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A Mg-Dependent Ecto-ATPase in Leishmania amazonensis and Its Possible Role in Adenosine Acquisition and Virulence
Abstract
The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear 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 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an 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. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.
... Therefore, Leishmania parasites employ ectonucleotidases on their surface to convert nucleotide triphosphates into corresponding nucleobases [28]. The ectonucleotidases are also acting as an adhesion molecule that facilitates the attachment of parasites to the host cell [208][209][210][211][212]. Since degradation of extracellular ATP is associated with suppression pro-inflammatory response, ectonucleotidases are considered as one of the virulent factors [204]. ...
... The ectonucleotidases are also acting as an adhesion molecule that facilitates the attachment of parasites to the host cell [208][209][210][211][212]. Since degradation of extracellular ATP is associated with suppression pro-inflammatory response, ectonucleotidases are considered as one of the virulent factors [204]. In line with this hypothesis, virulent Leishmania promastigote found to express higher ecto-NTPase activity than less virulent or avirulent strains [8,208,213]. Ecto-ATPase of Leishmania has high specificity for ATP compared to other nucleotide triphosphates, this is in consistent with the abundant occurrence of ATP and its derivatives in the extracellular milieu [208,213]. ...
... In line with this hypothesis, virulent Leishmania promastigote found to express higher ecto-NTPase activity than less virulent or avirulent strains [8,208,213]. Ecto-ATPase of Leishmania has high specificity for ATP compared to other nucleotide triphosphates, this is in consistent with the abundant occurrence of ATP and its derivatives in the extracellular milieu [208,213]. The degradation of eATP by Leishmania surface ectonucleotidases is hypothesized as one the mechanism to elude host immune system to avoid apoptosis of infected macrophages and suppression of local inflammatory response [28,201,202]. ...
Nucleotides are one of the most primitive extracellular signalling molecules across all phyla and regulate a multitude of responses. The biological effects of extracellular nucleotides/sides are mediated via the specific purinergic receptors present on the cell surface. In mammalian system, adenine nucleotides are the predominant nucleotides found in the extracellular milieu and mediate a constellation of physiological functions. In the context of host-pathogen interaction, extracellular ATP is recognized as a danger signal and potentiates the release of pro-inflammatory mediators from activated immune cells, on the other hand, its breakdown product adenosine exerts potential anti-inflammatory and immunosuppressive actions. Therefore, it is increasingly apparent that the interplay between extracellular ATP/adenosine ratios has a significant role in coordinating the regulation of the immune system in health and diseases. Several pathogens express ectonucleotidases on their surface and exploit the purinergic signalling as one of the mechanisms to modulate the host immune response. Leishmania pathogens are one of the most successful intracellular pathogens which survive within host macrophages and manipulate protective Th1 response into disease promoting Th2 response. In this review, we discuss the regulation of extracellular ATP and adenosine levels, the role of ATP/adenosine counter signalling in regulating the inflammation and immune responses during infection and how Leishmania parasites exploit the purinergic signalling to manipulate host response. We also discuss the challenges and opportunities in targeting purinergic signalling and the future prospects.
... Extracellular nucleoside triand diphosphates are hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) in the purine salvage pathway and produce nucleoside monophosphates. These nucleoside monophosphates are then converted to nucleosides by ecto-5 0 -nucleotidases and enter into the cells for intracellular purine nucleotide synthesis [6,53]. Purine salvage pathway enzymes may function as target for drug development in those pathogens which depend on this pathway [26]. ...
... ADP was also recognized as a substrate, indicating that this enzyme is an authentic nucleoside triphosphate diphosphohydrolase as described in other cells [2,13,20]. Trypanosoma brucei brucei and Leishmania amazonensis are pathogens which cannot synthesize purines de novo [6,51]; hence, it has been postulated that these ecto-ATPases in protozoa parasites play a role in the salvage of purines from the host cells [6,43] L. donovani might sequentially dephosphorylate ATP to adenosine: ATP ? ADP ? ...
... ADP was also recognized as a substrate, indicating that this enzyme is an authentic nucleoside triphosphate diphosphohydrolase as described in other cells [2,13,20]. Trypanosoma brucei brucei and Leishmania amazonensis are pathogens which cannot synthesize purines de novo [6,51]; hence, it has been postulated that these ecto-ATPases in protozoa parasites play a role in the salvage of purines from the host cells [6,43] L. donovani might sequentially dephosphorylate ATP to adenosine: ATP ? ADP ? ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... Ecto-nucleoside triphosphate diphosphohydrolases are glycoproteins present in the plasma membrane with their active sites facing the external environment, which suggests that these enzymes may be involved in surface membrane interactions between parasites and their host cells. Ectonucleoside triphosphate diphosphohydrolases have been described in several protozoa parasites including Toxoplasma gondii [9][10][11][12][13][14][15], Tetrahymena thermophila [16], Leishmania sp, [17][18][19][20][21][22][23][24][25], Entamoeba histolytica [26], Acanthamoeba sp [27], Balamuthia mandrillaris [28], Trichomonas vaginalis [29][30][31], Trichomonas foetus [32], Trichomonas gallinae [33], Giardia lamblia [34], Crithidia deanei [35], Herpetomonas sp [36,37] and Trypanosoma sp [38][39][40][41][42][43][44]. These enzymes are divalent cation-dependent. ...
... These enzymes are divalent cation-dependent. In trypanosomatide parasites such as Leishmania tropica [21], Leishmania amazonensis [20], Crithidia deanei [35], Trypanosoma cruzi [40] and Trypanosoma rangeli [39], the ecto-ATPase activities are stimulated by magnesium and manganese, but not by calcium [20,21,40]. In Trypanosoma brucei the ecto-ATPase activity is stimulated by magnesium and manganese, and also by calcium and zinc [38]. ...
... These enzymes are divalent cation-dependent. In trypanosomatide parasites such as Leishmania tropica [21], Leishmania amazonensis [20], Crithidia deanei [35], Trypanosoma cruzi [40] and Trypanosoma rangeli [39], the ecto-ATPase activities are stimulated by magnesium and manganese, but not by calcium [20,21,40]. In Trypanosoma brucei the ecto-ATPase activity is stimulated by magnesium and manganese, and also by calcium and zinc [38]. ...
Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), also known as ecto-ATPases and/or ecto- apyrases, are integral membrane glycoproteins or soluble enzymes that are dependent on divalent cations. These ecto- enzymes are important ecto-nucleotidases that are characterized by the ability to hydrolyze nucleoside triphosphates and nucleoside diphosphates to the monophosphate form. The hydrolysis of nucleoside monophosphates to nucleosides such as adenosine may then be catalyzed by the action of ecto-5´nucleotidases. The present study reviews the sequential hy- drolysis of ATPADPAMPadenosine catalyzed by these ecto-enzymes from different trypanosomatids. These reactions participate in the salvage of purines in these parasites and simultaneously interfere with the establishment of in- fection and changes in the host immune response.
... In trypanosomatids, the E-NTPDase is important to the salvation of purine nucleotides [14], since these parasites are unable to perform de novo synthesis of these nucleotides [15]. E-NTPDases have been extensively characterized in L. amazonensis [16] as well as in other species of protozoa, such as Toxoplasma gondii [17] and Trypanosoma cruzi [18,19]. Data from our research group have shown the importance of NTPDase activity in establishing Leishmania infection [20e22] and driving the clinical form of the disease [23]. ...
... Promastigotes of Leishmania (Leishmania) amazonensis, PH8 strain (IFLA/BR/67/PH8), and 1IIId clone of the same strain were cultured at 25 C, in Grace's insect medium (SigmaeAldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS e LGC, Cotia, SP, Brazil), 2 mM L-glutamine (SigmaeAldrich), and 100 U/mL penicillin G potassium (SigmaeAldrich), pH 6.5. The original strain was also maintained in the presence of adenine 5 mM, for a few passages, to reduce ectonucleotidase activity of the parasite, as described previously [16]. Metacyclic promastigotes were obtained by gradient centrifugation of parasites at the late log phase of culture (day 5) over Ficoll 400 (Sigma-eAldrich), as previously described [21]. ...
... Thus, we infected J774 cells with the original strain, which was preincubated for 30 min with 100 mM DIDS (4,4 0 -diisothiocyanatostilbene 2,2 0 -disulfonic acid). As previously shown [16], we observed that DIDS reduces the ectonucleotidase activity of E-NTPDase in L. amazonensis (Fig. 3A). As expected, inhibition of activity by DIDS decreased parasite survival (Fig. 3B) and prevented the reduction of NO production by stimulated J774 cells (Fig. 3C). ...
Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease.
Copyright © 2014. Published by Elsevier Masson SAS.
... In mammals, surfaceexpressed NTPDases function in inflammation and immunity, vascular hemostasis and purine salvage [14], while in the intracellular bacterial pathogen, Legionella pneumophila, a secreted NTPDase is required for full virulence in a mouse model of disease [15,16]. In Leishmania species, enzyme activity consistent with the presence of one or more surface-located NTPDases has been observed in both L. amazonensis and L. tropica, two species responsible for cutaneous leishmaniasis [17][18][19]. A number of lines of indirect evidence suggest that this surface NTPDase activity is important for virulence in the mammalian host. ...
... A number of lines of indirect evidence suggest that this surface NTPDase activity is important for virulence in the mammalian host. Specifically, surface NTPDase activity is elevated in virulent Leishmania strains and in the intracellular amastigote form of the parasite [17][18][19]; inhibition of surface NTPDase activity with chromium (III) adenosine 59-triphosphate complex, reduced promastigote attachment and entry into mouse macrophages [20]; treatment of parasites with an antibody to the human NTPDase CD39 also reduced the interaction of Leishmania with mouse macrophages [19]; finally, polyclonal antibodies raised against synthetic peptides derived from the amino acid sequences of a putative L. braziliensis NTPDase caused significant cytotoxicity in cultured L. braziliensis promastigotes [21]. While these studies suggest roles for NTPDases in parasite nutrition, surface/ secreted NTPDases could also contribute to pathogenesis by inducing host cell purinergic receptors. ...
