The RRM of the kRNA-editing protein TbRGG2 uses multiple surfaces to bind and remodel RNA.

Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process.

Maxi-circles, glycosomes, gene transposition, expression sites, transsplicing, transferrin receptors and base J.

This is a personal story of the author of his research on trypanosomatids, covering a period of 1970-2015. Some of the highlights include the discovery of new aspects of kDNA, the mini-circle heterogeneity and the maxi-circle; the glycosome; the discovery of gene transposition as a major mechanism for antigenic variation; trans-splicing as an essential step in the synthesis of all trypanosome mRNAs; Pulsed Field Gradient gels to size-fractionate chromosome-sized DNA molecules of protozoa; the sequence of trypanosome telomeres and their growth and contraction; the first ABC-transporter of trypanosomatids, LtpgpA; the variable transferrin receptor of T. brucei and its role in Fe uptake; and base J, its structure, biosynthesis and function.

Identification and structure solution of fragment hits against kinetoplastid N-myristoyltransferase.

Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis. Pyrazole sulfonamide (DDD85646), a potent inhibitor of TbNMT, has been identified in previous studies; however, poor central nervous system exposure restricts its use to the haemolymphatic form (stage 1) of the disease. In order to identify new chemical matter, a fragment screen was carried out by ligand-observed NMR spectroscopy, identifying hits that occupy the DDD85646 binding site. Crystal structures of hits from this assay have been obtained in complex with the closely related NMT from Leishmania major, providing a structural starting point for the evolution of novel chemical matter.

Kinetoplastid-specific histone variant functions are conserved in Leishmania major.

Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.

The paraflagellar rod of kinetoplastid parasites: from structure to components and function.

The role of the eukaryotic flagellum in cell motility is well established but its importance in many other aspects of cell biology, from cell signalling to developmental regulation, is becoming increasingly apparent. In addition to this diversity of function the core structure of the flagellum, which has been inherited from the earliest ancestor of all eukaryotes, is embellished with a range of extra-axonemal structures in many organisms. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. Here we discuss recent advances in the identification of further molecular components of the paraflagellar rod, how these impact on our understanding of its function and regulation and the implications for therapeutic intervention in a number of devastating human pathologies.

Kinetoplastid papain-like cysteine peptidases.

Cysteine peptidases are important for growth and survival of kinetoplastid parasites. The best characterised are those homologous to mammalian cathepsins B and L. To address a somewhat confusing terminology, we introduce a unifying nomenclature for kinetoplastid CATB and CATL peptidases. We review their evolutionary relatedness, genomic organisation, developmental expression, subcellular location and physiological functions. In addition, the applications of kinetoplastid CATB and CATL enzymes as vaccine candidates, diagnostic markers and drug targets are discussed.

A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania.

The single mitochondrion of kinetoplastids divides in synchrony with the nucleus and plays a crucial role in cell division. However, despite its importance and potential as a drug target, the mechanism of mitochondrial division and segregation and the molecules involved are only partly understood. In our quest to identify novel mitochondrial proteins in Leishmania, we constructed a hidden Markov model from the targeting motifs of known mitochondrial proteins as a tool to search the Leishmania major genome. We show here that one of the 17 proteins of unknown function that we identified, designated mitochondrial protein X (MIX), is an oligomeric protein probably located in the inner membrane and expressed throughout the Leishmania life cycle. The MIX gene appears to be essential. Moreover, even deletion of one allele from L. major led to abnormalities in cell morphology, mitochondrial segregation and, importantly, to loss of virulence. MIX is unique to kinetoplastids but its heterologous expression in Saccharomyces cerevisiae produced defects in mitochondrial morphology. Our data show that a number of mitochondrial proteins are unique to kinetoplastids and some, like MIX, play a central role in mitochondrial segregation and cell division, as well as virulence.

Binding and interaction of dinitroanilines with apicomplexan and kinetoplastid alpha-tubulin.

Despite years of use as commercial herbicides, it is still unclear how dinitroanilines interact with tubulin, how they cause microtubule disassembly, and why they are selectively active against plant and protozoan tubulin. In this work, through a series of computational studies, a common binding site of oryzalin, trifluralin, and GB-II-5 on apicomplexan and kinetoplastid alpha-tubulin is proposed. Furthermore, to investigate how dinitroanilines affect tubulin dynamics, molecular dynamics simulations of Leishmania alpha-tubulin with and without a bound dinitroaniline are performed. The results obtained provide insight into the molecular mechanism by which these compounds interact with tubulin and function to prevent microtubule assembly. Finally, to aid in the design of effective parasitic microtubule inhibitors, several novel dinitroaniline analogues are evaluated. The location of the binding site and the relative binding affinities of the dinitroanilines all agree well with experimental data.

