental forms adapted to each life cycle stage. Each point in the life cycle presents radically different challenges to parasite metabolism and physiology and distinct host interactions requiring remodeling of the parasite cell surface. Transcriptomic and proteomic studies of the related parasites T. brucei and T. congolense have shown how gene expression is regulated during their development. New methods for in vitro culture of the T. vivax insect stages have allowed us to describe global gene expression throughout the complete T. vivax life cycle for the first time. We combined transcriptomic and proteomic analysis of each life stage using RNA-seq and mass spectrometry respectively, to identify genes with patterns of preferential transcription or expression. While T. vivax conforms to a pattern of highly conserved gene expression found in other African trypanosomes, (e.g. developmental regulation of energy metabolism, restricted expression of a dominant variant antigen, and expression of 'Fam50' proteins in the insect mouthparts), we identified significant differences in gene expression affecting metabolism in the fly and a suite of T. vivax-specific genes with predicted cell-surface expression that are preferentially expressed in the mammal ('Fam29, 30, 42') or the vector ('Fam34, 35, 43'). T. vivax differs significantly from other African trypanosomes in the developmentally-regulated proteins likely to be expressed on its cell surface and thus, in the structure of the host-parasite interface. These unique features may yet explain the species differences in life cycle and could, in the form of bloodstream-stage proteins that do not undergo antigenic variation, provide targets for therapy.

Whipworm genome and dual-species transcriptome analyses provide molecular insights into an intimate host-parasite interaction.

Whipworms are common soil-transmitted helminths that cause debilitating chronic infections in man. These nematodes are only distantly related to Caenorhabditis elegans and have evolved to occupy an unusual niche, tunneling through epithelial cells of the large intestine. We report here the whole-genome sequences of the human-infective Trichuris trichiura and the mouse laboratory model Trichuris muris. On the basis of whole-transcriptome analyses, we identify many genes that are expressed in a sex- or life stage-specific manner and characterize the transcriptional landscape of a morphological region with unique biological adaptations, namely, bacillary band and stichosome, found only in whipworms and related parasites. Using RNA sequencing data from whipworm-infected mice, we describe the regulated T helper 1 (TH1)-like immune response of the chronically infected cecum in unprecedented detail. In silico screening identified numerous new potential drug targets against trichuriasis. Together, these genomes and associated functional data elucidate key aspects of the molecular host-parasite interactions that define chronic whipworm infection.

The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii.

Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa phylum. The Coccidia are obligate intracellular pathogens that establish infection in their mammalian host via the enteric route. These parasites lack a mitochondrial pyruvate dehydrogenase complex but have preserved the degradation of branched-chain amino acids (BCAA) as a possible pathway to generate acetyl-CoA. Importantly, degradation of leucine, isoleucine and valine could lead to concomitant accumulation of propionyl-CoA, a toxic metabolite that inhibits cell growth. Like fungi and bacteria, the Coccidia possess the complete set of enzymes necessary to metabolize and detoxify propionate by oxidation to pyruvate via the 2-methylcitrate cycle (2-MCC). Phylogenetic analysis provides evidence that the 2-MCC was acquired via horizontal gene transfer. In T. gondii tachyzoites, this pathway is split between the cytosol and the mitochondrion. Although the rate-limiting enzyme 2-methylisocitrate lyase is dispensable for parasite survival, its substrates accumulate in parasites deficient in the enzyme and its absence confers increased sensitivity to propionic acid. BCAA is also dispensable in tachyzoites, leaving unresolved the source of mitochondrial acetyl-CoA.

Resisting resistance.

This month's Genome Watch describes how knowledge of the malaria parasite genome can be used to better understand and mitigate the emergence of drug resistance.

Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B.

Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites.

A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii.

BACKGROUND: Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. RESULTS: We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. CONCLUSIONS: DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.

Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites.

BACKGROUND: Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. RESULTS: Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins--including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins--are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. CONCLUSIONS: Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum.