... It is also important to recognize that a number of studies have implicated general surface-located hydrolysis of ATP, ADP (and sometimes other NTPs and NDPs) in the virulence of both Leishmania and a number of other parasites [18,19,[58][59][60][61][62]. This observed activity has often been assumed to be due to the presence of NTPDases. ...
Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.
... The Mg-dependent ecto-ATPase activity was calculated from the total activity measured in the presence of 5 mM MgCl2 minus the basal activity measured in the absence of MgCl2. The ATPase activity was determined by measuring the hydrolysis of [γ −32 P]ATP (10 4 Bq/nmol ATP; specific activity of approximately 10 4 Bq/nmol ATP) (Berrêdo-Pinho et al., 2001). The experiments were started by the addition of living cells and terminated by the addition of 1.0 mL of ice-cold 25% charcoal in 1.0 M HCl. ...
... These studies also reported an increase in parasite load in mice and a marked increase in the production of IL-4, IL-10 and Arginase-1, which decreased the synthesis of nitric oxide by macrophages and facilitated survival of the parasite Vanaerschot et al., 2011). These data corroborate the link between the increase in ecto-ATPase activity and the increases in infectivity, virulence and adhesion in various microorganisms (Berrêdo-Pinho et al., 2001;Kiffer-Moreira et al., 2007;Meyer-Fernandes, 2002;Sansom et al., 2008;Santos et al., 2009). ...
... Both parameters were significantly different (p < 0.0001; n = 26).survival, proliferation, virulence and heat stress response in Leishmania(Berrêdo-Pinho et al., 2001;Meyer-Fernandes et al., 1997;Paletta-Silva et al., 2011;Peres-Sampaio et al., 2008). We analyzed the activity of these enzymes in resistant cells. ...
Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of Leishmania amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and severity of the disease caused by vinblastine-resistant L. amazonensis promastigotes. Taken together, these data suggest that ecto-enzymes could be potential therapeutic targets in the struggle against the spread of leishmaniasis, a neglected world-wide public health problem.
... Ecto-NTPDases (ENTPDases) are important ectonucleotidases found in parasites, including Toxoplasma gondii [13,14], Schistosoma mansoni [15], Leishmania amazonensis [16][17][18], Trypanosoma cruzi [19,20], L. braziliensis [21,22], Leishmania infantum [23], Leishmania major [24], and Plasmodium falciparum [25]. There are growing evidences pointing to the crucial roles of these ectonucleotidases in the parasite-host relationship, including processes such as adhesion and infection [17,18,20,23,26], generation of adenosine for parasite nutrition through the purine salvage pathway [16], and modulation of the host innate immune responses dependent on extracellular nucleotides [12,18,[27][28][29]. ...
... Ecto-NTPDases (ENTPDases) are important ectonucleotidases found in parasites, including Toxoplasma gondii [13,14], Schistosoma mansoni [15], Leishmania amazonensis [16][17][18], Trypanosoma cruzi [19,20], L. braziliensis [21,22], Leishmania infantum [23], Leishmania major [24], and Plasmodium falciparum [25]. There are growing evidences pointing to the crucial roles of these ectonucleotidases in the parasite-host relationship, including processes such as adhesion and infection [17,18,20,23,26], generation of adenosine for parasite nutrition through the purine salvage pathway [16], and modulation of the host innate immune responses dependent on extracellular nucleotides [12,18,[27][28][29]. ...
Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.
Graphical abstract
... Such sequential hydrolysis of extracellular ATP to adenosine was demonstrated in different parasite species, indicating that not only E-NTPDases but also ecto-5`-nucleotidases could be present in protozoa parasites, including Leishmania spp., Trypanosoma spp., Trichomonas vaginalis, Tritrichomonas foetus, Toxoplasma gondii, and Schistosoma spp. (Berredo-Pinho et al., 2001;Meyer-Fernandes, 2002;Sansom, 2012;Pimentel et al., 2016;Paes-Vieira et al., 2018). ...
... amazonensis, L. mexicana, L. tropica, L. braziliensis, L. donovani, L. infantum, L. major), two genes encoding putative NTPDases (LNTPDase 1 and LNTPDase 2) have been found in genome databases (Sansom, 2012;Paes-Vieira et al., 2018). In different Leishmania species, LNTPDase and ecto-5'-nucleotidase activities have been implicated with virulence and infectivity (Berredo-Pinho et al., 2001;Pinheiro et al., 2006;de Almeida Marques-da-Silva et al., 2008;Souza et al., 2011;Leite et al., 2012;Maia et al., 2013). ...
Belonging to the GDA1/CD39 protein superfamily, nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP) and inorganic phosphate (Pi). Several NTPDase isoforms have been described in different cells, from pathogenic organisms to animals and plants. Biochemical characterization of nucleotidases/NTPDases has revealed the existence of isoforms with different specificities regarding divalent cations (such as calcium and magnesium) and substrates. In mammals, NTPDases have been implicated in the regulation of thrombosis and inflammation. In parasites, such as Trichomonas vaginalis, Trypanosoma spp., Leishmania spp., Schistosoma spp. and Toxoplasma gondii, NTPDases were found on the surface of the cell, and important processes like growth, infectivity, and virulence seem to depend on their activity. For instance, experimental evidence has indicated that parasite NTPDases can regulate the levels of ATP and Adenosine (Ado) of the host cell, leading to the modulation of the host immune response. In this work, we provide a comprehensive review showing the involvement of the nucleotidases/NTPDases in parasites infectivity and virulence, and how inhibition of NTPDases contributes to parasite clearance and the development of new antiparasitic drugs.
... In the last 25 years, evidence has been accumulating that expression of E-NTPDase genes is required for virulence of many pathogens [93,[154][155][156][157]. Thus it appears likely that protozoan NTPDases, by altering the concentration of ATPe and other extracellular nucleotides accumulating on the cell surface of hosts may interfere with P signaling to suppress inflammatory responses and evade immune reactions [154]. ...
... In several Leishmania species ATP can be hydrolysed to adenosine at the cell surface, indicating the presence of NTPDase and 5′-ecto-nucleotidase activity [135]. Nucleotidase activity is higher in virulent strains than avirulent strains and is increased more than 10-fold in the obligate intracellular amastigote stage [155,156]. On the other hand, treatment of parasites with anti-CD39 antibody (i.e., cloned E-NTPDase 1) reduced the interaction of the parasites with mouse peritoneal macrophages [156], further suggesting a role for an NTPDase in pathogenesis. ...
In most animals, transient increases of extracellular ATP (ATPe) are used for physiological signaling or as a danger signal in pathological conditions. ATPe dynamics are controlled by ATP release from viable cells and cell lysis, ATPe degradation and interconversion by ecto-nucleotidases, and interaction of ATPe and byproducts with cell surface purinergic receptors and purine salvage mechanisms. Infection by protozoan parasites may alter at least one of the mechanisms controlling ATPe concentration. Protozoan parasites display their own set of proteins directly altering ATPe dynamics, or control the activity of host proteins. Parasite dependent activation of ATPe conduits of the host may promote infection and systemic responses that are beneficial or detrimental to the parasite. For instance, activation of organic solute permeability at the host membrane can support the elevated metabolism of the parasite. On the other hand ecto-nucleotidases of protozoan parasites, by promoting ATPe degradation and purine/pyrimidine salvage, may be involved in parasite growth, infectivity, and virulence. In this review, we will describe the complex dynamics of ATPe regulation in the context of protozoan parasite–host interactions. Particular focus will be given to features of parasite membrane proteins strongly controlling ATPe dynamics. This includes evolutionary, genetic and cellular mechanisms, as well as structural-functional relationships.
... The activity of ecto-nucleotidases has been demonstrated in Leishmania tropica, Leishmania amazonensis, and L. infantum (12,19,20). These enzymes play a role in virulence, cell adhesion, parasite release from infected cells, and in control of nucleotide concentrations inside cells and in the extracellular spaces (17,21,22). ...
... Leishmania infantum promastigotes were grown to the stationary phase and then incubated for 1 h with 250 µM suramin (Sigma Aldrich), an inhibitor of the ecto-nucleotidase activity (20). After incubation, promastigotes were washed twice and resuspended in saline solution (0.9% NaCl). ...
Ecto-nucleotidase activity is involved in the infection process of Leishmania and various other parasites that enables modulation of host immune responses to promote disease progression. One of the enzymes responsible for this activity is the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase). The enzyme hydrolyzes nucleotides tri- and/or di-phosphate into monophosphate products, which are subsequently hydrolyzed into adenosine. These nucleotides can serve as purinergic signaling molecules involved in diverse cellular processes that govern immune responses. Given the importance of the extracellular metabolism of these nucleotides during intracellular pathogen infections, this study evaluates the role of ecto-nucleotidase activity during Leishmania infantum (L. infantum) infection in human macrophages. E-NTPDase protein expression and activity was evaluated in L. infantum during purine starvation, adenosine-enriched medium, or in the presence of an inhibitor of ecto-nucleotidases. Results show that E-NTPDase is expressed in L. infantum parasites, including on the cell membrane. Furthermore, functional activity of the enzyme was modulated according to the availability of adenosine in the medium. Purine starvation increased the hydrolytic capacity of nucleotides leading to higher infectivity, while growth in adenosine-enriched medium led to lower infectivity. Moreover, inhibiting E-NTPDase function decreased L. infantum infection in macrophages, suggesting the enzyme may serve as a ligand. Taken together, the ability of L. infantum to hydrolyze nucleotides is directly associated with increased infectivity in macrophages.
... Adenosine is an immunosuppressive small molecule that has been gaining attention in leishmaniasis: studies show that increased adenosine production is related to increased lesion development and tissue parasitism (de Almeida Marques-da-Silva et al., 2008) and that a higher activity/expression of ectonucleotidases to produce adenosine correlates with a greater infection incidence and virulence of Leishmania strains (Berredo-Pinho et al., 2001;de Almeida Marques-da-Silva et al., 2008;de Souza et al., 2010;Peres et al., 2017;Vijayamahantesh et al., 2017). Therefore, adenosine can be a candidate to play a role in the autocrine modulation of macrophage response to infection and to favor Leishmania presence inside macrophages. ...
