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.     

Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca2+ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca2+ in both the cell types and reduced amastigote death, thus establishing a central role of Ca2+ in intracellular parasite clearance. This influx of Ca2+ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites.     

Activation of human immunodeficiency virus type 1 in monocytoid cells by the protozoan parasite Leishmania donovani.

In this study, we demonstrated that the protozoan parasite Leishmania donovani and one of its major surface molecules, the lipophosphoglycan (LPG), can induce human immunodeficiency virus type 1 (HIV-1) expression in U1 and OM-10.1, two cell lines of monocytoid origin latently infected with HIV-1. Treatment of U1 cells with various concentrations of LPG (1, 5, and 10 microM) resulted in a dose-dependent secretion of tumor necrosis factor alpha (TNF-alpha). Suppression of LPG-induced HIV-1 expression by polyclonal anti-TNF-alpha antibodies further confirmed the involvement of this cytokine. Results from these studies indicate that the protozoan parasite L. donovani can induce the secretion of TNF-alpha that will function in an autocrine or paracrine manner to upregulate HIV-1 expression. Our data suggest for the first time that this protozoan parasite can be viewed as a potential cofactor in the pathogenesis of AIDS.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

ABC transporters in the protozoan parasite Leishmania

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.     

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1)

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.     

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The present work is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP transfected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites were undergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the flagellum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lysosome (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 was identified in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phase of growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite. Therefore, this finding has importance to identify new target molecule like the proteasome for therapeutic intervention.     

Regulation of the Ca2+-inhibitable adenylyl cyclase type VI by capacitative Ca2+ entry requires localization in cholesterol-rich domains

The endogenous Ca(2+)-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca(2+) entry and not by a substantial elevation of [Ca(2+)](i) from either intracellular stores or via ionophore-mediated Ca(2+) entry (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). The present studies explored the role of cholesterol-rich domains in maintaining this functional association. The cholesterol-binding agent, filipin, profoundly inhibited adenylyl cyclase activity. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca(2+) entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca(2+) entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca(2+) entry. Adenylyl cyclase catalytic activity and immunoreactivity were extracted into buoyant caveolar fractions with Triton X-100. The presence of adenylyl cyclase in such structures was eliminated by depletion of plasma membrane cholesterol. Altogether, these data lead us to conclude that adenylyl cyclase must occur in cholesterol-rich domains to be susceptible to regulation by capacitative Ca(2+) entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.     

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.     

Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.     

Ca2+-dependent formation of an L-arginine-derived activator of soluble guanylyl cyclase in bovine lung

In a fraction of cytosolic proteins from bovine lung, soluble guanylyl cyclase was concentration-dependently stimulated by L-arginine but not by D-arginine. Stimulation was up to 20-fold with an EC50 of about 3 x 10(-5) M. Activation of guanylyl cyclase by L-arginine was dependent on NADPH (EC50 about 5 x 10(-7) M) and Ca2+ (EC50 about 1.4 x 10(-6) M). The activation by L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. The effect of L-arginine was dependent on the protein concentration and was not observed in preparations of purified gyanylyl cyclase. These results suggest that bovine lung contains a Ca2+-regulated enzyme or enzyme system which converts L-arginine into an activator of soluble guanylyl cyclase.     

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani

Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis. In this study we identify the L. donovani Rab5b gene, and demonstrate the localization of a Rab5b chimera to early endosomes. A full-length Rab5b protein was fused to green fluorescent protein (GFP) to generate a chimeric protein GFP::Rab5b. Transfected L. donovani promastigotes carrying this chimeric construct displayed GFP::Rab5b localization. Additionally, incubation of transfected promastigotes with the fluid-phase marker Texas Red dextran demonstrated anterior co-localization of GFP::Rab5b and dye. This suggests Rab5b may act as a marker for early endosomes in L. donovani. Note. Nucleotide sequence data reported in this paper are available in the GenBank^TM, EMBL and DDBJ databases under the accession numbers AY357217, AL359774, AF007547, BC032740.     

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.