Expression of Hexokinase in the Proteome Profile of Leishmania major and Crithidia

Objective: The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with Crithidia in Iran, this study aims to investigate the expression of HK in the proteome of Leishmania major and Crithidia using a proteomic approach. Methods: A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of L. major and Crithidia using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of L. major and Crithidia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS). Results: The PCR results showed 5 positive cases for Crithidia and 96 positive cases for L. major. MALDI TOF/TOF MS indicated HK as a common protein in the proteome of L. major and Crithidia. HK was up-regulated in the Crithidia proteome in comparison with the L. major proteome. Conclusion: Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in Crithidia might be related to the increased duration of the treatment period in CL patients co-infected with Crithidia.

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium.

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

Does pathogen spillover from commercially reared bumble bees threaten wild pollinators?

The conservation of insect pollinators is drawing attention because of reported declines in bee species and the 'ecosystem services' they provide. This issue has been brought to a head by recent devastating losses of honey bees throughout North America (so called, 'Colony Collapse Disorder'); yet, we still have little understanding of the cause(s) of bee declines. Wild bumble bees (Bombus spp.) have also suffered serious declines and circumstantial evidence suggests that pathogen 'spillover' from commercially reared bumble bees, which are used extensively to pollinate greenhouse crops, is a possible cause. We constructed a spatially explicit model of pathogen spillover in bumble bees and, using laboratory experiments and the literature, estimated parameter values for the spillover of Crithidia bombi, a destructive pathogen commonly found in commercial Bombus. We also monitored wild bumble bee populations near greenhouses for evidence of pathogen spillover, and compared the fit of our model to patterns of C. bombi infection observed in the field. Our model predicts that, during the first three months of spillover, transmission from commercial hives would infect up to 20% of wild bumble bees within 2 km of the greenhouse. However, a travelling wave of disease is predicted to form suddenly, infecting up to 35-100% of wild Bombus, and spread away from the greenhouse at a rate of 2 km/wk. In the field, although we did not observe a large epizootic wave of infection, the prevalences of C. bombi near greenhouses were consistent with our model. Indeed, we found that spillover has allowed C. bombi to invade several wild bumble bee species near greenhouses. Given the available evidence, it is likely that pathogen spillover from commercial bees is contributing to the ongoing decline of wild Bombus in North America. Improved management of domestic bees, for example by reducing their parasite loads and their overlap with wild congeners, could diminish or even eliminate pathogen spillover.

L-Proline uptake in Crithidia deanei is influenced by its endosymbiont bacterium.

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.

Episomally driven antisense mRNA abrogates the hyperinducible expression and function of a unique cell surface class I nuclease in the primitive trypanosomatid parasite, Crithidia luciliae.

Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3'-nucleotidase/nuclease (Cl 3'NT/NU) activity by approximately 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3'NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3'NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3'NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3'NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl alpha-tubulin) in these transfectants. Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3'NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.

Phosphatidylcholine synthesis in Crithidia deanei: the influence of the endosymbiont.

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.

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.

Influence of the endosymbiont of Blastocrithidia culicis and Crithidia deanei on the glycoconjugate expression and on Aedes aegypti interaction.

Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.

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.

Ectonucleotide diphosphohydrolase activity in Crithidia deanei.

In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.

A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.

Crithidia luciliae: functional expression of nucleoside and nucleobase transporters in Xenopus laevis oocytes.

The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Poly(A)+-mRNA from C. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected into X. laevis oocytes. For "purine-replete" mRNA, expression of adenosine and hypoxanthine uptake in microinjected X. laevis oocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with "purine-starved" mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast to C. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) in X. laevis oocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters in C. luciliae may not be under transcriptional control.

Stimulated transport of adenosine, guanosine and hypoxanthine in Crithidia luciliae: metabolic machinery in which the parasite has a distinct advantage over the host.

Nutritional insufficiency is a common environmental extreme to which parasitic protozoa are routinely exposed. In this study of purine salvage mechanisms we illustrate some successful adaptations of the parasite Crithidia luciliae to its environment, particularly in the case of purine stress. In purine-depleted conditions, the insect trypanosome C. luciliae has the ability to increase the rates of transport of adenosine, guanosine and hypoxanthine and the activity of the exoenzyme 3'nucleotidase (3'NTase) during the growth cycle. The dramatic increase in these activities appears after a 72-h period in culture. The increased activity of the purine transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine or hypoxanthine (100 microM). Under conditions where the concentration of purines in the medium could be closely regulated, C. luciliae grown in purine-replete medium (> or = 75 microM purine) exhibited low rates of purine transport and activity of 3'NTase. In comparison, parasites transferred to medium with a low purine source (< or = 7.5 microM adenosine) had levels of adenosine, guanosine and hypoxanthine transport elevated 25-40-fold. The results link the simultaneous increase in activity of the nucleoside and base transporters, 3'NTase activity and a general increase in the purine salvage of C. luciliae to the concentration of purines available at any time to the parasite.

