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F D Gillin

Publications and source records attributed to F D Gillin.

At least 55 records · Page 3Linked to original sources
PubMed1991

A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor.

Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment.

Acylation↗
PubMed1991

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.

Animals↗
PubMed1991

Organelles of protein transport in Giardia lamblia.

Giardia occupies a unique evolutionary position since it is considered to belong to the earliest known lineage to diverge from the eukaryotic line of descent. Although organelles of protein transport are thought to have evolved with the nuclear membrane, G. lamblia is reported to have no Golgi apparatus. Therefore, Frances Gillin, David Reiner and Michael McCaffery have investigated how it exports glycoproteins to the cyst wall during encystation and whether a Golgi might become evident during an active secretory phase. They have found both functional and morphological evidence of a Golgi in Giardia and have shown that trophozoites are capable of sophisticated protein recognition, sorting and trafficking. These studies suggest that membranous organelles of protein transport appeared early in the evolution of the eukaryotic cell.

Journal Article↗
PubMed1990

Isolation and expression of the gene for a major surface protein of Giardia lamblia.

To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we have cloned the gene that encodes the two major surface-labeled trophozoite protein species. Sequence analysis of this gene reveals a single open reading frame specifying a hydrophilic, cysteine-rich (11.8%) protein of 72.5-kDa molecular mass with an amino-terminal signal peptide and a postulated hydrophobic membrane-spanning anchor region near the carboxyl terminus. Most of the cysteine residues (58 of 84) are in the motif Cys-Xaa-Xaa-Cys, which is dispersed 29 times throughout the sequence. Antibodies against the recombinant protein react with the entire surface of live trophozoites, including flagella and adhesive disc. These antibodies inhibit trophozoite attachment, prevent growth, and immunoprecipitate the major approximately 66- and 85-kDa proteins from surface-labeled live trophozoites. The recombinant Escherichia coli also expresses polypeptides of approximately 66- and 85-kDa molecular mass, which are not fusion proteins. This suggests that the processing and/or conformational changes that lead to production of these two peptide species in E. coli reflect those that occur in Giardia. The abundance of cysteine residues suggests that the native proteins on the parasite surface may contain numerous disulfide bonds, which would promote resistance to intestinal fluid proteases and to the detergent activity of bile salts and would help to explain the survival of Giardia in the human small intestine.

Amino Acid Sequence↗
PubMed1990

Excystation of in vitro-derived Giardia lamblia cysts.

This is the first in-depth analysis of the excystation of Giardia lamblia cysts prepared in vitro. Its goals were both to achieve efficient excystation and to gain insights into this crucial but poorly understood process. To identify the critical elements of excystation, we tested the sequential low-pH induction and protease treatments which had been reported to be important for excystation of fecal cysts. The optimal pH for induction of excystation was 4.0. Emergence was greatly (approximately 10-fold) stimulated by subsequent exposure of in vitro-derived cysts to chymotrypsin, trypsin, or human pancreatic fluid. The stimulatory activity of each was abolished by soybean trypsin inhibitor, demonstrating that the activity of pancreatic fluid was due to these proteases. Excystation of in vitro-derived cysts was approximately 10 to 38%. Although the walls of in vitro-derived cysts were partially digested by protease treatment, trophozoites emerged only from one pole, as observed with fecal cysts. The conditions of encystation also determined the efficiency of excystation. Specifically, encystation in the presence of lactic acid, a major metabolite of colonic bacteria, stimulated excystation approximately fourfold, although it did not increase the total numbers of cysts. These experiments have shown that excystation of in vitro-derived cysts reflects that of cysts purified from human feces in that it is dependent upon conditions which simulate the passage of cysts through the human stomach (low pH) and into the small intestine (pancreatic proteases).

Animals↗
PubMed1990

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.

Acid Phosphatase↗
PubMed1989

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.

Animals↗
PubMed1989

Identification and localization of cyst-specific antigens of Giardia lamblia.