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.

RNA-binding domain proteins in Kinetoplastids: a comparative analysis.

RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

The structure and replication of kinetoplast DNA.

Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.

Diplonema spp. possess spliced leader RNA genes similar to the Kinetoplastida.

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

Transcription of the kinetoplastid spliced leader RNA gene.

In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA gene. The SL RNA gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

Genetic manipulation of kinetoplastida.

During the 1980s, many kinetoplastid genes were cloned and their function inferred from homology with genes from other organisms, location of the corresponding proteins or expression in heterologous systems. Up until 1990, before the availability of DNA transfection methodology, we could not analyze the function of kinetoplastid genes within the organisms themselves. Since then, it has become possible to create and complement mutants, to overexpress foreign proteins in the parasites, to knock out genes and even to switch off essential functions. However, these methods are not equally applicable in all parasites. Here, Christine Clayton highlights the differences and similarities between the most commonly used model organisms, and assesses the relative advantages of different approaches and parasites for different types of investigation.

Identification of carbohydrates on the surface membrane of pathogenic and nonpathogenic piscine haemoflagellates, Cryptobia salmositica, C. bullocki and C. catostomi (Kinetoplastida).

Carbohydrates and protein glycoconjugates on the cell membranes of Cryptobia salmositica, C. bullocki and C. catostomi were analyzed using 13 highly purified lectins (unlabelled or digoxigenin/biotin labelled). No agglutinations were observed with C. salmositica, C. bullocki and C. catostomi using lectin TPA (Tetragonolobus purpureas agglutinin, for alpha-L-fucose). C. salmositica was agglutinated by 3 of 12 lectins [Con A, for alpha-man and alpha-D-glc; PSA, for alpha-man; PWM, for (glcNAc)3], while C. bullocki was agglutinated by 8 lectins and C. catostomi was agglutinated by 10 lectins. Glycoconjugate analysis with digoxigenin or biotin labelled lectins showed a species-specific staining pattern in pathogenic and nonpathogenic Cryptobia spp. The nonpathogenic C. catostomi had the strongest reaction. These results indicate that the surface carbohydrate residues and glycoconjugate compositions on Cryptobia spp. are different between species they may be related to the virulence of the parasite.

Surface charge and surface carbohydrates of Cyptobia salmositica virulent and avirulent forms and of C. bullocki (Kinetoplastida: Cryptobiidae).

The surface charge and surface carbohydrate residues of the virulent (freshly isolated from the fish blood) and avirulent forms (from culture) of Cryptobia salmositica and one strain of C. bullocki were studied. Measurements of the zeta potential of parasites showed that C. bullocki and the virulent form of C. salmositica had a net negative surface charge of about -15 mV, whereas the attenuated form of C. salmositica showed a surface charge of -7.9 mV. Enzymatic treatments of parasites with neuraminidase, trypsin, or phospholipase C indicated the presence of sialic acid residues, phosphate groups, and protein glycoconjugates as components of the Cryptobia surface that accounted for their surface charge. Residues of alpha-D-man, alpha- and beta-D-gal, alpha-D-galNAc, alpha-L-fuc, and D-glcNAc could be detected on the surface of all parasites by specific fluorescein isothiocyanate (FITC)- and colloidal gold-labeled lectins. The cell surface of the avirulent form of C. salmositica showed the strongest reactivity to almost all lectins tested. A remarkable binding pattern of lectins in the anterior region of parasites was observed.

beta-D-glucosyl-hydroxymethyluracil, a novel base in African trypanosomes and other Kinetoplastida.

A novel base, beta-D-glucosyl-hydroxymethyluracil or J for short, was recently discovered in DNA of bloodstream form Trypanosoma brucei. The base is predominantly found in the hexameric repeat arrays of chromosome telomeres and in adjacent repetitive sub-telomeric DNA, and it is made by modification of specific thymines in DNA. J is present in inactive telomeric variant surface glycoprotein (VSG) genes, but not in active ones, suggesting a link between the presence of J and repression of the telomeric expression sites for VSG genes. The presence of J in DNA is specific for bloodstream form trypanosomes, as J is absent in insect form (procyclic) T. brucei. In addition to African trypanosomes, J has been found in DNA from other Kinetoplastida that do not undergo antigenic variation, such as Leishmania and Crithidia. The biological function of J remains to be deciphered.