... Adenosine may be formed by the host macrophage (Deussen et al., 1989;Schutz et al., 1981;Vijayamahantesh et al., 2017) or by the Leishmania parasites (Berredo-Pinho et al., 2001;Peres et al., 2017;Vasconcellos Rde et al., 2014). Therefore, Leishmania infected macrophages have the conditions to locally produce the endogenous adenosine needed to activate an adenosine receptor-mediated autocrine immunosuppression. ...
Background:
Adenosine is a purine, with an adenine group and a ribose sugar, formed endogenously by ATP catabolism both intracellularly and extracellularly. Among the medicinal features of adenosine and of its receptors (A1, A2A, A2B and A3), anticancer activity has been an intense field of research. The anticancer potential of adenosine receptor ligands has been brought forefront of research and evidenced in innumerous research articles and patents.
Objective:
The present review focuses on the patent literature from 2002 onwards (2002-2017).
Methods:
Patents were searched and downloaded from the open access patent data bases and available online.
Results:
A significant number of patents (65) have been published on adenosine receptor ligands claiming anticancer activity, or presenting new methods of preparation or of treatment thereof from 2002-2017 (May). From these, 35 were published highlighting the promising attributes of compounds/methods to fight cancer. Most of the compounds act as adenosine A3 receptor agonists, while others act as antagonists for the other adenosine receptor subtypes. The signaling events triggered by activation of adenosine A3 receptor or by blockade of adenosine A1, A2A and A2B receptors can reverse an environment pro-cancer to an anti-cancer in the body.
Conclusion:
The promising anticancer effects mediated by adenosine receptor ligands put them in the forefront as new drug candidates. The present compilation can be worthy to medicinal chemists, pharmacologists, biochemists and other researchers focusing on the putative anticancer activity of adenosine receptor ligands.
... The ecto-nucleoside triphosphate diphosphohydrolases are suggested to participate in the purine salvage pathway through the hydrolysis of extracellular nucleoside tri and diphosphates, leading to the production of nucleoside monophosphates. The ecto-59-nucleotidases can then convert the nucleoside monophosphates to nucleosides, which can be taken into the cells to be used in intracellular purine nucleotide synthesis [5,6]. In general, enzymes from the purine salvage pathway are considered good targets for rational drug design for diseases associated with pathogens that depend on this pathway [7]. ...
... The role of E-NTPDases in infectivity, virulence or purine acquisition of the pathogenic protozoan parasites has been investigated in Trypanosoma cruzi [8], Toxoplasma gondii [9] and the species of Leishmania that causes tegumental leishmaniasis [6,[10][11][12]. These studies suggested that the E-NTPDases have key roles in parasite infections through the subversion of extracellular nucleotide signaling pathways, particularly those involving ATP and ADP. ...
Background:
Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.
Methodology/principal findings:
We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP = UDP> ADP> UTP = ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.
Conclusions/significance:
In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.
... For the ATPase assay protocol, the standard reaction mixture consisted of 20-mM HEPES-Tris, pH 7.0, 10-mM MgCl 2 , 5-mM ATP, and ML or ML + BEA. The enzymatic hydrolysis of ATP was quantified by monitoring the liberation of inorganic phosphate from [γ 32 P]-ATP (10 4 Bq/nmol ATP) as previously documented (38). The reaction was initiated by the addition of plasma vesicles to achieve a final protein concentration of 0.5 mg/mL. ...
Leishmaniasis is a neglected tropical disease infecting the world’s poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.
... For instance, the surface of T. cruzi expresses an Mg 2+ -dependent ecto-ATPase enzyme (Mg-eATPase), which have higher activity in trypomastigotes, maybe promoting the host infection [115]. Another parasite with Mg-eATPase is L. amazonensis, whose virulent promastigotes are very efficient in hydrolyzing eATP and acquiring adenosine, which is used by the parasite [116]. ...
In mammalian cells, the purinergic signaling and inflammatory mediators regulate each other. During microbial infection, nucleotides and nucleosides from both dying host cells and pathogens may be recognized by the host receptors. These receptors include purinergic receptors such P2X, P2Y, and A2A, as well Toll-like receptors, and NOD-like receptors. The interaction with most of these receptors activates immune responses, including inflammasome activation, releasing of pro-inflammatory cytokines, reactive nitrogen and oxygen species production, apoptosis induction, and regulation of T cell responses. Conversely, activation of adenosine receptors is associated with anti-inflammatory responses. The magnitude of resultant responses may contribute not only to the host defense but also to the homeostatic clearance of pathogens, or even to the severe progression of infectious diseases. In this chapter, we discuss how the purinergic signaling activation upregulates or downregulates mechanisms in infectious diseases caused by the bacterial, parasite, and viral pathogens, including SARS-CoV-2. As a concluding remark, purinergic signaling can modulate not only infectious diseases but also cancer, metabolic, and cardiovascular diseases, constituting a strategy for the development of treatments.
... The Mgdependent ecto-ATPase activity was calculated from the total activity measured in the presence of 5 mM MgCl 2 minus the basal activity measured in the absence of MgCl 2 . The ATPase activity was determined by measuring the hydrolysis of [γ-32 P] ATP (10 4 Bq/nmol ATP; specific activity of approximately 10 4 Bq/nmol ATP) [4]. The experiments were started by the addition of the cells and stopped by the addition of 1.0 mL of ice-cold 25% charcoal in 0. 1 M HCl. ...
Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.
... Little is known about parasitic factors influencing the outcome of infection, but the activities of ecto-ATPase, ecto-ADPase and 5'nucleotidase enzymes involved in the hydrolysis of extracellular nucleotides have been thought to play a role in parasite survival and proliferation (Arora and Rai, 2019;Berrêdo-Pinho et al., 2001;Carvalho et al., 2019;Pinheiro et al., 2006). Furthermore, arginase is an enzyme expressed by parasites and is required for the production of ornithine, which generates polyamines (Aoki et al., 2019a) and it has been associated with the persistence of the parasite in macrophages and neutrophils, and also with the suppression or exhaustion of T cells, impairing the development of an effective immune response (Pessenda and Silva, 2020). ...
Leishmania (Leishmania) amazonensis is an important etiological agent of American cutaneous leishmaniasis (ACL) in Brazil. The species causes a large spectrum of clinical manifestations in humans and dogs, ranging from cutaneous, cutaneous diffuse, mucocutaneous, and visceral involvement, however, the factors that drive the development of different disease forms by the same species are not yet fully known. In the present work, it was systematically reviewed the studies addressing phenotypic and genotypic characteristics of Leishmania (L.) amazonensis isolates causing cutaneous and visceral clinical frames in humans and dogs, comparing the results observed. For this, four research databases were searched for the following keywords: (Leishmania amazonensis AND visceral leishmaniasis) AND (tropism OR virulence OR visceralization OR adaptations OR mutation OR clinical presentation OR resistance OR survival OR wide spectrum). The results revealed that the complexity disease seems to involve the combination of genetic factors of the parasite (as modifications in molecules related to the virulence and metabolism) and also of the host's immune background and status. Nonetheless, the exact mechanism that leads to different clinical manifestations between strains of the same species is still uncertain and future studies must be developed to better elucidate this phenomenon.
... In Legionella pneumophila, secreted NTPDases function as virulence factors (Sansom, 2012). In parasitic protozoa, NTPDases are involved in the infectivity and virulence, as previous reported to Toxoplasma gondii (Asai and Suzuki, 1990;Bermudes et al., 1994;Asai et al., 1995;Nakkar et al., 1998), Entameba histolytica (Bakker-Grunwald andParduhn, 1993;Barros et al., 2000), Tritrichomonas fetus (De Jesus et al., 2002), Leishmania (Leishmania) amazonensis (Berredo-Pinho et al., 2001;Ennes-Vidal et al., 2011) T. cruzi (Bernardes et al., 2000;Bisaggio et al., 2003;Meyer-Fernandes et al., 2004) and others. Most of these studies performed the biochemical characterization of the ecto-nucleotidases/NTPDases activity and investigated the role of the ecto-NTPDases in the parasite-host interaction, using enzyme inhibitors and/or polyclonal antibodies. ...
Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.
... It is worth mentioning that expression of CD26, which helps ADA to anchor on the surface of cells, has been found to be impaired in visceral leishmaniasis (Rai et al., 2012). The parasite Leishmania itself is also found to express surface ectonucleotidases to convert tri-nucleotides like ATP to adenosine (Berrêdo-Pinho et al., 2001;Meyer-Fernandes et al., 1997;Vasconcellos Rde et al., 2014). It has been shown that increased ectonucleotidase activity on the surface of these parasites is directly correlated with increased infectivity and virulence levels of the parasite (de Almeida Marques-da- Silva et al., 2008;Maioli et al., 2004;Peres et al., 2017). ...
Intracellular survival of Leishmania donovani demands rapid production of host ATP for its sustenance. However, gradual decrease in intracellular ATP in spite of increased glycolysis suggested ATP efflux during infection. Accordingly, extracellular ATP showed an increase and pannexin-1 was found to be the major ATP exporter in infected condition. Extracellular ATP also showed a gradual decrease after initial increase and analysing cell surface ATP-degrading enzymes revealed induction of the ectonucleotidases, CD39 and CD73. Ectonucleotidase mediated ATP degradation led to increased extracellular adenosine (eADO) and inhibiting CD39 and CD73 in infected cells decreased adenosine concentration and parasite survival, documenting importance of adenosine in infection. Inhibiting adenosine uptake by cells did not affect parasite survival implicating eADO might exert its effect through receptor-mediated signaling. Leishmania indeed induced expression of adenosine receptors A2AR and A2BR, both being important for anti-inflammatory responses. Treating infected BALB/c mice with CD39 and CD73 inhibitors resulted in decreased parasite burden and increased host-favourable cytokine production. Collectively, these observations indicate that infection-induced ATP is exported and after converting into adenosine exerts receptor-mediated signaling for propagating infection.
