Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania.

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.

Antidepressants cause lethal disruption of membrane function in the human protozoan parasite Leishmania.

The antidepressant compounds clomipramine and nitroimipramine were cidal to extracellular promastigotes of both human protozoan parasites Leishmania donovani and Leishmania major. Clomipramine also killed amastigotes of both species within murine macrophages with no apparent toxicity to the host cells. Further, amastigotes were more sensitive than promastigotes to clomipramine. Clomipramine (100 micromoles per liter or 0.2 nanomole per 1 X 10(6) cells) inhibited L-proline transport in promastigotes. Synergistic inhibition of L-proline transport was observed with clomipramine after addition of either of the ionophores valinomycin or nigericin. These observations suggest that the cytotoxic effects of clomipramine result from its disruption of the proton electrochemical gradient of the parasite surface membrane.

Stage-specific expression of the proline-alanine transporter in the human pathogen Leishmania.

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

A monoclonal antibody to rhesus erythrocyte band 3 inhibits invasion by malaria (Plasmodium knowlesi) merozoites.

Receptors on erythrocytes and malaria parasites mediate specific attachment and junction formation between these cells that lead to invasion of the erythrocytes. We identified monoclonal antibody A9 and its subclone A9D3 that bound to rhesus erythrocytes and blocked invasion of the erythrocytes by Plasmodium knowlesi merozoites. The monoclonal antibodies did not block attachment, the initial step in invasion, although swelling and crenation of the erythrocyte, which normally occur after attachment, were rarely observed in the presence of antibody. The monoclonal antibody immunoprecipitated rhesus erythrocyte band 3. It bound to erythrocytes of another Old World monkey, the kra monkey, but not to erythrocytes of New World monkeys, chimpanzees, or man. Since the antibody did not bind to human erythrocytes, we could test for nonspecific toxicity to the parasite by studying the effect of the ascites and purified antibody on invasion of human erythrocytes. The antibody caused a minimal reduction in invasion of human erythrocytes, a reduction no greater than that seen with an unrelated monoclonal antibody. Further evidence that the inhibition was specific came from study of Fab fragments of A9D3. Column-purified Fab fragments reduced invasion of rhesus erythrocytes without affecting invasion of human erythrocytes. Fab fragments preabsorbed with rhesus erythrocytes did not inhibit invasion. From the above data, we conclude that band 3 is involved in a stage in the invasion process after initial recognition.

Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation.

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarray-based expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up- and down-regulation during differentiation. Genes that were permanently up-regulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Down-regulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up- or down-regulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.

Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes.

Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes. Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L. donovani promastigotes. By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles. The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity. Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L. donovani promastigotes. Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells. This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes.

Regulation of L-proline transport in Leishmania donovani by extracellular pH.

We have previously shown that Leishmania donovani promastigotes adapted to long-term culture at acidic pH can serve as a model to study parasite development in a lysosomal-like environment. In this study we investigated the effect of growth pH on L. donovani L-proline transport systems. Reducing the pH of the growth medium causes an up to 7-fold decrease in the extent of L-proline transport. Transport resumes after switching the culture from pH 4.5 to pH 7 for 48 h by a protein synthesis-dependent process. The pH optimum for transport changes from 7.5 in promastigotes grown at pH 7 to 5.5 in cells grown at pH 4.5. In addition, kinetic analysis of L-proline transport showed that Vmax in pH 4.5-grown L. donovani promastigotes is one-tenth that of cells grown at pH 7 (4.5 and 44.7 nmol min-1 (10(8) cells)-1, respectively). The apparent Km for L-proline in pH 4.5 promastigotes is one-half of the Km in pH 7 cells (0.30 and 0.65 mM, respectively). In contrast to L-proline transport, D-glucose transport demonstrates a growth pH-independent activity: Km and Vmax as well as optimum pH of transport are similar in promastigotes grown at either pH 7 or pH 4.5. Taken together, the results indicate that in L. donovani, expression and activity of L-proline transport is regulated by culture pH. The pH-dependent expression of L-proline transporters may be of physiological significance during the promastigote-amastigote transition.

Maintenance of cytoplasmic pH and proton motive force in promastigotes of Leishmania donovani.

