scholarly journals Golgi-Disturbing Agents Lead to the Elimination of Intracellular Toxoplasma gondii

2009 ◽  
Vol 2 (1) ◽  
pp. 10-19 ◽  
Author(s):  
C. S. Carvalho ◽  
G. R. Figueiredo ◽  
E. J. T. de Melo
Keyword(s):  
Retinoic Acid ◽  
Toxoplasma Gondii ◽  
Golgi Apparatus ◽  
Okadaic Acid ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Host Cells ◽  
Parasite Development ◽  
Intracellular Medium

The Golgi apparatus is responsible for the genesis of secretory organelles of Toxoplasma gondii and lipid traffic to the vacuole. This study used anti-Golgi agents to demonstrate the importance of Golgi in Toxoplasma development. Monensin, Brefeldin A, Retinoic Acid and Okadaic Acid reduced the infection, leading to parasite elimination. Mon, BFA and RA affected secretory organelles and the Golgi Complex of the parasites, with faster parasite elimination in the presence of Monensin; in addition, the vesicular transit of host cell C6-NBD-ceramide metabolites was interrupted, but the GC of host cells was preserved. Our results suggest that several targets in the secretory pathway are affected in the intracellular Toxoplasma rather than in the host cells, resulting in interruption of parasite development and its elimination from the intracellular medium.

scholarly journals Chitinase Secretion by Encysting Entamoeba invadensand Transfected Entamoeba histolytica Trophozoites: Localization of Secretory Vesicles, Endoplasmic Reticulum, and Golgi Apparatus

1999 ◽  
Vol 67 (6) ◽  
pp. 3073-3081 ◽  
Author(s):  
Sudip K. Ghosh ◽  
Jessica Field ◽  
Marta Frisardi ◽  
Benjamin Rosenthal ◽  
Zhiming Mai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Entamoeba Histolytica ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Gene Promoter ◽  
Brefeldin A ◽  
Host Cells ◽  
Secretory Vesicles ◽  
Fluorescent Substrate

ABSTRACT Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

scholarly journals Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte

1994 ◽  
Vol 300 (3) ◽  
pp. 821-826 ◽  
Author(s):  
J Benting ◽  
D Mattei ◽  
K Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Brefeldin A ◽  
Cell Cytoplasm ◽  
Parasite Cell ◽  
Host Cell Cytoplasm

Plasmodium falciparum, a protozoan parasite of the human erythrocyte, causes the most severe form of malaria. During its intraerythrocytic development, the parasite synthesizes proteins which are exported into the host cell. The compartments involved in the secretory pathway of P. falciparum are still poorly characterized. A Golgi apparatus has not been identified, owing to the lack of specific protein markers and Golgi-specific post-translational modifications in the parasite. The fungal metabolite brefeldin A (BFA) is known to inhibit protein secretion in higher eukaryotes by disrupting the integrity of the Golgi apparatus. We have used the parasite-encoded glycophorin-binding protein (GBP), a soluble protein found in the host cell cytoplasm, as a marker to investigate the effects of BFA on protein secretion in the intracellular parasite. In the presence of BFA, GBP was not transported into the erythrocyte, but remained inside the parasite cell. The effect caused by BFA was reversible, and the protein could be chased into the host cell cytoplasm within 30 min. Transport of GBP from the BFA-sensitive site into the host cell did not require protein synthesis. Similar observations were made when infected erythrocytes were incubated at 15 degrees C. Incubation at 20 degrees C resulted in a reduction rather than a complete block of protein export. The relevance of our findings to the identification of compartments involved in protein secretion from the parasite cell is discussed.

Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1436-1454 ◽  
Author(s):  
RITA CARDOSO ◽  
SOFIA NOLASCO ◽  
JOÃO GONÇALVES ◽  
HELDER C. CORTES ◽  
ALEXANDRE LEITÃO ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Besnoitia Besnoiti ◽  
Microtubule Cytoskeleton ◽  
Cytoskeleton Organization ◽  
Evolutionary Paths ◽  
The Impact

SUMMARYBesnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

scholarly journals Caspase-resistant Golgin-160 Disrupts Apoptosis Induced by Secretory Pathway Stress and Ligation of Death Receptors

2005 ◽  
Vol 16 (6) ◽  
pp. 3019-3027 ◽  
Author(s):  
Rebecca S. Maag ◽  
Marie Mancini ◽  
Antony Rosen ◽  
Carolyn E. Machamer
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Brefeldin A ◽  
Resistant Mutant ◽  
Death Receptors ◽  
Drug Induced ◽  
Caspase Cleavage

