Sialoglycoconjugates in Herpetomonas megaseliae: role in the adhesion to insect host epithelial cells

Herpetomonas megaseliae is a monoxenic trypanosomatid isolated from the phorid fly Megaselia scalaris . In the present report, the expression of cell surface sialoglycoconjugates in this parasite was analyzed by Western blotting, flow cytometry and fluorescence microscopy analyses using lectins that specifically recognize sialic acid residues. A strong reaction was detected when parasites were treated with Limax flavus, Maackia amurensis and Sambucus nigra lectins. Analysis of crude protein extracts by Western blotting revealed that bands with molecular masses ranging from 19 to 80 kDa were reactive to these lectins, which showed a sugar-inhibited recognition with the parasite extract. These results indicated that molecules containing α2,3- and α2,6-sialylgalactosyl sequences are present in this protozoan. The role of the surface sialomolecules in the interaction with explanted guts from Aedes aegypti was assessed. The interaction of H. megaseliae with the insect gut was strongly inhibited in the presence of mucin (71%), fetuin (68%) and sialyllactose (68%). Collectively, our results suggest a possible involvement of sialomolecules in the interaction between this insect trypanosomatid and the invertebrate host.     

Programmed cell death in trypanosomatids: is it an altruistic mechanism for survival of the fittest?

The protozoan parasites Leishmania, Trypanosoma cruzi and Trypanosoma brucei show multiple features consistent with a form of programmed cell death (PCD). Despite some similarities with apoptosis of mammalian cells, PCD in trypanosomatid protozoans appears to be significantly different. In these unicellular organisms, PCD could represent an altruistic mechanism for the selection of cells, from the parasite population, that are fit to be transmitted to the next host. Alternatively, PCD could help in controlling the population of parasites in the host, thereby increasing host survival and favoring parasite transmission, as proposed by Seed and Wenk. Therefore, PCD in trypanosomatid parasites may represent a pathway involved both in survival and propagation of the species.     

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.     

Endocytosis and secretion in trypanosomatid parasites - Tumultuous traffic in a pocket

Trypanosomatids are flagellated protozoan parasites of invertebrates, vertebrates and plants. Some species, found in the subtropics and tropics, cause chronic diseases in humans and domestic animals. The surface of the trypanosomatid provides a shield against environmental challenges, ligands for interaction with host cells, as well as receptors and transporters for the uptake of nutrients. Communication between the parasite and its environment is confined to the flagellar pocket, an invagination of the plasma membrane around the base of the flagellum. In this review, the authors discuss endocytosis, secretion and membrane trafficking in Trypanosoma and Leishmania.     

The Limits on Trypanosomatid Morphological Diversity

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.     

Diversity of insect trypanosomatids assessed from the spliced leader RNA and 5S rRNA genes and intergenic regions

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.     

Biochemical properties of trypanosomatid lactate dehydrogenases

Lactate dehydrogenase (LDH, E.C.1.1.1.27) was found in supernatant (cytoplasmic enzyme) fractions of the trypanosomatid flagellates Trypanosoma conorhini and Crithidia fasciculata if 10 mm cysteine was present in the homogenizing medium. The T. conorhini LDH activity with pyruvate as substrate was increased 35% if 5 mm cysteine was also included in reaction mixtures. K(m) values for the T. conorhini enzyme were 3.3 x 10(-4)m with pyruvate, and 1.6 x 10(-4)m with alpha-ketobutyrate. Cysteine inhibited alpha-ketobutyrate reduction. Comparison of trypanosomatid and human serum LDH enzymes with respect to K(m), substrate activity and inhibition, pH optima, and K(i) values for oxalate and oxamate indicated that the trypanosomatid isoenzymes differed significantly from serum LDH. C. fasciculata LDH was extremely labile, since 59% of the activity was lost 90 min after isolation. The role of LDH enzymes in trypanosomatid metabolism is discussed, and the results are related to other trypanosomatid LDH enzymes. The comparison of homologous enzymes in host and parasite is discussed with regard to metabolic function and a possible model system for chemotherapy.     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.     

