Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

Reiner, David S.; McCaffery, J. Michael; Gillin, Frances D.

doi:10.1046/j.1462-5822.2001.00129.x

Cited by 46 publications

(53 citation statements)

References 57 publications

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“…To our knowledge, the observation that ER stress induces eIF2␣ phosphorylation and translational control is the first report that a UPR functions in Apicomplexa parasites. This is of significance because studies have suggested that Giardia lamblia, another early-branching eukaryotic cell, may have diverged before the appearance of this type of UPR (34). A transmembrane-containing eIF2 kinase has recently been identified in trypanosomes, but it localized to the flagellar pocket and not the ER (35).…”

Section: Figure 6 Localization Of Tgif2k-a and Tgif2k-bmentioning

confidence: 99%

Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

Narasimhan

Joyce

Naguleswaran³

et al. 2008

Journal of Biological Chemistry

117

203

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A key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2␣ (TgIF2␣) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2␣ remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Furthermore, we have characterized novel eIF2 kinases; one in the endoplasmic reticulum and a likely regulator of the unfolded protein response (TgIF2K-A) and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2␣ phosphorylation is employed by cells to maintain a latent state, and 3) endoplasmic reticulum and cytoplasmic stress responses evolved in eukaryotic cells before the early diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.A well characterized mechanism by which eukaryotic cells respond to environmental stress involves phosphorylation of eukaryotic initiation factor-2 (eIF2) 3 (1-3). The eIF2 combined with GTP delivers Met-tRNA i Met to the translational machinery during initiation of protein synthesis. In mammalian cells, four eIF2 kinases have been described that are each activated by unique stress arrangements. For example, in response to accumulation of malfolded protein in the lumen of the endoplasmic reticulum (so-called ER stress), PEK/Perk (EIF2KA3) phosphorylates the ␣ subunit of eIF2 at serine 51, causing this translation factor to become an inhibitor of its own guanine nucleotide exchange factor, eIF2B. The resulting repression in general translation prevents further synthesis of secretory proteins that would further overload the ER and allows cells sufficient time to trigger the unfolded protein response (UPR) (2). The UPR is a program of mRNA expression involving genes that function in the assembly and transport of secretory proteins (4). In addition to ER stress, three other eIF2 kinases have been described that recognize different forms of cytoplasmic stress in mammalian cells. These include: GCN2 (EIF2KA4), which responds to nutrient deprivation and is well conserved among eukaryotes (3, 5), HRI (EIF2KA1), which is reported to be activated by heme deficiency, oxidative stress induced by arsenite treatment, and heat shock (6, 7), and PKR (EIF2KA2), which is involved in the antiviral defenses (8, 9).Very little research has been performed on eIF2 kinase and related stress response pathways in early-diverging eukaryotes, including pathogenic eukaryotes. However, viability, pathogenesis, and transmission of many parasites hinges on their ability to recogn...

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Section: Figure 6 Localization Of Tgif2k-a and Tgif2k-bmentioning

confidence: 99%

Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

Narasimhan

Joyce

Naguleswaran³

et al. 2008

Journal of Biological Chemistry

117

203

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“…Functional analysis indicated that ESV neogenesis -formation of morphologically distinct post-ER organelles (Lujan et al, 1995a;Reiner et al, 2001;Stefanic et al, 2006) -is directed by a universally conserved machinery that is also responsible for the genesis of preGolgi intermediates and cis-Golgi cisternae in higher eukaryotes (reviewed by Stephens and Pepperkok, 2001). ER export of CWM and partitioning into ESVs appears to be completed by 5-8 h.p.i.…”

Section: Esvs Develop Into a Connected Post-er Network Of Organellesmentioning

confidence: 99%

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote

Štefanić

Morf

Kulangara

et al. 2009

Journal of Cell Science

115

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The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of `naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.

