Receptor-mediated endocytosis and trafficking between endosomal–lysosomal vacuoles in Giardia lamblia

The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.Fil: Rivero, Maria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Jausoro, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Lanfredi Rangel, Adriana. Centro de Pesquisas Gonçalo Moniz, Serviço de Microscopia Eletrônica; BrasilFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; Argentin

Exosome biogenesis in the protozoa parasite Giardia lamblia: A model of reduced interorganellar crosstalk

Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.Fil: Moyano, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Musso, Juliana Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Frontera, Lorena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Lanfredi-Rangel, Adriana. Centro de Pesquizas Gonzalo Monis. Fiocruz; BrasilFil: Lalle, Marco. Department Of Infectious Diseases, Foodborne And Neglec; ItaliaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Síntese, caracterização e estudo da atividade inibitória de novas dialquilfosforilarilidrazonas sobre o crescimento de tripanossomatídeos

A new series of dialkylphosphorylhydrazones was synthesized through the condensation of aromatic aldehydes with different phosphorylhydrazines. All synthesized compounds were characterized by IR, ¹H-NMR, 13C-NMR and 31P-NMR spectroscopies. The in vitro investigation of the activity of these compounds against Leishmania amazonensis promastigotes and epimastigotes of T. cruzi, showed an efficient inhibition of proliferation, at non toxic concentrations to mammalian cells. The results have shown some derivatives as potential antiparasitic agents against trypanosomatids

Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-12-12T12:19:04Z No. of bitstreams: 1 Meira CS Antiparasitic evaluation....pdf: 1441373 bytes, checksum: 57e6aa961c1ae67aa08d5af7ce78c3fc (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-12-12T12:43:14Z (GMT) No. of bitstreams: 1 Meira CS Antiparasitic evaluation....pdf: 1441373 bytes, checksum: 57e6aa961c1ae67aa08d5af7ce78c3fc (MD5)Made available in DSpace on 2016-12-12T12:43:14Z (GMT). No. of bitstreams: 1 Meira CS Antiparasitic evaluation....pdf: 1441373 bytes, checksum: 57e6aa961c1ae67aa08d5af7ce78c3fc (MD5) Previous issue date: 2016-07CNPq (grant number 562655/2010-7), PRONEX (grant number 0002/2014), FAPESB (grant number 0042/2013) and FINEP (grant number 01.04.0320-00).Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Paraíba. Laborat orio de Tecnologia Farmacêutica. João Pessoa, PB, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade do Estado da Bahia. Departamento de Ciências da Vida. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilBetulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent

Effects of a putrescine analog on Giardia lamblia

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-07-21T12:12:24Z No. of bitstreams: 1 Maia Effects of a Putrescine.pdf: 375890 bytes, checksum: b5c31f0233a709d591d0f216b2547c8d (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-07-21T12:29:14Z (GMT) No. of bitstreams: 1 Maia Effects of a Putrescine.pdf: 375890 bytes, checksum: b5c31f0233a709d591d0f216b2547c8d (MD5)Made available in DSpace on 2016-07-21T12:29:14Z (GMT). No. of bitstreams: 1 Maia Effects of a Putrescine.pdf: 375890 bytes, checksum: b5c31f0233a709d591d0f216b2547c8d (MD5) Previous issue date: 2008-07Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, BrasilFundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal do Rio de Janeiro. Bioquímica Médica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, BrasilFundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilThe protozoan Giardia lamblia is the most frequent intestinal parasite of first-world countries and a major cause of waterborne disorder often referred to as traveler's diarrhea. We have previously noticed that the putrescine analog 1,4-diamino-2-butanone (DAB) remarkably inhibits the growth of anaerobic trichomonad and Trypanosoma cruzi parasites. Here, we examined the role of polyamines in Giardia cells using this putrescine analog. DAB impaired parasite proliferation dose-dependently. The analog induced increased flagella numbers and sometimes four ventral disks as well as asymmetrical division, indicating truncated or deregulated cytokinesis. Electron microscopy analysis revealed that DAB also triggered the encystment process. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) detection. Trophozoites incubated either with 1 mM of DAB or putrescine for 18 h displayed increased lipoperoxide levels. Addition of 200 microM aminoguanidine, a polyamine/diamine oxidase inhibitor, partially reverted the DAB, but not the putrescine effects, indicating that the DAB effects are due, at least in part, to DAB oxidation end products. These data indicate that polyamines play a role in Giardia cell division, differentiation, and antioxidant defenses

Giardia lamblia : biochemical characterization of an ecto-ATPase activity

6 p. : il.In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi h 1 10 6 cells). The ATP hydrolysis was stimulated by MgCl2 in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent Km for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg2+-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1 (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl2. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology

The giardial ENTH protein participates in lysosomal protein trafficking and endocytosis

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-03-09T18:33:27Z No. of bitstreams: 1 Feliziani C The giardia....pdf: 2028133 bytes, checksum: 2ba0e20be87fcdfa8535761012ee5e0f (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-03-09T18:35:02Z (GMT) No. of bitstreams: 1 Feliziani C The giardia....pdf: 2028133 bytes, checksum: 2ba0e20be87fcdfa8535761012ee5e0f (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-03-09T18:52:58Z (GMT) No. of bitstreams: 1 Feliziani C The giardia....pdf: 2028133 bytes, checksum: 2ba0e20be87fcdfa8535761012ee5e0f (MD5)Made available in DSpace on 2015-03-09T18:52:58Z (GMT). No. of bitstreams: 1 Feliziani C The giardia....pdf: 2028133 bytes, checksum: 2ba0e20be87fcdfa8535761012ee5e0f (MD5) Previous issue date: 2015Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC, CONICET. Córdoba, ArgentinaUniversidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC, CONICET. Córdoba, ArgentinaUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Rosario, ArgentinaUniversidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC, CONICET. Córdoba, ArgentinaFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz.Serviço de Microscopia Eletrônica. Salvador, BA, BrasilUniversidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC, CONICET. Córdoba, ArgentinaIn the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3,was associatedwith the alpha subunit of AP-2, clathrin and ubiquitin andwas involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to- PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in themaintenance of PV homeostasis. In this study,we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family