... The absence of Ca+2 activation also indicate the absence of Ca+2 transport function usually associated with the Ca2+ activated, Mg+2 -dependent ATPase (Ghosh et al., 1990). Unlike mitochondria Mg+2 -ATPase (Berrêdo-Pinho et al., 2001) the ATPase of L. tropica was only partially sensitive to oligomycin at high inhibitor concentrations. The small inhibition by oligomycin may be due to contamination of membrane fraction with mitochondria and so it is possible to suggest that the origin of the major portion of ATPase activity in this was not mitochondrial. ...
A B S T RACT
The plasma membrane of cells contains enzymes whose active sites face the
external medium rather than the cytoplasm. The adenosine tri phosphatase
(ATP phosphohydrolase, EC 3.6.1.3.; ATPase) is membrane – bound enzyme
which transport protons across the plasma membrane using ATP as an enegy
source. In this work, we extracted the adenosine tri phosphatase from
promastigotes of Leishmania tropica by chloroform treatment and purified by
means of ammonium sulphate fractionation, gel filtration on sephadex G-200
and DEAE-Cellulose chromatography. Kinetic experiments demonstrated a
biphasic linear lineweaver - burk relationship (km= 0.25 and 1.1 mM) thus
revealing the existence of two substrate binding enzyme site and has an
apparent molecular weight of 365000 dalton by gel filtration. The result of
this study firmly provided the first direct evidence for the existence of Mg+2
-
dependent ATPase in L. tropica, a fact which is of great interest from the
phylogenetic point of view.
... amino acid, nucleobase/nucleoside, sucrose, and myo-inositol) transport, and energy generation [8,15,18,19,[90][91][92][93]. Recently, a Mg 2+ -dependent adenosine-translocating ecto-ATPase and two organellar Ca 2+ -transporting ATPases (Figure 1) have been identified as virulence determinants [35,94,95]. Similarly, two potential Mg 2+ transporters of the two-transmembrane domain (2-TM-GxN) type family of transporters, designated MGT1 and MGT2 (Figure 1), have been shown to be essential for parasite development and pathogenesis in mice [96]. ...
Leishmania are parasitic protozoa which infect humans and cause severe morbidity and mortality. Leishmania parasitise as extracellular promastigotes in the insect vector and as intracellular amastigotes in the mammalian host. Cycling between hosts involves implementation of stringent and co-ordinated responses to shifting environmental conditions. One of the key dynamic aspects of Leishmania biology is substrate acquisition and metabolism. Genomic analyses have revealed that Leishmania encode many putative membrane transporters, many of which are differentially expressed during the parasite life cycle. Only a small fraction of these transporters, however, have been functionally characterised. Currently, most information is available about nutrient transporters, mainly involved in carbohydrate, amino acid, nucleobase and nucleoside, cofactor, and ion acquisition. Several have apparent roles in Leishmania virulence and will be discussed in this perspective.
... 5,6 E-NTPDases play an important role in the control of biological functions, such as thromboregulation, 7,8 maintenance of vascular tone, 9,10 neurogenesis, 11 neurotransmission, 12 and regulation of immune cell function, of many cell types, including dendritic cells, lymphocytes, neutrophils, monocytes, and macrophages. [13][14][15] E-NTPDases are widely distributed in nature and have been studied in bacteria, 16 protozoa, 17 fish, 18,19 and many mammal tissues and cells. [20][21][22] Recently, studies have shown the activity of these enzymes in macrophages obtained from the peritoneal cavity. ...
This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+. A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.
... This adequately models the finding of Maioli et al. (2004) that no IL-4 producing cells could be detected during L. braziliensis or L. amazonensis C57BL/6 infection. An increase in Ado concentration to inhibit the activation of Th-cells was used to simulate L. amazonensis infection, in agreement with literature reports of incurable lesions in C57BL/6 mice infected with L. amazonensis, and with higher ecto-nucleotidase activity in the surface of this parasite (Berredo-Pinho et al., 2001;Ji et al., 2003;Maioli et al., 2004;Marques-da-Silva et al., 2008;de Souza et al., 2010;Gomes et al., 2015). This higher activity may lead to higher Ado and lower ATP concentration at the site of infection. ...
Infection by Leishmania protozoan parasites can cause a variety of disease outcomes in humans and other mammals, from single self-healing cutaneous lesions to a visceral dissemination of the parasite. The correlation between chronic lesions and ecto-nucleotidase enzymes activity on the surface of the parasite is addressed here using damage caused in epithelial cells by nitric oxide. In order to explore the role of purinergic metabolism in lesion formation and the outcome of the infection, we implemented a cellular automata/lattice gas model involving major immune characters (Th1 and Th2 cells, IFN-γ, IL-4, IL-12, adenosine−Ado−, NO) and parasite players for the dynamic analysis of the disease progress. The model were analyzed using partial ranking correlation coefficient (PRCC) to indicate the components that most influence the disease progression. Results show that low Ado inhibition rate over Th-cells is shared by L. major and L. braziliensis, while in L. amazonensis infection the Ado inhibition rate over Th-cells reaches 30%. IL-4 inhibition rate over Th-cell priming to Th1 independent of IL-12 are exclusive of L. major. The lesion size and progression showed agreement with published biological data and the model was able to simulate cutaneous leishmaniasis outcomes. The sensitivity analysis suggested that Ado inhibition rate over Th-cells followed by Leishmania survival probability were the most important characteristics of the process, with PRCC of 0.89 and 0.77 respectively. The simulations also showed a non-linear relationship between Ado inhibition rate over Th-cells and lesion size measured as number of dead epithelial cells. In conclusion, this model can be a useful tool for the quantitative understanding of the immune response in leishmaniasis.
... The absence of Ca+2 activation also indicate the absence of Ca+2 transport function usually associated with the Ca2+ activated, Mg+2 -dependent ATPase (Ghosh et al., 1990). Unlike mitochondria Mg+2 -ATPase (Berrêdo-Pinho et al., 2001) the ATPase of L. tropica was only partially sensitive to oligomycin at high inhibitor concentrations. The small inhibition by oligomycin may be due to contamination of membrane fraction with mitochondria and so it is possible to suggest that the origin of the major portion of ATPase activity in this was not mitochondrial. ...
... The main known roles of NTPDases in pathogens/host relationships are their actions as virulence molecules by modulating the immune response-dependent purinergic signaling and the action as pro-adhesion molecules that can facilitate the infection [18,26,28,29,31,40,44e48]. Another potential role of these enzymes is the participation in the purine salvage pathway, mainly in cells such as trypanosomatids that do not have the de novo pathway [23,28]. These important functions identify these enzymes as potential targets for biotechnological applications, such as target drug design, vaccines and antigens for diagnosis. ...
... A comparison of the Mg 2þ -ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes are less efficient at hydrolyzing eATP than the infective trypomastigote and amastigote stages [73]. Another parasite with Mg 2þ -dependent ecto-ATPase is L. amazonensis, where the avirulent promastigotes are less efficient than the virulent promastigotes in hydrolyzing eATP, suggesting that virulent strains acquire adenosine and utilize it in its favor [74]. ...
Despite recent advances unraveling mechanisms of host-pathogen interactions in innate immunity, the participation of purinergic signaling in infection-driven inflammation remains an emerging research field with many unanswered questions. As one of the most-studied oral pathogens, Porphyromonas gingivalis is considered as a keystone pathogen with a central role in development of periodontal disease. This pathogen needs to evade immune-mediated defense mechanisms and tolerate inflammation in order to survive in the host. In this review, we summarize evidence showing that purinergic signaling modulates P. gingivalis survival and cellular immune responses, and discuss the role played by inflammasome activation and cell death during P. gingivalis infection.
... and the ecto-5 0 -nucleotidase (EC 3.1.3.5) have been shown to exert important roles in the infection by Leishmania. E-NTPDase has been reported on Leishmania tropica [81] and L. amazonensis [82,83]. In addition to E-NTPDase, we also demonstrated that L. amazonensis presents ecto-5 0 -nucleotidase activity [84]. ...
Infection by protozoan parasites is part of the most common Tropical Neglected Diseases. In the case of leishmaniasis, several millions of people are at risk of contracting the disease. In spite of innumerous studies that elucidated the immune response capable of killing the parasite, the understanding of the evasion mechanisms utilized by the parasite to survive within the very cell responsible for its destruction is still incomplete. In this review, we offer a new approach to the control of the immune response against the parasite. The ability of the parasite to modulate the levels of extracellular ATP and adenosine either by directly acting on the levels of these molecules or by inducing the expression of CD39 and CD73 on the infected cell may influence the magnitude of the immune response against the parasite contributing to its growth and survival.
... This can cause the prevention of the activation of macrophages and leads to the increase of infection by Leishmania species [105][106][107][108][109]. Moreover, the virulence of L. amazonensis promastigotes could be due to its high ecto-nucleotidase activity [110] (Fig. 1d). Moreover, ecto-5-nucleotidase activity also has been seen in L. chagasi [105]. ...
Leishmaniasis is a neglected tropical disease affecting millions of people around the world caused by organisms of the genus Leishmania. Parasite escape mechanisms of the immune system confer the possibility of resistance and dissemination of the disease. A group of molecules that has become a target for Leishmania survival strategies are lipid mediators. Among them, leukotriene B4 (LTB4) has been described as a pro-inflammatory molecule capable of activating cells of the immune system to combat Leishmania. In an opposite way, prostaglandin E2 (PGE2) is a lipid mediator described as a deactivator of macrophages and neutrophils. The balance of these two molecules can be generated by extracellular nucleotides, such as adenosine 5'-triphosphate (ATP) and adenosine (Ado), which activate the purinergic receptors system. Herein, we discuss the role of extracellular nucleotides and the resulting balance of LTB4 and PGE2 in Leishmania fate, survival or death.