The more insect trypanosomatids under study-the more diverse Trypanosomatidae appears

From 10 trypanosomatids genera six comprise monogenetic parasites of insects and for the rest of four genera insects may serve as vectors. The invertebrate host is an essential element of trypanosomatids life cycle, but from more than 900 recognised vertebrate hosts only about 500 species of insects have been discovered to be the hosts of homoxenous trypanosomatids. Nothing or very little is known about insect trypanosomatids in many extensive areas such as South East Asia, Australia, Japan and some others. Each new region explored brings many new findings. Recently flagellates were found in new insect species and families. The border of parasites distribution was expanded till Central Asia, Far East and North over the Polar Circle. As paleogeographical events are now under contemplating in trypanosomatids phylogeny researches so northern insect trypanosomatids may attract some attention as the elements of postglacial fauna which is definitely young. Very broad host specificity of insect trypanosomatids and high probability to isolate non-specific parasite show causes that only the investigation of a culture may solve the question 'what parasite was really isolated?'. Examination of cell morphotypes in the host has clearly demonstrated that they are not sufficient for classification and may lead us to be mistaken. The number of insect trypanosomatid cultures is inadequate for characterisation of the diversity of insects trypanosomatids. Trypanosoma is actually the only trypanosomatid genus which is out of questions. Insect trypanosomatids comprise the most diversified part of trypanosomatids evolutionary tree. Recent ssrRNA phylogenetic analysis and morphological data show that three insect isolates represent new lineages on trypanosomatid evolutionary tree, as well as dendrograms derived from PCR data demonstrated some new groups of isolates. Therefore, the more insect trypanosomatids are involved in laboratory investigations--the more new clusters or/and new lineages are appearing on the tree.     

Honey bee and bumblebee trypanosomatids: specificity and potential for transmission

Abstract 1. Experimental studies of multihost parasite dynamics are scarce. Understanding the transmission dynamics of parasites in these systems is a key task in developing better models of parasite evolution and to make more accurate predictions of disease dynamics. 2. Bumblebee species (Bombus spp.) host the trypanosomatid parasite, Crithidia bombi. Its transmission in the field occurs through the shared use of flowers. Flowers are a perfect scenario for inter‐taxa transmission of diseases because they are used by a wide range of animals. 3. Honey bees host a poorly studied trypanosomatid, Crithidia mellificae. In this study, five questions have been experimentally addressed: (a) Can C. bombi infect honey bees? (b) Can C. mellificae infect bumblebees? (c) Can the honey bee act as a vector for C. bombi? (d) Are C. bombi cells present in honey‐bee faeces? (e) Does C. bombi have an effect on the mortality of honey bees after ingestion? 4. While both parasites were found to be specific to their hosts at the genus level, results suggest that honey bees may play a role in the epidemiology of C. bombi transmission.     

The genotypic structure of a multi-host bumblebee parasite suggests a role for ecological niche overlap

The genotypic structure of parasite populations is an important determinant of ecological and evolutionary dynamics of host-parasite interactions with consequences for pest management and disease control. Genotypic structure is especially interesting where multiple hosts co-exist and share parasites. We here analyze the natural genotypic distribution of Crithidia bombi, a trypanosomatid parasite of bumblebees (Bombus spp.), in two ecologically different habitats over a time period of three years. Using an algorithm to reconstruct genotypes in cases of multiple infections, and combining these with directly identified genotypes from single infections, we find a striking diversity of infection for both data sets, with almost all multi-locus genotypes being unique, and are inferring that around half of the total infections are resulting from multiple strains. Our analyses further suggest a mixture of clonality and sexuality in natural populations of this parasite species. Finally, we ask whether parasite genotypes are associated with host species (the phylogenetic hypothesis) or whether ecological factors (niche overlap in flower choice) shape the distribution of parasite genotypes (the ecological hypothesis). Redundancy analysis demonstrates that in the region with relatively high parasite prevalence, both host species identity and niche overlap are equally important factors shaping the distribution of parasite strains, whereas in the region with lower parasite prevalence, niche overlap more strongly contributes to the distribution observed. Overall, our study underlines the importance of ecological factors in shaping the natural dynamics of host-parasite systems.     

The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the cell surface of digestive or other organs, but the intracellular habit appears to have arisen independently in several groups of Protista.     