We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are specific to the human small intestinal milieu. We now show that encystation entails the appearance of two new groups of antigens detected in Western blots by rabbit antiserum against cysts which had been purified from human feces. A heterodisperse group of lower-molecular-mass antigens (approximately 21 to 39 kilodaltons) was expressed relatively early (less than 19 h) in encystation. In contrast, discrete bands at approximately 66, 78, 92, and 103 kilodaltons only appeared after 24 h of incubation under conditions which lead to production of large numbers of water-resistant cysts. We also describe for the first time the appearance of prominent cytoplasmic vesicles, which were the earliest morphologic change in encysting trophozoites observable by light microscopy. Early in encystation, cyst wall antigens were concentrated in these vesicles, as shown by immunocytochemistry, suggesting that the vesicles function in export of cyst wall constituents.

Animals↗
PubMed1988

Small-intestinal factors promote encystation of Giardia lamblia in vitro.

Bile salts and fatty acids stimulated differentiation of cultured Giardia lamblia trophozoites into water-resistant cysts at the slightly alkaline pH of the small intestinal lumen. Maximum encystation occurred at pH 7.8. Thus, specific small-intestinal factors may influence encystation in vivo as well as in vitro.

Animals↗
PubMed1987

Encystation and expression of cyst antigens by Giardia lamblia in vitro.

The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a common waterborne intestinal disease. In these studies, encystation of Giardia lamblia in vitro was demonstrated by morphologic, immunologic, and biochemical criteria. In the suckling mouse model, the jejunum was shown to be a major site of encystation of the parasite. Small intestinal factors were therefore tested as stimuli of encystation. An antiserum that reacted with cysts, but not with cultured trophozoites was raised in rabbits and used as a sensitive probe for differentiation in vitro. Cultured trophozoites that were exposed to bile salts showed a more than 20-fold increase in the number of oval, refractile cells that reacted strongly with anticyst antibodies, and in the expression of major cyst antigens. Exposure to primary bile salts resulted in higher levels of encystation than exposure to secondary bile salts. These studies will aid in understanding the differentiation of an important protozoan pathogen.

Animals↗
PubMed1987

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium.

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.

Culture Media↗
PubMed1987

Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk.

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed greater than 99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.

Animals↗
PubMed1987

A new method for purification of Giardia lamblia cysts.

One of the major problems in studying Giardia cysts is separating this form of the parasite from faecal debris and bacteria. We approached this problem by passing filtered stool suspensions over a column of Sephadex G-50 followed by low speed centrifugations. The Sephadex retained most of the faecal debris and the centrifugation reduced the bacterial flora more than 10(5)-fold to less than 3 X 10(4)/ml. Cyst recovery was 61% (+/- 28%). This new method of separating G. lamblia from faecal material is simple, rapid, and effective.

Centrifugation↗
PubMed1987

Intestinal mucus protects Giardia lamblia from killing by human milk.

We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.

Animals↗
PubMed1987

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

Animals↗
PubMed1986

Activation of complement by pathogenic and nonpathogenic Entamoeba histolytica.

Previous studies had demonstrated that strains of Entamoeba histolytica isolated from patients with colitis or amebic liver abscess were resistant to complement-mediated killing, whereas strains from asymptomatic patients were readily lysed by non-immune serum. Both serum-sensitive and serum-resistant strains of E. histolytica depleted complement rapidly as assessed by CH50, C3, and C7, and C5-9 hemolytic activities. Activation of the alternative pathway was important in lysis of nonpathogenic strains, as demonstrated by lysis by NHS (60.9 +/- 15.6%) and NHS + 5 mM EGTA (59.3 +/- 4.5%) as well as by C4-deficient guinea pig serum (72.8 +/- 7.1%) and C2-deficient human serum (64.4 +/- 11.1%), but not by NHS + 5 mM EDTA. Classical pathway activation also occurs as both pathogenic and nonpathogenic strains deplete greater than 98% of C4 activity, although it is not necessary for lysis. Pathogenic strains are not lysed by either the classical or the alternative pathway. These results suggest that pathogenic strains of E. histolytica activate complement but are able to evade an important host defense, complement-mediated lysis.

Animals↗