... NETosis is an innate response that may contribute to diminishing the parasitic burden at the Leishmania inoculation site (Guimaraes-Costa et al., 2009). Several microorganisms have nuclease activities that are able to degrade DNA filaments, allowing them to escape of the toxic effects of NETs (Sumby et al., 2005;Berends et al., 2010;Seper et al., 2013;Thammavongsa et al., 2013). Recently, it was observed that Leishmania infantum could escape NET-mediated killing through its 3 0 NT/NU activity (Guimaraes-Costa et al., 2014). ...
The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3′-nucleotidase/nuclease (3′NT/NU), which is able to hydrolyze extracellular 3′-monophosphorylated nucleosides and nucleic acids. 3′NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3′NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3′-nucleotidase (3′NT) activity of L. amazonensis by several nucleotides. It was observed that 3′NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5′-GMP on the 3′NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5′-GMP is able to reverse the stimulation promoted by 3′-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3′NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.
... Tais enzimas parecem ter uma função de bomba de extrusão de H + , além de ter um possível papel na acumulação de glicose e potássio no meio intracelular (Dutta et al., 1990;Mukherjee, Mandal & Bhaduri, 2001). Alterações na atividade dessas proteínas podem estar relacionadas com a virulência do parasito, participando da pré-adaptação dos promastigotas para sobrevivência dentro dos macrófagos (Berredo-Pinho et al., 2001;Lu et al., 1997). A gp63 é uma das principais glicoproteínas encontradas na superfície de promastigotas de Leishmania spp., com massa molecular aproximada de 63 kDa. ...
... Tais enzimas parecem ter uma função de bomba de extrusão de H + , além de ter um possível papel na acumulação de glicose e potássio no meio intracelular (Dutta et al., 1990;Mukherjee, Mandal & Bhaduri, 2001). Alterações na atividade dessas proteínas podem estar relacionadas com a virulência do parasito, participando da pré-adaptação dos promastigotas para sobrevivência dentro dos macrófagos (Berredo-Pinho et al., 2001;Lu et al., 1997). ...
No Brasil, ela já foi responsável por epidemias em diferentes cidades. Nos últimos dez anos, sua letalidade aumentou em diversas regiões do país. Em várias partes do mundo ela vem se expandindo, inclusive em lugares onde, anteriormente, não havia registro de transmissão. A infecção por parasitos do gênero Leishmania causa uma das doenças tropicais mais negligenciadas da atualidade. Estima-se que existam 350 milhões de pessoas em risco de contrair a infecção, sobretudo nas áreas mais pobres do planeta, e que dois milhões de novos casos de leishmanioses ocorram a cada ano. Esse grave cenário justifica o esforço empreendido pelos organizadores e demais pesquisadores do Instituto Oswaldo Cruz (IOC/Fiocruz) especialistas no assunto, assim como por profissionais de outras unidades da Fiocruz e instituições brasileiras: eles prepararam uma coletânea que compila o conhecimento já existente sobre o assunto, identifica os principais desafios e discute estratégias para enfrentá-los. ISBN: 978-85-7541-439-2. 2014. il., tab. Sumário: Parte I-Questões evolutivas, taxonômicas e ultraestruturais de parasitos do gênero LeishmaniaParte II-Bioquímica e metabolismo energético dos parasitos do gênero Leishmania Parte III-Aspectos bioquímicos e moleculares na interação parasito-hospedeiro-vetor Parte IV-Hospedeiros, reservatórios e manutenção dos ciclos de transmissão Parte V-Modelos experimentais no estudo das leishmanioses do continente americano Parte VI-Espectro clínico das leishmanioses Parte VII-Resposta imune na leishmaniose tegumentar americana Parte VIII-Tratamento e diagnóstico das leishmanioses Parte IX-Desafios para o controle das leishmanioses Sobre os organizadores: Fátima Conceição-Silva: Médica e doutora em ciências, com pós-doutorado na Universidade de Lausanne (Suíça). É pesquisadora titular do Instituto Oswaldo Cruz (IOC/Fiocruz) e professora da Escola de Medicina da Fundação Técnico-Educacional Souza Marques. Tem experiência na área de imunologia e parasitologia/micologia médica, com ênfase em leishmaniose tegumentar americana, esporotricose e outras doenças granulomatosas do tegumento cutâneo-mucoso, incluindo imunidade da pele. Carlos Roberto Alves: Biólogo e doutor em ciências. Atualmente é pesquisador titular do Instituto Oswaldo Cruz (IOC/Fiocruz). Tem experiência na área de parasitologia, com ênfase em bioquímica de protozoários parasitos. Desenvolve pesquisas com proteínas de Leishmania spp e seus mecanismos de ação na fisiologia destes protozoários e na interação com seus hospedeiros.
... In contrast, the exact function of NTPDases from microbial pathogens is less clear (7). In recent years, evidence has been accumulating that the expression of NTPDase genes is required for virulence of many pathogens (7)(8)(9)(10)(11). As host ATP exerts a proinflammatory action via P2 receptors and acts as a danger signal, it appears likely that microbial NTPDases interfere with this host response to suppress inflammatory responses and evade immune reactions (7). ...
Background: Disulfide-regulated NTPDases from T. gondii are related to the virulence of the parasite. Results: Crystal structures of the active and inactive state were determined. Conclusion: The 258 –268 disulfide bridge acts like a clamp, which upon reduction allows concerted motions of the subunits and domains of the tetrameric enzyme. Significance: First molecular model of the disulfide-regulated activation mode.
... Apyrase also exists widely in salivary glands of hematophagous arthropods, such as mosquitoes, ticks, sand flies (Lewis-Carl and Kirley 1997), fleas, triatome bugs, tsetse flies (De Jesus et al. 2002), black flies (Charlab et al. 1999) and cimex (Champagne et al. 1995). In contrast to the unknown function of apyrase in vertebrate animals, it has already been made clear that in bloody-sucking arthropods, apyrase can hydrolyze damaged cells in the bite suction area or ADP is released by the activated blood platelets, inhibiting the platelet aggregation and the formation of thrombus, facilitating blood-sucking process, and in the other hand, it can modulate host immune response in some parasitic helminthes (Berredo-Pinho et al. 2001). There are two types of apyrase: Ca 2+ -Mg 2+ ion-activated type and Ca 2+ type. ...
Apyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum
... are mainly eukaryotic enzymes widely described in mammals that catalyze the hydrolysis of several nucleosides tri and diphosphate producing their respective di and monophosphorylated counterparts (Knowles, 2011;Plesner, 1995). A series of evidences have suggested that these enzymes may be involved in eukaryotic events such as: virulence (Berredo-Pinho et al., 2001), purinergic signaling (Deaglio and Robson, 2011;Knowles, 2011), inflammation (Kannan, 2002), hemostasis (Bernardes et al., 2000;Jin et al., 2005;Marcus et al., 2005), purine salvage (de Souza Leite et al., 2007;Lorenz et al., 2007) and host-pathogen interactions (Bisaggio et al., 2003;Matin and Khan, 2008). ...
Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98%identity to the T. brucei homologous NTPDase gene.
Copyright © 2015. Published by Elsevier Inc.
... Apropos of the extracellular ATP metabolism, the formation of ADP and adenosine is regulated by three enzyme families: E-NTPDases (ectonucleoside triphosphate diphosphohydrolase), E-NPPs (ectonucleotide pyrophosphatase/phosphodiesterase), and ecto-5 -nucleotidases (Berrêdo-Pinho et al., 2001). The E-NTPDase family is a group of glycosylated membrane-bound enzymes that hydrolyze nucleoside tri-and di-phosphates. ...
... There are lines of evidence that NTPDases are related to virulence and infectivity in protozoan parasites [19][20][21][22][23]. However, most of the studies reported ecto-NTPDase enzymatic activities in intact parasites or plasma membrane fractions. ...
Background
Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri- and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock.FindingsComparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2¿±¿1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5¿±¿5.6 and 16.3¿±¿3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression.Conclusions
Our results suggest that TcNTPDase-1 plays an important role on T. cruzi infectivity and adaptation to stress conditions, such as long-term cultivation and heat shock.
... An ability to cleave ATP and/or ADP in the extracellular environment has been described in several pathogens including bacteria, protozoans, and nematodes [34][35][36][37][38][39][40]. This conserved feature of several pathogens is also found in schistosomes, here mediated by SmATPDase1. ...
Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm's tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes-SmATPDase1-actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri- and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms' ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.
... Parasite membrane ecto-ATPases are an important virulence factor in L. amazonensis (18). These ecto-ATPases control the balance of triphosphated nucleotides and their metabolites in the extracellular environment, and consequently control the signaling of receptors activated by these nucleotides. ...
Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7−/− mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning. © 2014 International Society for Advancement of Cytometry
... An ability to cleave ATP and/or ADP in the extracellular environment has been described in several pathogens. For example ecto-ATPase activity has been described in the protozoan parasites Toxoplasma gondii (Bermudes et al., 1994), Leishmania amazonensis (Berredo-Pinho et al., 2001), Trichomonas vaginalis (de Jesus et al., 2002) and Cryptosporidum parvum (Manque et al., 2012) and in several bacterial pathogens including Mycobacterium bovis (Zaborina et al., 1999), Vibrio cholera (Punj et al., 2000), Staphylococcus aureus (Thammavongsa et al., 2009), and Legionella pneumophilia (Vivian et al., 2010). Similarly, blood-feeding ectoparasites are known to release a repertoire of nucleotide-metabolizing enzymes in their saliva (Andersen et al., 2007;de Araujo et al., 2012). ...
Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms' ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.
... Parasites were then resuspended at 2 x 10 7 parasites/ml in RPMI minimum media pH 5.5 and incubated for 6 hours at 35°C under 5% CO 2 atmosphere. The viability of the parasites after 6 hours of incubation was assessed by the Trypan blue exclusion test of cell viability [Berredo-Pinho, 2001] and found to be over 97%. Following this incubation, the supernatant containing secreted/excreted proteins (LMES) was collected by centrifugation at 4000 x g for 20 min at 4°C then lyophilized using a speed-vaccum concentrator (Savant, Holbrook, NY). ...
