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Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells-glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host-pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron-glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae-Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.
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Ecto-5'-nucleotidase (CD73) hydrolyses 5'AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA-MB-231) express high CD73 levels, and this enzyme has been found to play a tumour-promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto-5'-nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF-10A cells, triple-negative breast cancer MDA-MB-231 cells had a higher ecto-5'-nucleotidase expression level and enzymatic activity. Although ecto-5'-nucleotidase activity in the MDA-MB-231 cell line showed no selectivity among monophosphorylated substrates, 5'AMP was preferred by the MCF-10A cell line. Compared to the MCF-10A cell line, the MDA-MB-231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,ßmethylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto-5-nucleotidase significantly reduced the migratory and invasive capacity of MDA-MB-231 cells, suggesting that ecto-5-nucleotidase activity might play an important role in metastatic progression.
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5'-Nucleotidase , Neoplasias de Mama Triplo Negativas , Humanos , 5'-Nucleotidase/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Movimento Celular , Adenosina/metabolismo , Adenosina/análogos & derivadosRESUMO
Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.
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Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3'-nucleotidase/nuclease (3'-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3'-AMP, and this activity is similar to that observed in other protists. The addition of 3'-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3'-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3'-AMP. We believe that ecto-3'-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3'-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.
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Acanthamoeba castellanii , Adenosina , Adesão Celular , Trofozoítos , Acanthamoeba castellanii/enzimologia , Adenosina/metabolismo , Linhagem Celular , Animais , Nucleotidases/metabolismo , Células Epiteliais/parasitologiaRESUMO
Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.
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Lipase , Rhodnius , Animais , Feminino , Lipase/genética , Lipase/metabolismo , Rhodnius/genética , Casca de Ovo/metabolismo , Mobilização Lipídica , Reprodução , Triglicerídeos/metabolismo , Locomoção , Insetos Vetores , Mamíferos/metabolismoRESUMO
Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cellsglial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of hostpathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuronglia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. lepraeSchwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. nzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR nalysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzymelinked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.
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Animais , Camundongos , Hanseníase/microbiologia , Viabilidade Microbiana , Gotículas Lipídicas , Camundongos Nus , Mycobacterium leprae/crescimento & desenvolvimentoRESUMO
Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3'-nucleotidase/nuclease (3'-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3'-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3'-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs.
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Ceratite por Acanthamoeba , Acanthamoeba castellanii , Armadilhas Extracelulares , Animais , Humanos , Trofozoítos/fisiologia , Ceratite por Acanthamoeba/parasitologiaRESUMO
Trypanosoma cruzi is the pathogen of Chagas disease, a neglected tropical disease that affects more than 6 million people worldwide. There are no vaccines to prevent infection, and the therapeutic arsenal is very minimal and toxic. The unique E-NTPDase of T. cruzi (TcNTPDase1) plays essential roles in adhesion and infection and is a virulence factor. Quercetin is a flavonoid with antimicrobial, antiviral, and antitumor activities. Its potential as a partial inhibitor of NTPDases has also been demonstrated. In this work, we synthesized the non-natural L-glycoside derivatives of quercetin and evaluated them as inhibitors of recombinant TcNTPDase1 (rTcNTPDase1). These compounds, and quercetin and miquelianin, a natural quercetin derivative, were also tested. Compound 16 showed the most significant inhibitory effect (94%). Quercetin, miquelianin, and compound 14 showed inhibition close to 50%. We thoroughly investigated the inhibitory effect of 16. Our data suggested a competitive inhibition with a Ki of 8.39 µM (± 0.90). To better understand the interaction of compound 16 and rTcNTPDase1, we performed molecular dynamics simulations of the enzyme and docking analyses with the compounds. Our predictions show that compound 16 binds to the enzyme's catalytic site and interacts with important residues for NTPDase activity. As an inhibitor of a critical T. cruzi enzyme, (16) could be helpful as a starting point in the developing of a future treatment for Chagas disease. Furthermore, the discovery of (16) as an inhibitor of TcNTPDase1 may open new avenues in the study and development of new inhibitors of E-NTPDases.
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Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: Km = 2.12 ± 0.54 mM p-NPP and Vmax = 26.12 ± 2.53 nmol p-NP × h-1 × 10-6 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process.
