Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets

The protozoan parasites Trypanosoma, Leishmania and Crithidia, which belong to the order kinetoplastidae, emerge from the most ancient eukaryotic lineages. The diversity found in the life cycle of these organisms must be directed by genetic events, wherein topoisomerases play an important role in cellular processes affecting the topology and organization of intracellular DNA. Topoisomerases are valuable as potential drug targets because they have indispensable function in cell biology. This review summarizes what is known about topoisomerase genes and proteins of kinetoplastid parasites and the roles of these enzymes as targets for therapeutic agents.     

Cell-to-flagellum attachment and surface architecture in kinetoplastids

A key morphological feature of kinetoplastid parasites is the position and length of flagellum attachment to the cell body. This lateral attachment is mediated by the flagellum attachment zone (FAZ), a large complex cytoskeletal structure, which is essential for parasite morphogenesis and pathogenicity. Despite the complexity of the FAZ only two transmembrane proteins, FLA1 and FLA1BP, are known to interact and connect the flagellum to the cell body. Across the different kinetoplastid species, each only has a single FLA/FLABP pair, except in Trypanosoma brucei and Trypanosoma congolense where there has been an expansion of these genes. Here, we focus on the selection pressure behind the evolution of the FLA/FLABP proteins and the likely impact this will have on host–parasite interactions.     

Unravelling the kinetoplastid paraflagellar rod.

Researchers who study human pathogens are often interested in unique and essential aspects of the biology of the pathogen. Recent progress has been made in understanding such a target in kinetoplastid parasites. The paraflagellar rod is a unique cytoskeletal structure that plays a key role in the life-cycle of these fascinating organisms. This review discusses the protein components and structure of the paraflagellar rod and its function in cell motility.     

Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis

Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.     

The evolution of kinetoplastid glycosomes

The available data on carbohydrate metabolism in Kinetoplastida have been reviewed. Based on the metabolic pattern of different kinetoplastid organisms, on the subcellular distribution of their glycolytic enzymes, and on the structural and regulatory properties of these proteins, we propose that the glycosome developed from an endosymbiont, as a specific manner to control carbohydrate and energy metabolism. It is discussed how the enzymes were subcellularly recompartmentalized during evolution as adaptation to the environment encountered by the organisms.     

MOLECULAR EVOLUTION OF EUGLENOZOAN PARAXONEMAL ROD GENES par1 AND par2 COINCIDES WITH PHYLOGENETIC RECONSTRUCTION BASED ON SMALL SUBUNIT rDNA DATA1

Emergent flagella of Euglenozoa consist of two prominent structural elements: the axoneme built by microtubules with motor proteins to enable the movement of the flagellum and a highly organized protein structure of unknown function, called the paraxonemal rod (PAR), which consists of two major proteins paralleling the axoneme of euglenid and kinetoplastid emergent flagella. These flagellar structures are considered apomorphic characters of Euglenozoa. We examined the evolution of the genes par1 and par2 encoding the two major proteins, where we could show that these proteins are encoded by two very similar genes found in kinetoplastids and euglenids. The branching pattern indicated a gene duplication before the diversification into euglenids and kinetoplastids. In the clades of the genes, subtrees of euglenid and kinetoplastid monophyla arose. Both genes showed strong genetic diversity with biased GC content at taxon rather than at gene level. We also examined phylogenies inferred from PAR genes that are well in agreement with established small subunit rDNA analyses. Both showed further separation of the euglenid subtree into primary osmotrophs and a phototrophic clade, including secondarily derived osmotrophs.     

DNA repair mechanisms and Toxoplasma gondii infection

Lately, we can observe significant progress in understanding mechanism of DNA repair owing to fast methods of DNA sequence analysis from different organisms the revealing of structure and function of DNA repair proteins in prokaryota and eukaryota. The protozoan parasites survival depends on DNA repair systems. Better understanding of DNA repair systems can help in new antipathogen drug development. This review is aimed at updating our current knowledge of the various repair pathways by providing an overview of DNA repair genes regarding Toxoplasma gondii infections and the corresponding proteins, participating either directly in DNA repair, or in checkpoint control and signaling of DNA damage.     

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.     