The present invention relates to new proteins of Leishmania major and to therapeutical and diagnostic applications thereof. More particularly, the present invention relates to excreted/secreted polypeptides and polynucleotides encoding same, compositions comprising the same, and methods of diagnosis, vaccination and treatment of Leishmaniasis.
Hypochlorous acid (HClO) and its ionic form (ClO⁻), as important components of reactive oxygen species, were produced mainly in mitochondria and participated in various biological processes, and they abnormal level could cause various diseases and threaten human health. Hence, a “turn‐on” phosphorescent probe Ir‐DAMN based on Ir (III) complex was synthesized to monitor HClO/ClO⁻ in DMF/PBS solution and living cells. Due to isomerization of C=N bond, probe Ir‐DAMN exhibited very weak phosphorescence initially, which displayed obvious phosphorescent enhancement at 620 nm after upon addition with ClO⁻. Confocal bioimaging experiments implied that probe Ir‐DAMN possessed good cell membrane permeability, specific localization of mitochondria and could achieve detection of exogenous and endogenous HClO/ClO⁻ in the living cells.
Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).
Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.
Metastasis is a major cause of death in patients with breast cancer. A growing body of evidence has demonstrated the antitumour effects of resveratrol, a non‐flavonoid polyphenol. Resveratrol inhibits metastatic processes such as the migration and invasion of cancer cells. In several cancer types, the importance of inorganic phosphate (Pi) for tumour progression has been demonstrated. The metastatic process in breast cancer is associated with Na⁺‐dependent Pi transporters. In this study, we demonstrate, for the first time, that resveratrol inhibits the Na⁺‐dependent Pi transporter. Results from kinetic analysis shows that resveratrol inhibits Na⁺‐dependent Pi transport non‐competitively. Resveratrol also inhibits adhesion/migration in MDA‐MB‐231 cells, likely related to inhibition of the Na⁺‐dependent Pi transporter.
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Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. . We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The K m of SmATPDase1 for ATP is 0.4 ±0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.)
ENTPDases are enzymes known for hydrolyzing extracellular nucleotides and playing an essential role in controlling the nucleotide signaling via nucleotide/purinergic receptors P2. Moreover, ENTPDases, together with Ecto-5´-nucleotidase activity, affect the adenosine signaling via P1 receptors. These signals control many biological processes, including the immune system. In this context, ATP is considered as a trigger to inflammatory signaling, while adenosine (Ado) induces anti-inflammatory response. The trypanosomatids Leishmania and Trypanosoma cruzi, pathogenic agents of Leishmaniasis and Chagas Disease, respectively, have their own ENTPDases named "TpENTPDases," which can affect the nucleotide signaling, adhesion and infection, in order to favor the parasite. Besides, TpENTPDases are essential for the parasite nutrition, since the Purine De Novo synthesis pathway is absent in them, which makes these pathogens dependent on the intake of purines and nucleopurines for the Salvage Pathway, in which TpENTPDases also take place. Here, we review information regarding TpNTPDases, including their known biological roles and their effect on the purinergic signaling. We also highlight the roles of these enzymes in parasite infection and their biotechnological applications, while pointing to future developments.
Leishmania infected macrophages have conditions to produce adenosine. Despite its known immunosuppressive effects, no studies have yet established whether adenosine alter Leishmania parasitic burden upon macrophage infection. This work aimed at investigating whether endogenous adenosine exerts an autocrine modulation of macrophage response towards Leishmania infection, identifying its origin and potential pharmacological targets for visceral leishmaniasis (VL), using THP-1 differentiated macrophages. Adenosine deaminase treatment of infected THP-1 cells reduced the parasitic burden (29.1 ± 2.2%, P < 0.05). Adenosine A2A and A2B receptor subtypes expression was confirmed by RT-qPCR and by immunocytochemistry and their blockade with selective adenosine A2A and A2B antagonists reduced the parasitic burden [14.5 ± 3.1% (P < 0.05) and 12.3 ± 3.1% (P < 0.05), respectively; and 24.9 ± 2.8% (P < 0.05), by the combination of the two antagonists)], suggesting that adenosine A2 receptors are tonically activated in infected THP-1 differentiated macrophages. The tonic activation of adenosine A2 receptors was dependent on the release of intracellular adenosine through equilibrative nucleoside transporters (ENT1/ENT2): NBTI or dipyridamole reduced (∼25%) whereas, when ENTs were blocked, adenosine A2 receptor antagonists failed to reduce and A2 agonists increase parasitic burden. Effects of adenosine A2 receptors antagonists and ENT1/2 inhibitor were prevented by L-NAME, indicating that nitric oxide production inhibition prevents adenosine from increasing parasitic burden. Results suggest that intracellular adenosine, released through ENTs, elicits an autocrine increase in parasitic burden in THP-1 macrophages, through adenosine A2 receptors activation. These observations open the possibility to use well-established ENT inhibitors or adenosine A2 receptor antagonists as new therapeutic approaches in VL.
ATP-diphosphohydrolases (EC 3.6.1.5), also known as ATPDases, NTPases, NTPDases, E-ATPases or apyrases, are enzymes that hydrolyze a variety of nucleoside tri- and diphosphates to their respective nucleosides, being their activities dependent on the presence of divalent cations, such as calcium and magnesium. Recently, ATP-diphosphohydrolases were identified on the surface of several parasites, such as Trypanosoma sp, Leishmania sp and Schistosoma sp. In parasites, the activity of ATP-diphosphohydrolases has been associated with the purine recuperation and/or as a protective mechanism against the host organism under conditions that involve ATP or ADP, such as immune responses and platelet activation. These proteins have been suggested as possible targets for the development of new antiparasitic drugs. In this review, we will comprehensively address the main aspects of the location and function of ATP-diphosphohydrolase in parasites. Also, we performed a detailed research in scientific database of recent developments in new natural and synthetic inhibitors of the ATP-diphosphohydrolases in parasites.
Nucleoside Triphosphate Diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.
Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5'-α,β-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4- and IL-10-producing CD4(+) T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle-brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.
A suspension of intact guinea pig polymorphonuclear leukocytes hydrolyzed added ATP, AMP, and p-nitrophenyl phosphate under physiologically appropriate conditions. These enzymatic activities were not due to artifacts such as breakage of the cells during the incubation period. It thus seemed possible that the hydrolyses were being catalyzed by ecto-enzymes, i.e. enzymes on the plasma membrane with their active sites facing the external medium. Three types of experiment were designed to test this hypothesis.
First, the activities of intact cells were compared to those of homogenates, sonicates, and cells treated with detergent. Disruption of cells resulted in an approximately 2-fold increase in maximal ATPase and p-nitrophenyl phosphatase activities, suggesting that the plasma membrane was acting as a permeability barrier to the substrates involved. Disruption did not increase AMPase activity, leaving open the possibility that an ecto-enzyme is the only protein in polymorphonuclear leukocytes capable of hydrolyzing AMP.
Second, the products of ATPase, AMPase, and p-nitrophenyl phosphatase activities of intact cells were localized by using radioactively labeled substrates. The concentration of inorganic phosphate produced by these reactions was 18 to 100 times greater in the extracellular medium than in the intracellular milieu. This suggests that the substrates are cleaved outside the cells, or that they are cleaved inside and the products are transported out. The latter possibility was militated against by the following experiment. Cells were loaded with inorganic [³³P]phosphate, then allowed to hydrolyze substrates labeled with ³²P. The distributions of the two isotopes were compared. Almost all of the inorganic [³²P]phosphate was found outside of the cells, while 90% of the inorganic [³³P]phosphate remained inside.
Third, the cells were treated with the diazonium salt of sulfanilic acid, a reagent known not to penetrate into intact erythrocytes. This treatment rapidly and dramatically inhibited the intact-cell ATPase, AMPase, and p-nitrophenyl phosphatase, while lactate dehydrogenase, a soluble cytoplasmic enzyme, was unaffected. Control experiments demonstrated that in sonicates lactate dehydrogenase was as susceptible to inhibition by the diazonium salt as were the other three activities.
Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.
A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.
The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 ± 0.5 nmol Pi x mg-1 x min-1. Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mM mannitol, the (Na++K+)ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na++K+)ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na++K+)ATPase activity in hyperosmotic medium. Furthermore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca concentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10-6 M Ca. No effect was observed in hyperosmotic medium. The inhibitory effect of Ca2+ on the (Na++K+)ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na++K+)ATPase activity of the Malpighian tubule is regulated by both increasing Ca2+ concentration and by the osmolality of the medium by different and integrative ways.
Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.
Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.
The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.
We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II.
The two isozymes had similar molecular masses of approximately 240–270 kDa by gel filtration and contained four identical subunits of molecular mass 66–67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP).
The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.
Rat renal mitochondrial glutaminase (GA) is synthesized as a 74-kDa cytosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-kDa subunits, respectively. The 66-kDa subunit is derived by removal of a 72-amino-acid presequence. The structural determinants necessary for translocation and proteolytic processing were further delineated by characterizing the processing of different chimeric constructs formed by fusing various segments of the N-terminal sequence of the GA precursor to chloramphenicol acetyl transferase (CAT). GA1-118 CAT is translocated and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an intermediate peptide and two mature subunits that are analogous to the products of processing of the GA precursor. The two reactions also occur with kinetics which are similar to those observed for processing of the GA precursor. Thus, all of the information required for the translocation and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GA1-72 CAT, a construct that contains the GA presequence fused to CAT, is apparently translocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addition, GA1-31 CAT associates with mitochondria but is not proteolytically processed and GA1-31,73-118 CAT is slowly translocated and processed to a single peptide that is analogous to the 66 kDa form of GA. The latter results suggest that the MPP cleavage reactions which yield the GA intermediate and the 66-kDa subunit depend primarily on information that is present C-terminal to the respective sites of cleavage.