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Ceratite por Acanthamoeba , Acanthamoeba castellanii , Humanos , Animais , Manose/metabolismo , Vanadatos , Adesão Celular/fisiologia , Sódio , TrofozoítosRESUMO
The ecto-phosphatases belong to a group of enzymes closely associated with the cell surface that has its catalytic site facing the extracellular environment, where different phosphorylated substrates can be hydrolyzed. In the present work, we biochemically characterized the ecto-phosphatase activity of the freshwater microalgae Euglena gracilis, a model microorganism, ubiquitously distributed and resistant to several environmental stressors. The ecto-phosphatase activity is acidic, stimulated by copper and presents the following apparent kinetic parameters: Km = 2.52 ± 0.12 mM p-NPP and Vmax = 3.62 ± 0.06 nmol p-NP × h-1 × 106 cells. We observed that zinc, orthovanadate, molybdate, fluoride, and inorganic phosphate inhibit the ecto-phosphatase activity with different magnitudes. Fluoroaluminate complexes are also inhibitors of this ecto-phosphatase activity. They can be formed in the enzyme reaction conditions and are likely to occur in a natural environment where E. gracilis can be found. The ecto-phosphatase activity is constant through the culture growth phases and is negatively modulated after continuous subculturing in the dark when a shift from phototrophic to the heterotrophic metabolism is likely. The analysis of those biochemical parameters may contribute to understanding the role of E. gracilis ecto-phosphatase activity in natural environments.
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Euglena gracilis , Monoéster Fosfórico Hidrolases , Monoéster Fosfórico Hidrolases/metabolismo , Euglena gracilis/metabolismo , Membrana Celular/metabolismoRESUMO
ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.
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Rhodnius , Feminino , Animais , Rhodnius/genética , Rhodnius/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo Energético , Triglicerídeos/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Mucosal-associated parasites, such as Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis, have significant clinical relevance. The pathologies associated with infection by these parasites are among those with the highest incidence of gastroenteritis (giardiasis and amoebiasis) and sexually transmitted infections (trichomoniasis). The treatment of these diseases is based on drugs that act on the anaerobic metabolism of these parasites, such as nitroimidazole and benzimidazole derivatives. One interesting feature of parasites is their ability to produce ATP under anaerobic conditions. Due to the absence of enzymes capable of producing ATP under anaerobic conditions in the vertebrate host, they have become interesting therapeutic targets. This review discusses anaerobic energy metabolism in mucosal-associated parasites, focusing on the anaerobic metabolism of pyruvate, the importance of these enzymes as therapeutic targets, and the importance of treating their infections.
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Antiprotozoários , Entamoeba histolytica , Parasitos , Trichomonas vaginalis , Animais , Humanos , Parasitos/metabolismo , Anaerobiose , Metabolismo Energético , Trifosfato de Adenosina/metabolismo , Entamoeba histolytica/metabolismoRESUMO
Tumor cells develop a high demand for inorganic phosphate (Pi) due to their high growth rates and energy requirements. Serum Pi concentrations in cancer patients have been found to be two to four times higher than baseline levels in healthy individuals. Twofold Pi accumulation was observed in breast cancer cells in the mouse tumor microenvironment. In the breast tumoral microenvironment, ectonucleotidases and ectophosphatases-presenting catalytic sites facing the extracellular environment-could be involved in the extracellular release of Pi to be internalized by Pi transporters to fuel the high energy requirement typical of cancer cells. Two Pi transporters were characterized in breast cancer cells (Na+-dependent and H+-dependent) with strong associations with tumor processes such as proliferation, migration, adhesion, and epithelium-mesenchymal transition (EMT). Moreover, a high extracellular Pi concentration stimulates ROS production in triple-negative breast cancer cells by Pi transport stimulation. Several compounds show a potent ability to inhibit ectonucleotidases, ectophosphatases, Pi transporters, and Pi-modulated signal pathways in breast cancer cells and regulate proliferation, migration, adhesion, and EMT. This review article aimed to gather the relevant experimental records regarding Pi's effects on the breast cancer microenvironment and points to possible inhibitors for ectonucleotidases, ectophosphatases, Pi transporters, and Pi-modulated signal pathways as potential chemotherapeutic agents or Pi acting as a potent enhancer of classical chemical-induced cytotoxicity in triple-negative breast cancer cells.
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Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/patologia , Fosfatos/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (Pi) availability. Pi is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H+:Pi-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, Pi transport was linear for up to 15 min, and the cell density was 3 × 107 cells/ml. Physiological variations in pH (6.4-8.0) did not influence Pi uptake. This Pi transporter had a high affinity, with K0.5 = 67.7 ± 7.1 µM Pi. SCH28080 (inhibitor of H+, K+-ATPase), bafilomycin A1 (inhibitor of vacuolar H+-ATPase), and FCCP (H+ ionophore) were able to inhibit Pi transport, indicating that an H+ gradient in the cell powered uphill Pi movement. PAA, an H+-dependent Pi transport inhibitor, reduced cell proliferation, Pi transport activity, and GdPHO48 mRNA levels. Pi starvation stimulated membrane potential-sensitive Pi uptake coupled to H+ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize Pi as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized Pi can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits Pi influx. Together, these results reinforce the hypothesis that Pi is a crucial nutrient for G. duodenalis energy metabolism.
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Giardia lamblia , Giardíase , Trifosfato de Adenosina , Animais , Giardia lamblia/genética , Proteínas de Transporte de Fosfato , Saccharomyces cerevisiae/genética , TrofozoítosRESUMO
Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.
