Dramatic changes in gene expression in different forms of Crithidia fasciculata reveal potential mechanisms for insect-specific adhesion in kinetoplastid parasites.

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.

Screening of the MMV and GSK open access chemical boxes using a viability assay developed against the kinetoplastid Crithidia fasciculata.

Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis.

An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Cinética multiplicativa de clones de Crithidia fasciculata (Kinetoplastida: Trypanosomatidae) en un sistema de cultivo in vitro / Multiplicative kinetics of Crithidia fasciculata (Kinetoplastida: Trypanosomatidae) clones in an in vitro culture system

La cinética multiplicativa de la cepa parental y cinco clones de Crithidia fasciculata (Kinetoplastida: Trypanosomatidae) fue estudiada en agar sangre 40% difásico. Los clones fueron obtenidos de cultivos en agar sangre 40% y denominados Ia, IIIa, IVa, Va y CAA. En cada caso, tubos de agar sangre 40% difásico con infusión cerebro corazón 3,7 % como fase líquida fueron inoculados con 103 coanomastigotos de cada clon y la cepa parental. La concentración de parásitos fue estimada semanalmente durante 12 semanas expresando las concentraciones en coanomastigotos/mm3. La variabilidad fue evaluada por análisis de variancia (ANDEVA) para mediciones repetidas (a:0,05) realizadas durante cada día de observación. Electroforesis en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE) fueron también efectuadas para evaluar los perfiles proteicos de los clones y la cepa parental. Los resultados mostraron una fase de crecimiento logarítmico muy corta que antecedió a una fase estacionaria prolongada. (p > 0,05). No hubieron diferencias estasdísticamente significativas entre la concentración de coanomastigotos/mm3 entre los sistemas estudiados durante cada día de observación. Los perfiles proteicos mostraron patrones altamente homogéneos en todos los sistemas estudiados. Los resultados sugieren que las menores presiones evolutivas que sufre naturalmente C. fasciculata en su ciclo propagativo podrían estar relacionadas con la homogeneidad observada, pero es requerida más información en tópicos como inmunología, biología y genética para demostrar dicha homogeneidad.

Fellowship of the rings: the replication of kinetoplast DNA.

Kinetoplastid protozoa such as trypanosomes and Leishmania are important because they cause human disease. These parasites are named after one of their most unusual features, a mitochondrial DNA known as kinetoplast DNA (kDNA). Unlike all other DNA in nature, kDNA comprises a giant network of interlocked DNA rings with a topology resembling that of medieval chain mail. The replication of the kDNA network is more complex than previously thought, and the discovery of new proteins involved in this process is currently the best approach for illuminating the replication mechanism.

Growth-phase dependent substrate adhesion in Crithidia fasciculata.

Crithidia fasciculata is a trypanosomatid flagellate that parasitizes several species of mosquito. Within the alimentary tract of its host, C. fasciculata exists in two forms: one is a non-motile form, attached in clusters to the lining of the gut, the other a more elongated form swimming freely in the gut lumen. We have developed an in vitro culture system that reproduces the appearance of these two distinct morphological forms. Using two different cultivation methods, shaking and stationary incubations, we have demonstrated that adherence phenotypes are growth-phase dependent. Organisms in the logarithmic phase of growth possess the ability to adhere to substrates; this ability is lost when the organism enters a stationary growth phase. Parasite adherence was independent of cultivation method or substrate. Furthermore, adherent forms of Crithidia maintained their adhesive properties following their removal from substrates. Our data reveal a growth-phase-regulated process of cell attachment that may influence the transmission and dissemination of this parasitic flagellate.

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata.

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Extremely rapid turnover of S-adenosylmethionine decarboxylase in Crithidia fasciculata.

The activity of S-adenosylmethionine decarboxylase (AdoMetDC) in Crithidia fasciculata was shown to be correlated to the growth of the parasite. An increase in activity was observed during exponential growth. Inhibition of protein synthesis induced an extremely rapid decay of AdoMetDC activity. The half-life of the enzyme was estimated to be about 3 min, which is the shortest half-life ever recorded for an eukaryotic AdoMetDC. The reduction in AdoMetDC activity was correlated with a decrease in AdoMetDC protein content, demonstrating a rapid turnover of the enzyme. No polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite.