The obligate intracellular parasite Toxoplasma gondii produces a nucleoside triphosphate hydrolase (NTPase) (nucleoside-triphosphatase, EC 3.6.1.15) activable by dithiol-containing compounds. We have isolated the genomic DNA for the NTPase from the RH strain of Toxoplasma and determined the nucleotide sequence of three tandemly arranged open reading frames termed NTP1, NTP2, and NTP3. We have also isolated and sequenced cDNAs for NTP1 and NTP3; no cDNA for NTP2 was obtained. The two cDNA clones encode proteins that are more than 97% identical at the amino acid level but significantly differ within two small domains, indicating the presence of NTPase isoforms. Both possess N-terminal signal sequences and two regions with partial homology to certain known ATP binding motifs: the glycine-rich loop common to many ATP binding proteins and the beta-phosphate binding domain found in the hexokinase-actin-hsp70 family. Antiserum against a NTP1-fusion protein immunoprecipitated NTPase activity from extracellular parasites that was increased in activity by treatment with dithiothreitol, confirming the identity of the cloned genes. By immunofluorescence, the NTPase is located in vesicular structures within the parasite, and in infected cells it is secreted into the vacuolar space and becomes partially associated with the parasitophorous vacuolar membrane. Since the vacuolar membrane is freely permeable to small molecules of < 1300 Da, host cell ATP may serve as a substrate for the NTPase and supply the energy for parasite-directed processes in the vacuolar space.
Trehalose, the disaccharide of glucose, inhibits both initial rate and maximal capacity of ATP-dependent Ca2+ transport in inside-out vesicles of basolateral membrane from kidney proximal tubules. This inhibition (I0.5 = 60 mM) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by trehalose. In the presence of 600 mM trehalose, Ca2+ uptake was almost completely inhibited, but the Ca(2+)-stimulated ATPase activity was unaffected; thus trehalose uncouples the Ca2+ transport from the ATPase activity. The Ca2+ transport inhibition by trehalose is reversible, since the inhibition disappeared when the vesicles were pre-incubated with 600 mM trehalose and then diluted in reaction medium to measure Ca2+ accumulation. Other mono- and disaccharides such as glucose, fructose, galactose, sucrose, maltose and lactose were tested but were not so effective as trehalose. The uncoupling of Ca2+ transport from hydrolysis can be explained by an interaction of trehalose with the phospholipid environment of the enzyme that induces conformational changes in specific domains of the enzyme so as to impair the coupling process.
Cell-CAM105 proteins (also called C-CAM) are epithelial cell adhesion molecules of the immunoglobulin (Ig) superfamily. The sequences of C-CAM are highly homologous to those of human carcinoembryonic antigen (CEA)-family proteins. In previous studies using baculoviral vectors, we showed that expression of the L-form cell-CAM105 (also called C-CAM1) in insect cells resulted in cell aggregation (Cheung, P. H., Thompson, N. L., Earley, K., Culic, O., Hixson, D., and Lin, S. H. (1993) J. Biol. Chem. 268, 6139-6146). This result indicates that the insect-cell system is suitable for studying the adhesion function of C-CAM. Since C-CAM1 contains four extracellular Ig-domains, the structural features directly responsible for C-CAM1 adhesion function were investigated by site-directed deletion and expression in the baculovirus/insect cell system. Results from these studies indicated that the first Ig domain located in the NH2-terminal of C-CAM plays a crucial role in intercellular adhesion. Site-directed deletion producing mutants lacking the second, third, or fourth Ig domains had no effect on the adhesion function. In addition, adhesion function was retained when both the third and fourth Ig domains were deleted, although the adhesion activity was reduced to half that in control cells. However, simultaneous deletion of the second, third, and fourth domains abolished adhesion, suggesting that these domains affect the accessibility of the binding site localized in the first domain. In our previous studies, we showed that the cytoplasmic domains of C-CAM play a significant role in the isoforms' adhesion activity since expression of a C-CAM isoform containing only 6 instead of 71 amino acids intracellularly failed to show the adhesion phenotype (Cheung, P. H., Culic, O., Qiu, Y., Earley, K., Thompson, N., Hixson, D. C., and Lin, S.-H. (1993) Biochem. J. 295, in press). These results together suggest that both the cytoplasmic domain and the first N-terminal Ig-like domain are required for C-CAM-mediated cell adhesion activity.
La leishmaniose cutanee, pathologie transmise par les phlebotomes, est une des trois entites connues parmi les manifestations cliniques causees par les protozoaires du genre leishmania. S'y ajoute la leishmaniose cutaneomuqueuse et la leishmaniose viscerale, et une variante de la leishmaniose cutanee: la forme cutanee diffuse. La periode d'incubation longue, la generalisation des voyages transcontinentaux, et la decouverte de foyers aux USA vont amener les dermatologues a diagnostiquer de plus en plus frequemment cette pathologie. Le probleme du traitement se pose alors, la forme cutanee diffuse etant totalement refractaires aux traitements, et les traitements les plus efficaces pour les autres formes n'ayant pas recu d'autorisation de mise sur le marche aux USA
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote,Tetrahymena,at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme inTetrahymena.It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+or Mg2+dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain,N-ethylmaleimide andp-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ectoATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein inTetrahymenaplasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase ofTetrahymenamay play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker
of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca,Mg)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression
and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the
observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide
triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPJ to ADP and inorganic phosphate, is expressed upon cell activation, Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label, 5′-p-(fluorosulfonyl)benzoyl adenosine (5′-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide ant agonists results in the inhibition of antigen induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung.
The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.
Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non–covalent interactions.
Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent dephosphorylation of ATP. Ca2+/Mg2+-dependent ecto-ATPase was active over a limited range of 0.01–1.0 mM ATP, while Ca2+/Mg2+-independent ATPase activity was active over a range of 0.1–30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0–9.0), but increased in a relatively linear manner with: (1) time of reaction (0–90 min), (2) number of cells (1–20 oocytes), and (3) temperature (10–37C). Ecto-ATPase activity was unaffected by ouabain (100 M), sodium azide (100 M), and oligomycin (5 g/ml) (as inhibitors of endo-ATPases) and -glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP,-methylene ATP, ,-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg2+-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPe) to ADP and inorganic phosphate, is expressed upon cell activation. Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide antagonists results in the inhibition of antigen-induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.
Nutritional requirements of promastigotes of Leishmania donovani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. "Continuous growth," considered as propagation through 10 successive passages, was supported by inorganic salts, 14 l-amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine), glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the above species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculata, for which glucose, proline and glutamine were found to be nonessential. It is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher levels of folic acid than L. tarentolae, probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as "minimal essential" media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthetic capabilities of Trypanosomatids.
Although there are several reports of Leishmania parasitizing cells other than macrophages or even existing extracellularly in mammalian tissue, it is generally acknowledged that in the vertebrate host these organisms normally reside within cells of the macrophage lineage. The first description of this particular host cell–parasite interaction can probably be attributed to Cunningham, who examined biopsy material, stained with gentian violet, from a patient with “Delhi boil.” Given the now well-documented multifaceted role of macrophages in the immune response, the parasite's interplay with its host cell poses many intriguing problems for researchers. This chapter intends to update the progress that has been made in recent years in understanding the biology of the leishmania–macrophage interaction and how the parasite can attach to and enter the host cell and survive in the intracellular environment. The chapter also discusses the implications of these interactions in the induction of specific immune responses and on disease progression. It also describes those areas where future research may be of value in the rational development of new chemical and immunological therapies and vaccines.
Cell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector.
pp120/HA4, a membrane protein found in hepatocyte plasma membranes and a substrate for the insulin receptor tyrosine kinase, was purified to homogeneity, subjected to partial proteolysis, and peptides were sequenced by Edman degradation. Six amino acid sequences were obtained, and they matched the deduced amino acid sequences of six regions of a hepatocyte membrane protein called ecto-ATPase.
Cell-CAM 105 (C-CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto-ATPase. Detailed biochemical analyses confirmed the identity between C-CAM and the ecto-ATPase. C-CAM/ecto-ATPase is a transmembrane protein having 4 immunoglobulin-like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.
Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.
1. The effects of some possible inhibitors of ectonucleotidases on the breakdown of extracellular ATP by strips of guinea-pig urinary bladder were investigated. 2. Suramin and ethacrynic acid (10 mM) both inhibited ATP breakdown significantly, and difluorodinitrobenzene (10 mM) inhibited it slightly whereas N-ethylmaleimide, adenosine 5'-(gamma-thiotriphosphate) (ATP-gamma-S) and reactive blue-2 (10 mM) were without effect. 3. The inhibitory effects of suramin on ATP breakdown were non-competitive. 4. Ethacrynic acid (1 mM) irreversibly inhibited contractions of the guinea-pig bladder induced by ATP, substance P, histamine, non-adrenergic, non-cholinergic nerve stimulation or KCl, whereas suramin (100 microM) had no inhibitory effect. 5. The results suggest that suramin might provide a starting point for the design of selective inhibitors of ectonucleotidases.
A human hepatoma cell line (Li-7A) possesses ectoATPase activity which is activated by either Mg2+ or Ca2+. Both ectoMg2+-ATPase and ectoCa2+-ATPase hydrolyze other nucleoside triphosphates, are inactive with ADP and AMP, and are inhibited by both p-chloromercuriphenyl sulfonate (pCMPS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Different Km values for ATP and pH curves are obtained for ectoMg2+-ATPase and ectoCa2+-ATPase. The specific activities of the two ATPases remain relatively constant through several days of cell growth after an initial decrease. In contrast, the specific activities of the two ATPases, especially the ectoCa2+-ATPase, increases continuously in Li-7A cells cultured in the presence of EGF, cholera toxin, and hydrocortisone. The ATPases of the factor-treated cells are also indiscriminate with respect to nucleoside triphosphate substrates; however, the kinetic constants for substrates are altered when compared to that of the untreated cells. Most strikingly, the sensitivity to inhibitors is greatly reduced. It is concluded that the long-term effect of EGF, cholera toxin, and hydrocortisone on the Li-7A cells is the induction or activation of a new or minor component of the ectoATPases, which is preferentially activated by Ca2+ and insensitive to pCMPS.