Three methods were used to measure intracellular pH (pHi) of Leishmania donovani promastigotes: (a) measurement of the fluorescence of the pH indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein; (b) pH null point assays; and (c) determination of the distribution across the promastigote plasma membrane of the fluorescent amine acridine orange and of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. The three methods gave similar results and showed that promastigotes of L. donovani maintain pHi at a narrow range of 6.4-6.7, throughout an extracellular pH (pHo) range of 5.5-7.4. L-Proline transport in L. donovani promastigotes, which is known to be coupled to proton translocation, was used to estimate the proton electrochemical gradient across parasite plasma membrane. While proline uptake is optimal at pHo 7.5, an outward-directed concentration gradient is obtained at steady state throughout a pHo range of 5-8. The calculated electrochemical gradient of proline across the parasite plasma membrane at steady state is 90-100 mV within a pHo range of 5-8, suggesting an almost constant proton electrochemical gradient at this pHo range. Taken together, the results show that the parasites regulate both pHi and the size of the chemiosmotic energy required to drive active transport of nutrients.

Glucose transport in Leishmania donovani promastigotes.

Mid-log phase Leishmania donovani promastigotes accumulated 2-deoxy-D-glucose (2-DOG) via a carrier mediated transport system, maintaining an apparent Km of 24.4 microM and a Vmax of 3.12 nmol mg-1 protein min-1. D-Glucose but not L-glucose competitively inhibited the 2-DOG transport with an apparent Ki of 18.7 microM. Transport of 2-DOG was inhibited by 2,4-dinitrophenol and NaN3. The parasites maintained a 2-DOG gradient of at least 79 fold across the surface membrane, demonstrating the active nature of the transport system.

Kinetic correlation of the acquisition of resistance to immune attack in schistosomula of Schistosoma mansoni with a developmental change in membrane potential.

When Schistosoma mansoni cercariae penetrate the skin of the mammalian host they rapidly pass from fresh water to a high salt physiologic environment and transform into schistosomula. Following this transition, the parasites migrate from the skin to the lungs during which time they change from being highly susceptible to immune attack to being refractory, as measured by in vitro cytotoxicity assays. In this study, in vivo or in vitro schistosomula of different ages were examined for developmentally linked changes in membrane function which might correlate with the attainment of the resistant state. In particular, alterations in the distribution of tetraphenylphosphonium (TPP+), a synthetic lipophilic cation which shows a potential dependent partition across membranes, were followed. Three-hour-old schistosomula, which are greater than 75% susceptible to antibody-dependent complement-mediated attack or lymphokine-activated macrophage-mediated cytotoxicity, acquired TPP+ at a similar rate and steady state level to 5-day-old lung worms, which were completely resistant to both these effector mechanisms. The addition of ouabain, a Na+/K+-ATPase inhibitor, caused a 50% decrease in both the rate and steady state of TPP+ uptake by 3 h parasites but had little effect on these parameters in lung worms. Valinomycin, a K+-ionophore, completely inhibited TPP+ influx in both stages. The characteristics of TPP+ efflux from 3-h and 5-day-old parasites preloaded with the cation were found to be dissimilar. Whereas 30% of acquired TPP+ was lost from lung worms within 2 h, only 10% of acquired cation was released from 3-h schistosomula during the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani.

Leishmania donovani is an obligatory intracellular parasite which cycles between the midgut of sand flies (extracellular promastigote) and the phagolysosomes of mammalian macrophages (intracellular amastigote). Promastigotes have been readily cultured, whereas axenic cultures of amastigotes have only recently been developed. A new method for in vitro differentiation of L. donovani promastigotes into amastigotes is presented, in which promastigotes are exposed to environmental changes that mimic the in vivo process. First, promastigotes are subjected to 37 degrees C + 5% CO2 for 24 h, and then are shifted to pH 5.5. Under these conditions, differentiation is completed within 120 h. In the reverse process, amastigotes are induced to differentiate back to promastigotes by transferring them to promastigote growth conditions (medium 199 at pH 7.4 and 26 degrees C). Axenic amastigotes closely resemble animal-derived amastigotes. They manifest all seven proteins of the amastigote-specific A2 gene family. They down-regulate lipophosphoglycan (LPG) synthesis and do not express it on their surface. LPG is up-regulated 2 h after inducing amastigotes to differentiate to promastigotes. Within 6 h, parasites resume the promastigote level of this molecule, although differentiation is completed only after 48 h. Axenic amastigotes also express amastigote-like metabolic activities of proline uptake, as well as thymidine and proline incorporation. In conclusion, the results indicate that the method developed for in vitro differentiation of L. donovani promastigotes to amastigotes is efficient and yields organisms resembling animal-derived amastigotes. Being able to induce in vitro differentiation of L. donovani provides us with an excellent tool to study Leishmania development and differentiation.