Golgin-160 is a coiled-coil protein on the cytoplasmic face of the Golgi complex that is cleaved by caspases during apoptosis. We assessed the sensitivity of cell lines stably expressing wild-type or caspase-resistant golgin-160 to several proapoptotic stimuli. Cells expressing a caspase-resistant mutant of golgin-160 were strikingly resistant to apoptosis induced by ligation of death receptors and by drugs that induce endoplasmic reticulum (ER) stress, including brefeldin-A, dithiothreitol, and thapsigargin. However, both cell lines responded similarly to other proapoptotic stimuli, including staurosporine, anisomycin, and etoposide. The caspase-resistant golgin-160 dominantly prevented cleavage of endogenous golgin-160 after ligation of death receptors or induction of ER stress, which could be explained by a failure of initiator caspase activation. The block in apoptosis in cells expressing caspase-resistant golgin-160 could not be bypassed by expression of potential caspase cleavage fragments of golgin-160, or by drug-induced disassembly of the Golgi complex. Our results suggest that some apoptotic signals (including those initiated by death receptors and ER stress) are sensed and integrated at Golgi membranes and that golgin-160 plays an important role in transduction of these signals.

scholarly journals Characterization of Class I and II ADP-Ribosylation Factors (Arfs) in Live Cells: GDP-bound Class II Arfs Associate with the ER-Golgi Intermediate Compartment Independently of GBF1

2008 ◽  
Vol 19 (8) ◽  
pp. 3488-3500 ◽  
Author(s):  
Justin Chun ◽  
Zoya Shapovalova ◽  
Selma Y. Dejgaard ◽  
John F. Presley ◽  
Paul Melançon
Keyword(s):  
Golgi Complex ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Live Cells ◽  
Adp Ribosylation ◽  
Rapid Release ◽  
Intermediate Compartment ◽  
Adp Ribosylation Factor

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2–5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf · GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf · GTP, rather than the formation of an Arf · GDP · BFA · GBF1 complex.

scholarly journals Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of Toxoplasma gondii

2008 ◽  
Vol 7 (4) ◽  
pp. 664-674 ◽  
Author(s):  
Fabien Brossier ◽  
G. Lucas Starnes ◽  
Wandy L. Beatty ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Invasion ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Growth ◽  
Adhesive Proteins

ABSTRACT Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.

scholarly journals Structural Integrity of the Golgi Stack Is Essential for Normal Secretory Functions of Rat Parotid Acinar Cells: Effects of Brefeldin A and Okadaic Acid

2002 ◽  
Vol 50 (12) ◽  
pp. 1611-1623 ◽  
Author(s):  
Hideaki Tamaki ◽  
Shohei Yamashina
Keyword(s):  
Golgi Apparatus ◽  
Okadaic Acid ◽  
Cathepsin D ◽  
Structural Integrity ◽  
Brefeldin A ◽  
Acinar Cells ◽  
Ultrastructural Organization ◽  
Golgi Stack ◽  
Parotid Acinar Cells ◽  
Rat Parotid

We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (β-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and β-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules.

scholarly journals O-fucosylation of thrombospondin-like repeats is required for processing of MIC2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

2018 ◽  
Author(s):  
Giulia Bandini ◽  
Deborah R. Leon ◽  
Carolin M. Hoppe ◽  
Yue Zhang ◽  
Carolina Agop-Nersesian ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Cell Attachment ◽  
Host Cells ◽  
Type I ◽  
Human Foreskin Fibroblasts ◽  
Genes Encoding ◽  
Host Cell Attachment ◽  
Glycosylation Pathway

AbstractToxoplasma gondii is an intracellular parasite that causes disseminated infections which can lead to neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2)2, a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals.Here we used mass spectrometry to show that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either pofut2 or nucleotide sugar transporter 2 (nst2), the putative GDP-fucose transporter, results in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulates earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts is reduced in these knockouts, which both show defects in attachment to and invasion of host cells comparable to the phenotype observed in the Βmic2.These results, which show O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that P. falciparum Δpofut2 has decreased virulence and support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Recovery of protein secretion after brefeldin A treatment of rat lacrimal glands: effect of cAMP

1996 ◽  
Vol 271 (3) ◽  
pp. C783-C793 ◽  
Author(s):  
P. Robin ◽  
B. Rossignol ◽  
M. N. Raymond
Keyword(s):  
Golgi Apparatus ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Specific Protein ◽  
Dependent Manner ◽  
Lacrimal Glands ◽  
Extracellular Stimuli ◽  
Few Data

In exocrine cells, the discharge of secretory granule contents in response to extracellular stimuli has been widely documented. However, few data are available concerning the effect of these stimuli on the steps of the secretory pathway preceding protein exocytosis. To obtain more data on this subject, we used brefeldin A (BFA) to perturb intracellular protein transit. When, after exposure of the lacrimal gland lobules to 10 microM BFA, which led to a complete dismantling of the Golgi apparatus and fully inhibited the secretion of newly synthesized proteins, the drug concentration was lowered to 100 nM, a restoration of protein secretion was observed in a secretagogue-dependent manner. Secretagogues increasing the adenosine 3',5'-cyclic monophosphate (cAMP) level facilitated the recovery of protein secretion and Golgi apparatus restructuring, whereas other secretagogues, involving the calcium pathway, did not. Furthermore, the cAMP effect was prevented by H-89, a specific protein kinase A inhibitor. These effects of cAMP are due to neither BFA degradation nor BFA excretion from the cells. We conclude from these results that in rat lacrimal glands the recovery from the dramatic damage caused by BFA is promoted by a cAMP-dependent mechanism and further suggest a role of cAMP in the regulation of the Golgi structure and/or function.

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