Volume-regulatory amino acid release from the protozoan parasite Crithidia luciliae.

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue alpha-aminoisobutyrate. alpha-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of alpha-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated alpha-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for alpha-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated alpha-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mM. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells.

Crithidia luciliae: regulation of purine nucleoside transport by extracellular purine concentrations.

During the growth cycle of the protozoan parasite Crithidia luciliae, there was a dramatic concomitant increase in the rate of adenosine and guanosine transport and 3' nucleotidase (3'NTase) activity after 72-94 hr. The simultaneous increased activities of the nucleoside transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine (100 microM). C. luciliae grown in purine-replete medium (> or = 75 microM adenosine) exhibited low rates of adenosine and guanosine transport whilst parasites transferred to a defined serum-free medium containing < or = 7.5 microM adenosine demonstrated elevated levels of both adenosine and guanosine transport up to 25- to 40-fold. The increased activity of the nucleoside transporters was inhibited by cycloheximide (10 microM). Under conditions of purine depletion 3'AMP and 3'GMP inhibited the adenosine and guanosine transporters, respectively. However, in the presence of a purine supplement (100 microM), neither 3'AMP nor 3'GMP was an effective inhibitor of nucleoside transport. Our results link the increased activity of the nucleoside transporters to the increased activity of the 3'NTase, indicating the activation of a purine salvage system not previously reported in other organisms.

Putrescine active uptake system in the Trypanosomatid Crithidia fasciculata.

Using the insect Trypanosomatid Crithidia fasciculata as a model parasite of mammalian pathogenic flagellates, i.e. Leishmania and Trypanosoma spp., we have studied the kinetic and regulatory characteristics of the polyamine uptake system. Putrescine transport was age-dependent with maximum expression values at the proliferative logarithmic phase. Putrescine transport in Crithidia fasciculata was energy-dependent and against a putrescine concentration gradient. The integrity of the membrane sulfhydryl groups was absolutely required for optimum transport rates. The specificity of this mechanism was studied in the presence of a series of different chain length aliphatic diamines, showing the high specificity for putrescine and the poor effect of this series at the highest concentration analyzed as well as the higher polyamines spermidine and spermine. Finally, the well-known inhibitor of polyamine biosynthesis, DFMO, led to an upward regulation of putrescine uptake correlating with the depletion of intracellular polyamine pool. In addition, the presence of high concentrations of putrescine in the culture medium produced a downward regulation of this system.

Enhanced acquisition of purine nucleosides and nucleobases by purine-starved Crithidia luciliae.

The effects of purine starvation on the ability of the trypanosomatid Crithidia luciliae to accumulate purines were determined. Kinetic studies showed that the uptake of the nucleoside adenosine by purine-starved organisms was approximately 7-fold faster than by nutrient-replete cells. Further, these studies demonstrated that purine-starved organisms accumulated the nucleobases hypoxanthine and adenine at a rate > 100-fold faster than organisms cultivated under replete conditions. Activities of several intracellular purine-salvage enzymes were measured in organisms from both culture conditions. Of those measured, the activities of adenine deaminase and hypoxanthine phosphoribosyltransferase were elevated approximately 4-fold and approximately 11-fold, respectively, in purine-starved organisms. Competitive substrate specificity studies suggested that these elevated enzyme activities were not responsible for the increased rates of uptake by purine-starved cells. The results are consistent with the induction of novel surface membrane purine transporters expressed in response to purine starvation. These studies using C. luciliae may provide insights into the mechanisms of trypanosomatid adaptation to altered environments encountered during the course of the life cycle.

Crithidia luciliae: effect of purine starvation on S-adenosyl-L-methionine uptake and protein methylation.

The utilization of S-adenosyl-L-[methyl-3H]methionine ([3H-methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate approximately 10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an approximately 10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [3H-methyl]AdoMet, S-adenosyl-L-[carboxyl-14C]methionine, L-[methyl-3H]methionine and L-[35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [3H-methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, approximately 15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [3H-methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.

Nucleoside transport in Crithidia luciliae.

Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent Km values for adenosine and guanosine were 9.34 +/- 1.30 and 10.6 +/- 2.60 microM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, L-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.