Evolutionary Relationships and Protein Domain Architecture in an Expanded Calpain Superfamily in Kinetoplastid Parasites

Employing whole-genome analysis we have characterized a large family of genes coding for calpain-related proteins in three kinetoplastid parasites. We have defined a total of 18 calpain-like sequences in Trypanosoma brucei, 27 in Leishmania major, and 24 in Trypanosoma cruzi. Sequence characterization revealed a well-conserved protease domain in most proteins, although residues critical for catalytic activity were frequently altered. Many of the proteins contain a novel N-terminal sequence motif unique to kinetoplastids. Furthermore, 24 of the sequences contain N-terminal fatty acid acylation motifs indicating association of these proteins with intracellular membranes. This extended family of proteins also includes a group of sequences that completely lack a protease domain but is specifically related to other kinetoplastid calpain-related proteins by a highly conserved N-terminal domain and by genomic organization. All sequences lack the C-terminal calmodulin-related calcium-binding domain typical of most mammalian calpains. Our analysis emphasizes the highly modular structure of calpains and calpain-like proteins, suggesting that they are involved in diverse cellular functions. The discovery of this surprisingly large family of calpain-like proteins in lower eukaryotes that combines novel and conserved sequence modules contributes to our understanding of the evolution of this abundant protein family. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor : Dr. John Oakeshott]     

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

Base J or beta-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals approximately 25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi.     

Distinct genes encode type II Topoisomerases for the nucleus and mitochondrion in the protozoan parasite Trypanosoma brucei

Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2alpha and TbTOP2beta. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned approximately 1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2alpha encodes an ATP-dependent topoisomerase that appears as a single approximately 170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2beta is unclear. Although transcribed in trypanosomes, TbTOP2beta is not detected by beta-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2alpha as a promising target for the development of much-needed new therapies.     

Features of autophagic cell death in Plasmodium liver-stage parasites

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.     

Characterization of Endotrypanum (Kinetoplastida: Trypanosomatidae), a unique parasite infecting the neotropical tree sloths (Edentata)

This article reviews current concepts of the biology of Endotrypanum spp. Data summarized here on parasite classification and taxonomic divergence found among these haemoflagellates come from our studies of molecular characterization of Endotrypanum stocks (representing an heterogenous population of reference strains and isolates from the Brazilian Amazon region) and from scientific literature. Using numerical zymotaxonomy we have demonstrated genetic diversity among these parasites. The molecular trees obtained revealed that there are, at least, three groups (distinct species?) of Endotrypanum, which are distributed in Central and South America. In concordance with this classification of the parasites there are further newer molecular data obtained using distinct markers. Moreover, comparative studies (based on the molecular genetics of the organisms) have shown the phylogenetic relationships between some Endotrypanum and related kinetoplastid lineages.     

Trypanosomes and mammalian sperm: one of a kind?

Flagellar-mediated motility is an indispensable function for cell types as evolutionarily distant as mammalian sperm and kinetoplastid parasites, a large group of flagellated protozoa that includes several important human pathogens. Despite the obvious importance of flagellar motility, little is known about the signalling processes that direct the frequency and wave shape of the flagellar beat, or those that provide the motile cell with the necessary environmental cues that enable it to aim its movement. Similarly, the energetics of the flagellar beat and the problem of a sufficient ATP supply along the entire length of the beating flagellum remain to be explored. Recent proteome projects studying the flagella of mammalian sperm and kinetoplastid parasites have provided important information and have indicated a surprising degree of similarities between the flagella of these two cell types.     

Functions of LIM-homeobox genes

Homeobox genes play fundamental roles in development. They can be subdivided into several subfamilies, one of which is the LIM-homeobox subfamily. The primary structure of LIM-homeobox genes has been remarkably conserved through evolution. Have their functions similarly been conserved? A host of new data has been derived from mutational analysis in diverse organisms, such as nematodes, flies and vertebrates. These studies have revealed a prominent involvement of LIM-homeodomain proteins in tissue patterning and differentiation, and their function in neural patterning is evident in all organisms studied to date. Here, we summarize the recent findings on LIM-homeobox gene function, compare the function of these genes from different organisms and describe specific co-factor requirements.     

The evolution and diversity of kinetoplastid flagellates

Five years ago, little was known about kinetoplastid evolution. Recent improvements in the taxon sampling for nuclear rRNA genes and several protein markers have transformed this understanding. Parasitism evolved at least four times in kinetoplastids. Obligate parasitic trypanosomatids are a relatively 'derived' group within kinetoplastids; their closest relative is likely to be the free-living Bodo saltans, and the ancestral trypanosomatids were probably parasites of insects. Although subject to recent controversy, trypanosomes (genus Trypanosoma) probably constitute a monophyletic group. Several unusual features of trypanosomatid genomes (e.g. trans-splicing, mitochondrial RNA editing and intron poverty) are common in kinetoplastids and pre-date the adoption of parasitism. The framework of relationships is becoming robust enough for real comparative approaches to be used to understand kinetoplastid biology.     

Genome-wide detection of serpentine receptor-like proteins in malaria parasites

Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7-TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.