A Lipoprotein-Cholesterol-Albumin Serum Substitute Stimulates Giardia lamblia Encystation Vesicle Formation

ABSTRACT. We found previously that the A6 clone of Giardia lamblia strain WB that did not encyst in vitro was blocked at an early stage in differentiation, as it did not form encystation secretory vesicles (ESV) efficiently or express cyst antigens, in comparison with the related clone C6. We now report that A6 formed ESV normally in the suckling mouse model. Therefore, we asked whether our serum-containing encystation media might lack a stimulus or component or contain an inhibitor of ESV formation to which A6 was especially sensitive. We found that replacing bovine serum with a lipoprotein-cholesterol solution and bovine serum albumin (LPC) in pre-encystation and encystation media increased ESV formation by both A6 and C6. The % of A6 cells with ESV increased from 8% in BS medium to 48% in LPC medium, compared with 64% and 98% for C6. Similarly, the average number of ESV/positive cell increased from 1.5 in BS medium to 7.7 in LPC medium for A6, and from 13.3 to 19.7 for C6. Moreover, in LPC encystation media, A6 expressed the cyst wall epitope recognized by monoclonal GCSA-1. Although formation of water-resistant cysts by A6 was increased >60 fold in LPC media, the numbers of cysts remained only ∼3–15% that of C6. This suggests that LPC may primarily affect early events in encystation and that A6 may require additional factors later in encystation.     

Naegleria fowleri: enolase is expressed during cyst differentiation

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.     

Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro

Encystation of Giardia lamblia is required for survival outside the host, as well as for initiation of new infections. Previously, we induced cultured G. lamblia trophozoites to encyst in vitro for the first time. During encystation, we observed the appearance of a new class of large secretory vesicle (encystation-specific vesicle; ESV) within which cyst antigens are concentrated and transported to the nascent wall. The present kinetic and physiologic studies now show that ESV are the earliest morphologic change observed in encystation. Expression of ESV, as well as subsequent encystation, are regulated by exposure to bile at the slightly alkaline pH which is typical of the human intestinal tract. ESV formation appears to be less stringently regulated than formation of water-resistant cysts because omission of either encystation stimuli or alkaline pH preferentially inhibits encystation. Since cysts do not attach, we asked when in encystation this physiologic transition occurs. We found that most encysting trophozoites remain attached until they begin to round up (greater than 24 hr). However, if they are made to detach, as early as 12 hr in encystation, well before they round up, they are defective in the ability to reattach. If trophozoites also become less able to reattach to the intestinal epithelium early in encystation in vivo, this would increase their exposure to lumenal encystation stimuli and promote encystation. These studies have provided new insights into the complex sequence of morphologic and physiologic alterations which occur during encystation of G. lamblia in vitro and their regulation by host intestinal factors.     

How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection

Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.     

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora,Hypotrichia), with preliminary notes on encystation and various types of excystation

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.     

Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro

Large numbers (10(4) to greater than 10(5)/ml) of Type I water-resistant Giardia lamblia cysts were produced in vitro under conditions that are characteristic of the human intestinal lumen. We define Type I cyst morphology as oval shaped, smooth, and refractile, with cyst wall, axostyle, and median body visible in relief by Normarski differential interference contrast optics. Human and porcine bile induced higher levels of encystation than bovine bile at the alkaline pH (7.8) which occurs in the human lower small intestine. High-pressure liquid chromatography analysis showed that the porcine bile had a preponderance of hyocholate, rather than cholate, while bovine bile had less chenodeoxycholate and more deoxycholate than human bile. Lactic acid, a major product of bacterial metabolism in the human colon, further stimulated encystation. Growth of the preencystation culture without bile also increased subsequent encystation. More than 90% of Type I cysts produced with porcine bile plus lactic acid were viable as indicated by the uptake and retention of fluorescein diacetate and exclusion of propidium iodide. Biological activity of in vitro-derived water-resistant cysts was demonstrated by the observation that 1 to 9.5% excysted in vitro. The percentage of excystation was greatly decreased following encystation at pH 7.0 or by omission of bile or lactic acid. This is the first quantitative in vitro demonstration of the complete life cycle of G. lamblia from humans.     

Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia

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.     

Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan,Giardia lamblia

The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Mining the Giardia lamblia genome for new cyst wall proteins

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.     

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).     

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.