Molecular dissection of the functional domains of a unique, tartrate-resistant, surface membrane acid phosphatase in the primitive human pathogen Leishmania donovani

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L. donovani enzyme. In addition, we characterized its structure, demonstrated its constitutive expression in both parasite developmental forms, and determined the cell surface membrane localization of its translated protein product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters in a homologous leishmanial expression system to dissect the functional domains of this unique, tartrate-resistant, surface membrane enzyme.     

First description of Lotmaria passim and Crithidia mellificae haptomonad stages in the honeybee hindgut

The remodelling of flagella into attachment structures is a common and important event in the trypanosomatid life cycle. Lotmaria passim and Crithidia mellificae can parasitize Apis mellifera, and as a result they might have a significant impact on honeybee health. However, there are details of their life cycle and the mechanisms underlying their pathogenicity in this host that remain unclear. Here we show that both L. passim promastigotes and C. mellificae choanomastigotes differentiate into haptomonad stages covering the ileum and rectum of honeybees. These haptomonad cells remain attached to the host surface via zonular hemidesmosome-like structures, as revealed by transmission electron microscopy. This work describes for the first known time the haptomonad morphotype of these species and their hemidesmosome-like attachments in A. mellifera, a key trait used by other trypanosomatid species to proliferate in the insect host hindgut.     

Plant derived therapeutics for the treatment of Leishmaniasis

Diseases caused by insect borne trypanosomatid parasites are significant, yet remain a neglected public health problem. Leishmania, a unicellular protozoan parasite is the causative organism of Leishmaniasis and is transmitted by female phlebotamine sandflies affecting millions of people worldwide. In the wake of resistance to pentavalent antimonial drugs, new therapeutic alternatives are desirable. The plant kingdom has in the past provided several affordable compounds and this review aims to provide an overview of the current status of available leishmanicidal plant derived compounds that are effective singly or in combination with conventional anti-leishmanial drugs, yet are non toxic to mammalian host cells. Furthermore, delineation of the contributory biochemical mechanisms involved in mediating their effect would help develop new chemotherapeutic approaches.     

Invasion factors are coupled to key signalling events leading to the establishment of infection in apicomplexan parasites

Invasion of host cells by apicomplexan parasites is initiated when specialized secretory organelles called micronemes discharge protein complexes onto the parasite surface in response to a rise in parasite intracellular calcium levels. The microneme proteins establish interactions with host cell receptors, engaging the parasite with the host cell surface, and signal for the immediate exocytosis of another set of secretory organelles named the rhoptries. The rhoptry proteins reprogram the invaded host cell and participate in the formation of the parasitophorous vacuole in which the intracellular parasite resides and replicates. Disengagement of the invading parasite from the host cell receptors involves the action of at least one parasite plasma membrane rhomboid protease, which is concomitantly implicated in a checkpoint that signals the parasite to switch from an invasive to a replicative mode.     

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by ³H- palmitic acid and phosphate (³²Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC–MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

Experimental pathogenicity of a presumed monoxenous trypanosomatid isolated from humans in a murine model

Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV-infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow-derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 10(7) promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post-infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross-reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.     

Host chaperones are recruited in membrane-bound complexes by Plasmodium falciparum.

The ability of malarial parasite to deploy proteins at the surface of infected erythrocytes is well known. After their synthesis within the parasite, the cargo proteins are exported from the parasite and carried across the erythrocyte cytoplasm to be delivered at the erythrocyte surface. Our knowledge about the mechanisms involved in this complex trafficking path is limited. We have addressed the involvement of chaperones in traffic across erythrocyte cytoplasm. Our analyses of the chaperones available to the parasite indicated that none of the reported chaperones of the parasite origin are present in the erythrocyte cytoplasm. The chaperones of the host (Hsp70, Hsp90, Hop60), on the other hand, were readily detected in the erythrocyte cytosol. Hypotonic lysis and detergent solubilization experiments indicated that unlike their soluble nature in normal erythrocytes, host chaperones are recruited in membrane-bound, detergent-resistant complexes in infected cells. The association of host-Hsp70 with detergent-resistant complexes was ATP-dependent. Importantly, host chaperones could be detected in knob-enriched fractions and could be cross-linked to the knob subunit, PfHRP1, in a large complex at the surface of the infected erythrocytes. Our results implicate host chaperones in the assembly of parasite proteins such as knob subunits at the erythrocyte surface.