An M(r) 145,000 low-density lipoprotein (LDL)-binding protein is conserved throughout the Kinetoplastida order.

In view of the importance of the low-density lipoprotein (LDL)-receptor in Trypanosoma brucei, we have examined whether other bloodstream trypanosomes of medical and veterinary importance (T.b. rhodesiense, T. equiperdum, T. vivax, T. congolense), but also related parasites developing in mammalian (Leishmania donovani) and non-mammalian hosts (Crithidia luciliae and Phytomonas sp. isolated from Euphorbia), would possess an LDL-receptor of their own. (1) All these parasites specifically accumulate human 125I-LDL with a relatively 2.5-fold higher rate for bloodstream trypanosomes. (2) A mixture of monoclonal antibodies raised against T.b. brucei LDL-receptor inhibit binding of LDL to all species but with different efficiency. (3) A single glycoprotein of similar M(r) (gp145) is isolated by LDL-affinity chromatography from all the above species, as well as from both human serum-resistant and sensitive strain of T.b. rhodesiense, and from the bodonid member of the Kinetoplastida Trypanoplasma borelli. (4) Several control experiments including 35S-metabolic labeling of procyclic T.b. brucei and of C. luciliae followed by LDL-affinity chromatography or immunoprecipitation demonstrate that gp145 is indeed synthesised by the parasites and is not a contaminant of the experimental system. (5) In immunoblots and ELISA, these gp145 cross-react with the polyclonal and monoclonal antibodies raised against the LDL-receptor of T.b. brucei, the highest degree of cross-reactivity being found among the members of the Trypanozoon subgroup. (6) Finally, immunisation of mice with the purified LDL-receptor from one strain of T.b. brucei is not sufficient to confer durable protection against another strain of this parasite.

Phylogenetic position of kinetoplastid protozoa inferred from the protein phylogenies of elongation factors 1alpha and 2.

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a kinetoplastid protozoan, Trypanosoma cruzi, were amplified by means of polymerase chain reaction and their primary structures were analyzed. The deduced amino acid sequence was aligned with those of other eukaryotic and archaebacterial EF-2s, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) method. ML analyses of EF-2 phylogeny using six different stochastic models of amino acid substitutions consistently suggested that the phylogenetic position of T. cruzi is likely to be closer to higher eukaryotes than that inferred from the phylogeny of small subunit ribosomal RNA (SrRNA). These results are consistent with those for the elongation factor 1alpha (EF-1alpha) phylogeny. When the EF-1alpha and EF-2 phylogenies were totally evaluated, it became much clearer that the divergence of T. cruzi occurred later than that of a mitochondrion-lacking protozoan, Entamoeba histolytica, although this is not conclusive.

Kinetoplastid membrane protein-11 (KMP-11) is differentially expressed during the life cycle of African trypanosomes and is found in a wide variety of kinetoplastid parasites.

An abundant 11-kDa membrane protein was purified from African trypanosomes by organic solvent extraction and octyl-Sepharose chromatography. This protein cross-reacts with monoclonal antibodies originally generated against the lipophosphoglycan-associated protein of Leishmania donovani. Immunoblot analysis showed that the 11-kDa molecule was present in a variety of species of kinetoplastids. It was found in several species and subspecies of African trypanosomes and was present in low amounts in bloodstream forms and in larger amounts in procyclic, epimastigote and metacyclic life cycle stages. Expression of the 11-kDa molecule rapidly increased during transformation from bloodstream forms to procyclic forms, paralleling expression of the major surface glycoprotein of Trypanosoma congolense, the glutamic acid/alanine-rich protein, an analogue of T. brucei procyclin. The molecule was present in procyclic trypanosome membranes at approximately 2 x 10(5)-1 x 10(6) molecules per cell, suggesting it may have an important role in parasite membrane organization and function. Amino-acid analysis of the trypanosome 11-kDa protein showed it had a different composition than that of its leishmania counterpart. Its wide distribution in kinetoplastids and its membrane disposition suggest a name for this class of molecules: kinetoplastid membrane protein-11 (KMP-11).