Plasmodium falciparum possesses two GRASP proteins that are differentially targeted to the Golgi complex via a higher- and lower-eukaryote-like mechanism.

Plasmodium falciparum, the causative agent of malaria, relies on a complex protein-secretion system for protein targeting into numerous subcellular destinations. Recently, a homologue of the Golgi re-assembly stacking protein (GRASP) was identified and used to characterise the Golgi organisation in this parasite. Here, we report on the presence of a splice variant that leads to the expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies on a well-conserved myristoylation motif, the variant (GRASP2) displays a different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses between GRASP proteins of numerous taxa point to an independent evolution of the unusual N-terminus that could reflect unique requirements for Golgi-dependent protein sorting and organelle biogenesis in P. falciparum. Golgi association of GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor, leading to a unique mode of Golgi targeting and membrane attachment.

Evolution of malaria parasite plastid targeting sequences.

The transfer of genes from an endosymbiont to its host typically requires acquisition of targeting signals by the gene product to ensure its return to the endosymbiont for function. Many hundreds of plastid-derived genes must have acquired transit peptides for successful relocation to the nucleus. Here, we explore potential evolutionary origins of plastid transit peptides in the malaria parasite Plasmodium falciparum. We show that exons of the P. falciparum genome could serve as transit peptides after exon shuffling. We further demonstrate that numerous randomized peptides and even whimsical sequences based on English words can also function as transit peptides in vivo. Thus, facile acquisition of transit peptides from existing sequence likely expedited endosymbiont integration through intracellular gene transfer.

Phylogenetic analysis to uncover organellar origins of nuclear-encoded genes.

Most proteins that are located in mitochondria or plastids are encoded by the nuclear genome, because the organellar genomes have undergone severe reduction during evolution. In many cases, although not all, the nuclear genes encoding organelle-targeted proteins actually originated from the respective organellar genome and thus carry the phylogenetic fingerprint that still bespeaks their evolutionary origin. Phylogenetic analysis is a powerful in silico method that can yield important insights into the evolutionary history or molecular kinship of any gene or protein and that can thus also be used more specifically in the context of organellar targeting as one means to recognize protein candidates (e.g., from genome data) that may be targeted to mitochondria or plastids. This chapter provides protocols for creating multiple sequence alignments and carrying out phylogenetic analysis with the robust and comprehensive software packages Clustal and PHYLIP, which are both available free of charge for multiple computer platforms. Besides presenting step-by-step instructions on how to run these computer programs, this chapter also covers topics such as data collection and presentation of phylogenetic trees.

New insights into myosin evolution and classification.

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases.

The Plasmodium falciparum genome contains genes encoding three alpha-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain alpha-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial alpha-ketoglutarate dehydrogenase and branched chain alpha-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.

The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.

The relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl-CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1alpha, E1beta, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear-encoded proteins are targeted to the apicoplast courtesy of two-part N-terminal leader sequences, and the presence of such N-terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two-part leader sequences from the E1alpha and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1alpha and E1beta PDH subunits in blood-stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl-CoA in the apicoplast, possibly for use in fatty acid biosynthesis.

Molecular and functional aspects of parasite invasion.

Apicomplexan parasites have evolved an efficient mechanism to gain entry into non-phagocytic cells, hence challenging their hosts by the establishment of infection in immuno-privileged tissues. Gliding motility is a prerequisite for the invasive stage of most apicomplexans, allowing them to migrate across tissues, and actively invade and egress host cells. In the late 1960s, detailed morphological studies revealed that motile apicomplexans share an elaborate architecture comprising a subpellicular cytoskeleton and apical organelles. Since 1993, the development of technologies for transient and stable transfection have provided powerful tools with which to identify gene products associated with these structures and organelles, as well as to understand their functions. In combination with access to several parasite genomes, it is now possible to compare and contrast the strategies and molecular machines that have been selectively designed by distinct life stages within a species, or by different apicomplexan species, to optimize infection.