Tricyclic drugs reduce proton motive force in Leishmania donovani promastigotes.

Tricyclic compounds have been suggested as potential anti-leishmanial drugs. We have studied the effect of tricyclic drugs on several cellular functions in L. donovani promastigotes. Imipramine inhibits proline transport and reduces delta pH and cellular ATP at relatively high concentrations (IC50 = 50-80 microM). High concentrations of imipramine are also required to kill L. donovani promastigotes (LD50 greater than 50 microM). The presence of a chlorine atom in the side ring of either imipramine or promazine results in a three-fold increase in both IC50 and LD50 values. Tricyclic compounds in which the nitrogen in the middle ring was substituted with a carbon atom (amitryptyline and chlorprothixene) are most effective in causing cell death and in decreasing proline transport and delta pH (IC50 congruent to 5 microM), whereas depletion of cellular ATP requires a higher drug concentration (IC50 = 12 microM). Transchlorprothixene has IC50 values for proline transport, delta pH and cellular ATP that are similar to those of amitriptyline, whereas the cis isomer is less active. Imipramine, chlomipramine and chlorpromazine decrease the membrane potential in promastigotes. There is a direct correlation between inhibition of membrane transport of proline and the size of the membrane potential at various concentrations of the drugs. Taken together, the multiple effects of the tricyclic drugs on cellular functions in Leishmania suggest that the drugs cause cellular death by non-specific mechanisms, probably involving a general increase in membrane permeability.

The role of pH and temperature in the development of Leishmania parasites.

Protozoans of the genus Leishmania are obligate intracellular parasites that cycle between the midgut of sandflies and the phagolysosomes of mammalian macrophages and therefore are exposed to extreme environmental changes. Recent evidence obtained from in vitro experiments indicate that such environmental changes trigger a developmental program in the parasites. Thus, following heat shock, promastigotes from certain Leishmania species differentiate to amastigotes. Promastigotes also respond to acidification of their environment by changing the expression of a number of genes. However, the combination of both low pH and high temperature induces the transformation of the promastigote to the amastigote in all Leishmania species examined to date. This review discusses the role of pH and heat shock in gene regulation and its contribution to the differentiation processes in Leishmania spp. Cycling between cold-blooded insect vectors and the warm-blooded mammalian host is not unique to Leishmania spp., but typical to most parasitic protozoa. It is therefore likely that the mechanism of stress-induced differentiation is shared by other mammalian parasites.

Regulation of intracellular calcium in promastigotes of the human protozoan parasite Leishmania donovani.

By using the fluorescent Ca2+ indicator fura 2, we show that the concentration of free calcium in the cytoplasm of Leishmania donovani promastigotes is maintained at very low levels (73.5 +/- 10-94 +/- 8 nM at a [Ca2+]i range of 0-1 mM). The maintenance of low [Ca2+]i is energy-dependent as it is disrupted by KCN, H+-ATPase inhibitors, and ionophores. KCN, nigericin, and N,N'-dicyclohexylcarbodiimide increase cytosolic free calcium by mobilizing calcium from intracellular pools. Monensin and oligomycin increase [Ca2+]i by allowing influx of calcium from the external medium through the plasma membrane, but they have no effect on intracellular pools. Intracellular traffic of calcium was examined by measuring the transport of 45Ca2+ in digitonin-permeabilized promastigotes. Two transport systems for calcium were identified in these cells. One is respiration-dependent, suggesting a mitochondrial localization. A second system is respiration-independent but requires either endogenous or externally added ATP. The ATP-dependent Ca2+ transport is optimal at pH 7.1, has high affinity for calcium (Km = 92 nM, Vmax = 1 nmol/min/mg of protein), and is sensitive to orthovanadate. These properties suggest the presence of a Ca2+-ATPase similar to that of mammalian endoplasmic reticulum. Taken together, the results indicate that [Ca2+]i in L. donovani promastigotes is regulated at low concentration by mechanisms similar to those found in higher eukaryotic cells.