The formation of [32P]pyrophosphate from acetyl phosphate and [32P]orthophosphate was studied under conditions in which phosphate-metal salts or acetyl phosphate-metal salts precipitate. In the absence of precipitates in purely aqueous media, the initial rate constant of transphosphorylation (kobs) was extremely small and the formation of pyrophosphate was detected only in the presence of calcium. In various combinations, conditions such as high pH, high concentrations of reactants, and the presence of dimethyl sulfoxide caused three types of precipitates to form. In completely aqueous solution with an excess of orthophosphate, the crystals formed at high pH contained 3 mol of calcium for 2 mol of phosphate and they were poorly effective at promoting phosphorolysis. In the presence of dimethyl sulfoxide, the ratio of calcium to phosphate in the sediment was 1:1 and phosphorolysis proceeded at a high rate. In either solvent, an excess of acetyl phosphate caused precipitation of a complex containing 1 mol of acetyl phosphate to 1 mol of calcium. In aqueous media the rate constant of phosphorolysis increased with increasing precipitation of the acetyl phosphate-calcium complex. With destabilization of the anions by dimethyl sulfoxide the increase in kobs for a given amount of acetyl phosphate-calcium precipitated was 200-fold higher. Magnesium did not form precipitates and was ineffective in promoting transphosphorylation in completely aqueous media, either in the presence of excess phosphate or in the presence of excess acetyl phosphate. However, when precipitation of phosphate-magnesium or acetyl phosphate-magnesium was promoted by addition of dimethyl sulfoxide, phosphorolysis was observed with rate constants as high as those found in the presence of calcium. These results indicate that phosphorolysis of acetyl phosphate occurs at higher rates on the surface of solid structures, through highly specific interactions involving acetyl phosphate, orthophosphate, and divalent cations.
ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.
Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
Midlogarithmic phase Leishmania donovani promastigotes accumulate 2-deoxy-D-glucose (2-dGlc) and L-proline, maintaining concentration gradient factors across the surface membrane of 78.7 and 60, respectively. Cyanide (1 mM) and iodoacetate (0.5 mM) inhibited the transport of both substrates. L-proline uptake was also inhibited by 2-dGlc (10 mM). Transport of neither substrate was affected by Na+, phlorizin, or ouabain, indicating the sodium-independent transport of both systems. However, N',N'-dicyclohexylcarbodiimide (DCCD; 20 microM) significantly inhibited the transport of both 2-dGlc and L-proline (70% and 90%, respectively). The ionophores valinomycin (1 microM) and nigericin (5 microM) each partially inhibited the uptake of both substrates. In parallel experiments, nigericin and valinomycin were added concomitantly to promastigotes, each at a concentration that individually inhibited the transport of 2-dGlc and L-proline by less than 30%. Under such conditions, the transport of 2-dGlc and L-proline was inhibited by 69% and 78%, respectively. However, these ionophores had no significant effect on the promastigotes cellular ATP level. Carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM) inhibited 2-dGlc (79%) and L-proline (85%) transport, whereas ATP levels of such cells were diminished by only 20%. Symport of D-glucose/H+ and L-proline/H+ was measured directly in cells pretreated with KCN and DCCD. Upon addition of D-glucose to such cells, a rapid movement of protons into the organisms occurred and was reversed upon addition of FCCP. Conversely, no proton movement was observed when L-glucose was added to such cells. L-proline, as D-glucose, caused a rapid influx of protons into the promastigotes, indicating that both substrates were cotransported with protons. We conclude that transport of D-glucose and L-proline in L. donovani promastigotes is protonmotive force-driven and is coupled to both delta pH and delta psi.
Amastigotes and log-phase promastigotes of Leishmania mexicana mexicana contained distinct acid phosphatase, 3'-nucleotidase and 5'-nucleotidase activities, distinguishable by their response to pH and inhibitors. Both tartrate-sensitive and tartrate-resistant acid phosphatase were present in the two forms, amastigotes possessed less tartrate-resistant acid phosphatase than promastigotes. A tartrate-sensitive acid phosphatase was secreted into the medium in large amounts during the growth in vitro of L. m. mexicana promastigotes. The 5'-nucleotidase activity of both parasite forms was inhibited by ammonium molybdate, sodium tartrate and, to less extent, by sodium fluoride whereas 3'-nucleotidase was inhibited by EDTA. All three activities were shown to be present on the external surface of both amastigotes and promastigotes. The three phosphomonoesterase activities were also detected in extracts of L. m. amazonensis, L. donovani, L. tarentolae, Crithidia fasciculata, Herpetomonas muscarum muscarum, H.m. ingenoplastis and Trichomitus batrachorum whereas 5'-nucleotidase was not detected in Trypanosoma brucei brucei extract and 3'-nucleotidase was absent from extracts of Trichomonas vaginalis and Tritrichomonas foetus.
Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca2+ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca2+ exceeds 0.3 to 0.4 μm, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg2+ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca2+ concentration dependence of Ca uptake (K
0.5, 0.5 to 0.8 μm; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.
The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.
The glycoconjugate of Leishmania major recognized by the monoclonal antibody WIC-79.3 exists in two forms. The cellular form associated with the promastigote is a population of amphipathic molecules consistent with membrane insertion. In contrast, the extracellular form mainly consists of hydrophilic molecules, and probably arises by cleavage of the cellular form by an endogenous phospholipase. The hydrophilic population of extracellular glycoconjugate molecules binds specifically to macrophages but not to T or B lymphoid cells. Binding of the glycoconjugate and also intact promastigotes to macrophages in vitro is specifically inhibited by Fab fragments of WIC-79.3. These data indicate that the L. major glycoconjugate is the parasite receptor for macrophages, and hence the molecule directly involved in the initiation of infection.
A suspension of intact guinea pig polymorphonuclear leukocytes hydrolyzed added adenosine triphosphate (ATP), adenosine monophosphate (AMP) and p nitrophenyl phosphate under physiologically appropriate conditions. These enzymatic activities were not due to artifacts such as breakage of the cells during the incubation period. It thus seemed possible that the hydrolyses were being catalyzed by ecto enzymes, i.e. enzymes on the plasma membrane with their active sites facing the external medium. Three types of experiment were designed to test this hypothesis. First, the activities of intact cells were compared to those of homogenates, sonicates, and cells treated with detergent. Disruption of cells resulted in an approximately 2 fold increase in maximal ATPase and p nitrophenyl phosphatase activities suggesting that the plasma membrane was acting as a permeability barrier to the substrates involved. Disruption did not increase AMPase activity, leaving open the possibility that an ecto enzyme is the only protein in polymorphonuclear leukocytes capable of hydrolyzing AMP. Second, the products of ATPase, AMPase, and p nitrophenyl phosphatase activities of intact cells were localized by using radioactively labeled substrates. The concentration of inorganic phosphate produced by these reactions was 18 to 100 times greater in the extracellular medium than in the intracellular milieu. This suggests that the substrates are cleaved outside the cells, or that they are cleaved inside and the products are transported out. The latter possibility was militated against by the following experiment. Cells were loaded with inorganic [33P]phosphate, then allowed to hydrolyze substrates labeled with 32P. The distributions of the two isotopes were compared. Almost all of the inorganic [32P]phosphate was found outside of the cells, while 90% of the inorganic [33P]phosphate remained inside. Third, the cells were treated with the diazonium salt of sulfanilic acid, a reagent known not to penetrate into intact erythrocytes. This treatment rapidly and dramatically inhibited the intact cell ATPase, AMPase, and p nitrophenyl phosphatase, while lactate dehydrogenase, a soluble cytoplasmic enzyme, was unaffected. Control experiments demonstrated that in sonicates lactate dehydrogenase was as susceptible to inhibition by the diazonium salt as were the other three activities.
A method is described for preparing /sup 32/P-labeled ATP of high
specific activity by an excbange reaction. The method is simple and requires
only substrates and enzymes that are available commercially. Hydrolysis of the
labeled ATP with heavy meromyosin indicates that 98 to 99% of the /sup 32/P is in
the 1 -phosphate group. (auth)
A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.
A surface membrane fraction isolated from Leishmania donovani promastigotes contained distinct 5'- and 3'-nucleotidase activities. These were distinguished from each other, and from a previously described surface membrane nonspecific acid phosphomonoesterase, on the basis of several properties. The 5'- and 3'-nucleotidases had p' optima of 6.5 and 8.5, respectively. In contrast to the 3'-nucleotidase, the 5'-nucleotidase was inhibited by both ammonium molybdate and fluoride ions; the latter inducing a biphasic response. Neither divalent cations nor chelators affected the 5'-enzyme activity whereas the 3'-enzyme was inactivated by EDTA. This inactivation was fully reversed following removal of the chelator, either by resuspension of the membranes in EDTA free medium or by addition of certain divalent cations in excess; Co2+ being the most effective. The 5'-nucleotidase had activity with both ribo- and deoxyribonucleotide substrates, whereas the 3'-nucleotidase did not hydrolyse deoxyribonucleotides.
Plasma membranes isolated from the protozoan parasite Leishmania donovani were enriched in acid phosphatase (E.C. 3.1.3.2)
activity. Cytochemically, the enzyme was distributed uniformly on the surface of intact cells and was localized on the external
face of isolated membranes. Physical characteristics and orientation of the membrane-bound enzyme suggest that the organism
is adapted for existence in hydrolytic environments.
The secretion of nitric oxide (NO) was stimulated by the addition of ATP or ADP, but not by AMP or adenosine, in cultured bovine aorta endothelial cells. Inhibitors of ecto-ATPase, NaN3 and Ap5A, significantly inhibited the stimulation, while an inhibitor of P2Y-purinoceptor and ecto-ATPase, RB2, completely suppressed it. A non-hydrolyzable ATP analogue, AMP-PNP, stimulated NO release; the stimulation was completely suppressed by RB2 but not by NaN3 and Ap5A. Therefore, only P2Y-purinoceptor was involved in the stimulation by AMP-PNP, while both ecto-ATPase and P2Y-purinoceptor were involved in the stimulation by ATP and ADP. It is not clear whether the stimulation is dependent on the dephosphorylation activity of ecto-ATPase or not, but the enzyme appears to act as an ATP and ADP receptor for signal transduction through adenine nucleotides.
To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.