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Acanthamoeba castellanii , Acanthamoeba castellanii/genética , Trifosfato de Adenosina/farmacologia , Anaerobiose , Animais , Proteínas de Transporte de Fosfato , Fosfatos , Trofozoítos/fisiologiaRESUMO
In addition to their standard inorganic phosphate (Pi) nutritional function, Pi transporters have additional roles in several cells, including Pi sensing (the so-called transceptor) and a crucial role in Pi metabolism, where they control several phenotypes, such as virulence in pathogens and tumour aggressiveness in cancer cells. Thus, intracellular Pi concentration should be tightly regulated by the fine control of intake and storage in organelles. Pi transporters are classified into two groups: the Pi transporter (PiT) family, also known as the Pi:Na+ symporter family; and the Pi:H+ symporter (PHS) family. Highly proliferative cells, such as protozoan parasites and cancer cells, rely on aerobic glycolysis to support the rapid generation of biomass, which is equated with the well-known Warburg effect in cancer cells. In protozoan parasite cells, Pi transporters are strongly associated with cell proliferation, possibly through their action as intracellular Pi suppliers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Similarly, the growth rate hypothesis (GRH) proposes that the high Pi demands of tumours when achieving accelerated proliferation are mainly due to increased allocation to P-rich nucleic acids. The purpose of this review was to highlight recent advances in understanding the role of Pi transporters in unicellular eukaryotes and tumorigenic cells, correlating these roles with metabolism in these cells.
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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.
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Leishmania , Parasitos , Trichomonas vaginalis , Trypanosoma , Trifosfato de Adenosina , Animais , Interações Hospedeiro-ParasitaRESUMO
Protozoans of the genus Leishmania are the causative agents of an important neglected tropical disease referred to as leishmaniasis. During their lifecycle, the parasites can colonize the alimentary tract of the sand fly vector and the parasitophorous vacuole of the mammalian host, differentiating into distinct stages. Motile promastigotes are found in the sand fly vector and are transmitted to the mammalian host during the insect blood meal. Once in the vertebrate host, the parasites differentiate into amastigotes and multiply inside macrophages. To successfully establish infection in mammalian hosts, Leishmania parasites exhibit various strategies to impair the microbicidal power of the host immune system. In this context, stage-specific class I nucleases play different and important roles related to parasite growth, survival and development. Promastigotes express 3'-nucleotidase/nuclease (3'-NT/NU), an ectoenzyme that can promote parasite escape from neutrophil extracellular traps (NET)-mediated death through extracellular DNA hydrolysis and increase Leishmania-macrophage interactions due to extracellular adenosine generation. Amastigotes express secreted nuclease activity during the course of human infection that may be involved in the purine salvage pathway and can mobilize extracellular nucleic acids available far from the parasite. Another nuclease expressed in amastigotes (P4/LmC1N) is located in the endoplasmic reticulum of the parasite and may be involved in mRNA stability and DNA repair. Homologs of this class I nuclease can induce protection against infection by eliciting a T helper 1-like immune response. These immunogenic properties render these nucleases good targets for the development of vaccines against leishmaniasis, mainly because amastigotes are the form responsible for the development and progression of the disease. The present review aims to present and discuss the roles played by different class I nucleases during the Leishmania lifecycle, especially regarding the establishment of mammalian host infection.
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Armadilhas Extracelulares , Leishmania , Leishmaniose , Parasitos , Psychodidae , Animais , HumanosRESUMO
The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.
Assuntos
Doença de Chagas/parasitologia , Mitocôndrias/metabolismo , Prolina Oxidase/metabolismo , Rhodnius/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Diferenciação CelularRESUMO
According to the growth rate hypothesis (GRH), tumour cells have high inorganic phosphate (Pi) demands due to accelerated proliferation. Compared to healthy individuals, cancer patients present with a nearly 2.5-fold higher Pi serum concentration. In this work, we show that an increasing concentration of Pi had the opposite effect on Pi-transporters only in MDA-MB-231 when compared to other breast cell lines: MCF-7 or MCF10-A (non-tumoural breast cell line). Here, we show for the first time that high extracellular Pi concentration mediates ROS production in TNBC (MDA-MB-231). After a short-time exposure (1 h), Pi hyperpolarizes the mitochondrial membrane, increases mitochondrial ROS generation, impairs oxygen (O2) consumption and increases PKC activity. However, after 24 h Pi-exposure, the source of H2O2 seems to shift from mitochondria to an NADPH oxidase enzyme (NOX), through activation of PKC by H2O2. Exogenous-added H2O2 modulated Pi-transporters the same way as extracellular high Pi, which could be reversed by the addition of the antioxidant N-acetylcysteine (NAC). NAC was also able to abolish Pi-induced Epithelial-mesenchymal transition (EMT), migration and adhesion of MDA-MB-231. We believe that Pi transporters support part of the energy required for the metastatic processes stimulated by Pi and trigger Pi-induced H2O2 production as a signalling response to promote cell migration and adhesion.