Spermidine is essential for normal proliferation of trypanosomatid protozoa.

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of alpha-difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis-cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.

Disruption of the Crithidia fasciculata KAP1 gene results in structural rearrangement of the kinetoplast disc.

The mitochondrial DNA (kinetoplast DNA) in trypanosomatids exists as a highly organized nucleoprotein structure with the DNA consisting of thousands of interlocked circles. Four H1 histone-like proteins (KAP1, 2, 3 and 4) are associated with the kinetoplast DNA in the trypanosomatid Crithidia fasciculata. We have disrupted both alleles of the KAP1 gene in this diploid protozoan and shown that expression of the KAP1 protein is eliminated. The mutant strain is viable but has substantial rearrangement of the kinetoplast structure. Expression of the KAP1 protein from an episome restored expression of the KAP1 protein in the mutant strain and also restored a normal kinetoplast structure. These studies provide evidence that the KAP1 protein is involved in kinetoplast DNA organization in vivo but is nonessential for cell viability.

Sensitivity of trypanosomatid protozoa to DFMO and metabolic turnover of ornithine decarboxylase.

alpha-Difluoromethylornithine (DFMO), the specific and irreversible inhibitor of ornithine decarboxylase (ODC), was able to induce the arrest of proliferation in Leishmania mexicana and ODC-transformed Trypanosoma cruzi cultures grown in a semi-defined medium essentially free of polyamines. Conversely, Crithidia fasciculata and Phytomonas 274 were not affected by the inhibitor. The drug-resistance of Crithidia and Phytomonas was neither caused by an impairment of DFMO uptake nor by a decrease of the enzyme affinity for the inhibitor. We were also able to rule out the possibility of ODC overexpression in the drug-tolerant parasites. The measurements of ODC metabolic turnover indicated that the enzymes from Crithidia and Phytomonas have a short half-life of 20-40 min, while ODC from Leishmania and transgenic Trypanosoma cruzi are rather stable with a half-life longer than 6 hours. Analyses of polyamine internal pools under different growth conditions have shown that DFMO was able to markedly decrease the levels of putrescine and spermidine in all parasites, but the depletion of spermidine was higher in trypanosomatids containing an ODC with slow turnover. Our results suggest that in these parasites cultivated in the presence of the drug, spermidine might decrease below critical levels needed to maintain trypanothione concentrations or other conditions essential for normal proliferation.

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei.

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.

Effects of cationic diamidines on polyamine metabolism in the trypanosomatid Crithidia fasciculata.

The effect of a series of five recently synthesized cationic diamidines on cell proliferation and polyamine metabolism was studied on cultures of the model Trypanosomatid Crithidia fasciculata. Compounds displaying two arylic moieties (CGP039937A and CGP040215A) were ten fold more cytostatic than those displaying only one arylic residue (CGP033829A, CGP035753A and CGP036958A). The depletion of intracellular polyamine, putrescine and spermidine, pools and the effect of these compounds on S-adenosylmethionine decarboxylase and putrescine uptake suggest the requirement of two arylic groups in their chemical structure to obtain measurable effects on both polyamine metabolism and cell growth.

The structure of replicating kinetoplast DNA networks.

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.

A microtitre plate method for measuring biopterin with cryopreserved Crithidia fasciculata.

The assay of biopterin derivatives in dried blood spots is used by us in initial screening for inherited defects in tetrahydrobiopterin synthesis. The previously described method (1) required aseptic technique and microbiological facilities. The modification detailed here has the advantages of antibiotic cover, which overcomes these needs and microtitre plate technology allowing the incubation time to be halved with precision and accuracy retained. Data reduction facilities may be applied.

Ornithine decarboxylase from Crithidia fasciculata is metabolically unstable and resistant to polyamine down-regulation.

Ornithine decarboxylase (ODC) of Crithidia fasciculata extracts shows maximal activity during exponential growth of the parasite and decreases markedly in the stationary phase. The inhibition of protein synthesis by cycloheximide evoked a rapid loss of enzyme activity with a half-life of about 30 min. Upon removal of DFMO from Crithidia cultures treated with the drug for 24 h, the ODC activity increased at the same rate as total protein synthesis. The addition of putrescine at high concentrations to parasites cultivated in a synthetic medium showed that Crithidia ODC levels were not reduced by polyamines.