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1.
The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.  相似文献   

2.
In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5' flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3' flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1-GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.  相似文献   

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Giardia lamblia is a flagellate protozoan that infects humans and other mammals and the most frequently isolated intestinal parasite worldwide. Giardia trophozoites undergo essential biological changes to survive outside the intestine of their host by differentiating into infective cysts. Cyst formation, or encystation, is considered one of the most primitive adaptive responses developed by eukaryotes early in evolution and crucial for the transmission of the parasite among susceptible hosts. During this process, proteins that will assemble into the extracellular cyst wall (CWP1 and CWP2) are transported to the cell surface within encystation-specific secretory vesicles (ESVs) by a developmentally regulated secretory pathway. Cyst wall proteins (CWPs) are maintained as a dense material inside the ESVs, but after exocytosis, they form the fibrillar matrix of the cyst wall. Little is known about the molecular mechanisms involved in granule biogenesis and discharge in Giardia, as well as the assembly of the extracellular wall. In this work, we provide evidences that a novel 54-kDa protein that exclusively localizes to the ESVs is induced during encystation similar to CWPs, proteolytically processed during granule maturation, and able to bind calcium in vitro. The gene encoding this molecule predicts a novel protein (called gGSP for G. lamblia Granule-specific Protein) without homology to any other protein reported in public databases. Nevertheless, it possesses characteristics of calcium-sequestering molecules of higher eukaryotes. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this secretory granule protein regulates Ca(2+)-dependent degranulation of ESVs during cyst wall formation.  相似文献   

4.
The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by approximately 50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.  相似文献   

5.
Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.  相似文献   

6.
To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid ( t 1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.  相似文献   

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Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.  相似文献   

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During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.  相似文献   

12.
Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.  相似文献   

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In electrophoretic analyses, extracts of Xenopus laevis neurulae exhibited activities digesting yolk proteins maximally at pH4.8. These activities were completely inhibited by a mixture of pepstatin A and Z-Phe-Phe-CHN2, thus being identifiable as cathepsin D and cysteine proteinase. The electrophoretic profiles of yolk proteins cleaved by embryonic extracts changed at gastrula stages; the profile before stage 13 was the same as that given by cathepsin D treatment and the profile at stage 13 was a combination of the profile given by cathepsin D treatment and that given by cysteine proteinase treatment. Quantitative measurement of enzyme activities showed that the cathepsin D activity that was preserved from the beginning of development increased from stages 13 to 25 and decreased thereafter, whereas the cysteine proteinase activity appeared at stage 13, gradually increased until stage 35 and strongly increased thereafter. Immunoblot analyses showed that the 43 kDa form of cathepsin D was processed to its 36 kDa form, presumably by cysteine proteinase. This change can explain the increase of cathepsin D activity at stage 13 and thereafter. Immunofluorescent staining with the antibody against cysteine proteinase occurred in mesodermal and ectodermal cells other than neural ones at stages 13–24, and in the endodermal cells at stages 24–36. Faint staining in the neural ectoderm persisted from stages 18 to 36. Immunoelectron microscope observation showed that what stained was the superficial layer of yolk platelets. All these results indicate that cysteine proteinase plays a key role in the initiation of yolk digestion during embryonic development.  相似文献   

15.
Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative micro1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.  相似文献   

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BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.  相似文献   

19.
Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant-negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant-negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.  相似文献   

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