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“…The hallmark of encystation in Giardia is the synthesis of ESVs that transport CWPs to the cell surface (16). During encystation, three encystation-specific CWPs (CWP-1, -2, and -3) are expressed and concentrated within ESVs before they are targeted to the cyst wall (17,36,44). Because the expression of the gglct-1 gene is upregulated (Fig.…”

Section: Vol 76 2008mentioning

confidence: 99%

“…During encystation, various molecular and cellular changes take place that allow this protozoan to transport cyst wall proteins (CWPs) through regulatory secretory pathways (35). In encysting cells, three encystation-specific CWPs (CWP-1, -2, and -3 encoded by cwp-1, -2, and -3, respectively) are synthesized and concentrated within encystation-specific vesicles (ESVs) before targeting into the cyst wall (17,36,44).Recent studies suggest that all three CWPs are essential for forming ESVs and that CWP-2 functions as an aggregation factor to regulate ESV formation by interacting with CWP-1 and CWP-3 via conserved regions (17). It has been proposed that transient Golgi body-like membranes synthesized during encystation are involved in modifying the CWPs and other membrane proteins (27-29).…”

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Novel Role of Sphingolipid Synthesis Genes in Regulating Giardial Encystation

et al. 2008

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Although encystation (cyst formation) is important for the survival of Giardia lamblia outside its human host, the molecular events that prompt encystation have not been fully elucidated. Here, we demonstrate that sphingolipids (SLs), which are important for the growth and differentiation of many eukaryotes, play key roles in giardial encystation. Transcriptional analyses showed that only three genes in the SL biosynthesis pathways are expressed and transcribed differentially in nonencysting and encysting Giardia trophozoites. While the putative homologues of giardial serine palmitoyltransferase (gSPT) subunit genes (gspt-1 and -2) are differentially expressed in nonencysting and encysting trophozoites, the giardial ceramide glucosyltransferase 1 gene (gglct-1) is transcribed only in encysting cells. L-Cycloserine, an inhibitor of gSPT, inhibited the endocytosis and endoplasmic reticulum/perinuclear targeting of bodipy-ceramide in trophozoites, and this could be reversed by 3-ketosphinganine. On the other hand, D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthesis, blocked karyokinesis and reduced cyst production in culture. PPMP also altered the expression of cyst wall protein transcripts in encysting cells. Phylogenetic analyses revealed that the gspt genes are paralogs derived from an ancestral spt sequence that underwent gene duplication early in eukaryotic history. This ancestral sequence, in turn, was probably derived from prokaryotic aminoacyl transferases. In contrast, gglct-1 is found in both prokaryotes and eukaryotes without any evidence of gene duplication. These studies indicate that SL synthesis genes are involved in key events in giardial biology and could serve as potential targets for developing new therapies against giardiasis.Giardiasis, a clinical syndrome caused by the intestinal protozoan Giardia lamblia, is a reemerging waterborne infectious illness worldwide. Giardia exists in two morphological forms: (i) actively dividing trophozoites and (ii) relatively inactive cysts. The water-resistant dormant cysts are responsible for transmission of giardiasis via contaminated water and undergo excystation, a stage of the giardial life cycle in which a cyst differentiates into two trophozoites in the stomach. Newly emerged trophozoites then move down the small intestine and colonize below the bile duct (2). Components of the intestinal milieu, including dietary lipids, bile salts, intestinal pH (pH ϳ7.8), and lactic acid, among others, trigger the process of encystation to complete the life cycle of Giardia in the small intestine (10, 16). During encystation, various molecular and cellular changes take place that allow this protozoan to transport cyst wall proteins (CWPs) through regulatory secretory pathways (35). In encysting cells, three encystation-specific CWPs (CWP-1, -2, and -3 encoded by cwp-1, -2, and -3, respectively) are synthesized and concentrated within encystation-specific vesicles (ESVs) before targeting into the cyst wall (17,3...

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Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

Cited by 46 publications

References 57 publications

Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondii

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote

Novel Role of Sphingolipid Synthesis Genes in Regulating Giardial Encystation

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