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F D Gillin

Publications and source records attributed to F D Gillin.

At least 37 records · Page 2Linked to original sources
PubMed1996

Developmentally regulated transcripts and evidence of differential mRNA processing in Giardia lamblia.

Although encystation and excystation are crucial to transmission of Giardia lamblia, little is known about the regulation of these very distinct differentiation processes. Fingerprinting of giardial mRNA populations throughout the time course of differentiation demonstrated complex patterns in mRNA differential display. Certain transcripts appeared or increased, while others decreased or disappeared at specific times, in response to physiologic stimuli that mimic key stages in parasite descent through the host gastrointestinal tract. This approach has allowed the direct identification of critical stages in differentiation, as well as isolation of genes which may be crucial to the development of G. lamblia. One stage-specific single copy gene (ENC6) whose transcript is greatly upregulated during encystation was analyzed further. Partial sequence analysis revealed no correspondence with known genes. 3'-rapid amplification of cDNA ends (3'-RACE) analysis of ENC6 transcripts at various times of encystation revealed two polyadenylation sites. The more proximal site, 10 nucleotides past the single classic AGTAAA sequence, was utilized only during encystation and its transcript increased approximately 16-fold during the first 24 h of encystation. In contrast, a slightly divergent polyadenylation site 288 nucleotides downstream from the open reading frame (ORF) was used during both vegetative growth and encystation, although its transcript was present at low levels. These studies are the first evidence of differential mRNA processing in G. lamblia and suggest a potential role of the 3'-untranslated region (3'-UTR) in modulating gene expression during differentiation of this primitive eukaryote.

Amino Acid Sequence↗
PubMed1996

Giardia lamblia: increased UDP-N-acetyl-D-glucosamine and N-acetyl-D-galactosamine transferase activities during encystation.

The cyst wall of Giardia lamblia is essential for survival of the parasite outside the host. N-acetyl-D-glucosamine (GalNAc) has been reported as a major terminal sugar of cyst wall glycoproteins and N-acetyl-D-galactosamine (GalNAc) as the major sugar of the fibrous insoluble cyst wall fraction. Therefore, we measured UDP-glycosyltransferase activities as the incorporation of [3H]UDP-sugar into an alcohol-insoluble product. We found that during encystation only UDP-GlcNAc and UDP-GalNAc transferase (UDP-GT) activities increased approximately three- to five-fold compared to nonencysting trophozoites. These activities were distributed approximately equally in the pellet and soluble fractions. The apparent K(m) and V(max) of UDP-GT in these fractions were similar. The activities from both fractions were dependent on Mn2+; however, the pellet enzymes were also partially activated by other metal ions. Both pUDP-GT and sUDP-GT were inhibited by uridine, UDP, and UDP sugars, but not by GlcNAc or GalNAc. Isolation and analysis of the reaction products suggest that pUDP-GT incorporate GlcNAc and GalNAc into glycoproteins, since the products were proteinase sensitive. In contrast, sUDP-GT products were resistant to proteinase treatment. Hydrolysis of the product of UDP-GlcNAc-T incorporation by trifluoroacetic acid released only glucosamine, while both glucosamine and galactosamine were released from UDP-GalNAc-T products, supporting the presence of an epimerase in Giardia which can convert GalNAc to GlcNAc during incorporation. This study suggests that at least two UDP-GT activities are induced during encystation, which are responsible for the transfer of GlcNAc and GalNAc from UDP-GlcNAc or UDP-GalNAc into both proteinase-sensitive and proteinase-resistant components of the Giardia cyst wall.

Animals↗
PubMed1996

Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin.

Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II). Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ agglutinin (WGA) inhibited excystation by > 90%. Adsorption of either ligand with PCWs eliminated inhibition, demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid, which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA- and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall glycoproteins during stage II.

Animals↗
PubMed1996

Cell biology of the primitive eukaryote Giardia lamblia.

Giardia lamblia is an extremely primitive or early-diverging eukaryote that has been considered to have no typical ER or Golgi apparatus, although it is a complex and highly developed cell. Both the trophozoite and cyst have unusual surface proteins that enable these stages to survive in very different and hostile environments. We found that G. lamblia forms novel encystation-specific secretory vesicles and can sort cyst wall proteins to a regulated secretory pathway distinct from the constitutive pathway used to transport the variable cysteine-rich protein to the trophozoite surface. Our studies, utilizing novel ultrastructural methods that preserve the endomembranes, as well as IEM, support the idea that G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells and that these appeared very early in the evolution of eukaryotic cells.

Animals↗
PubMed1995

Giardicidal activity of lactoferrin and N-terminal peptides.

Human and bovine lactoferrins and their derived N-terminal peptides were giardicidal in vitro. Fe3+, but not Fe2+, protected trophozoites from both native lactoferrin and peptides, although the latter lack iron-binding sites. Other divalent metal ions protected only against native lactoferrin. Log-phase cells were more resistant to killing than stationary-phase cells. These studies suggest that lactoferrin, especially in the form of the N-terminal peptides, may be an important nonimmune component of host mucosal defenses against Giardia lamblia.

Animals↗
PubMed1995

Specialized surface adaptations of Giardia lamblia.

Although Giardia lamblia trophozoites were first described by Von Leeuwenhoek in his own diarrheic stool, relatively little is known of the basic biology of this common parasite or the pathophysiology of giardiasis. In particular, there is little specific information about trophozoite properties that cause diarrhea, as neither toxins nor conventional virulence factors have been identified. Therefore, parasite adaptations that promote cyst survival in the external environment and infection and trophozoite persistence in the small intestine, may be viewed as key virulence properties. This review focuses on unusual surface structures of the trophozoite and cyst forms that enable Giardia to be such a successful parasite.

Adaptation, Physiological↗
PubMed1994

Giardia lamblia: ultrastructural basis of protein transport during growth and encystation.

Giardia, an early diverging eukaryote, is reported to have no Golgi apparatus. Moreover, the structural basis for its ability to sort key proteins and transport them to the trophozoite plasma membrane or to the extracellular wall during encystation is not well-understood. Therefore, we have used ultrastructural techniques that enhance the endomembrane system to evaluate the presence and relationships of cytoplasmic organelles and structures that correspond to those present in higher cells. In addition to the perinuclear cisternae, we found rough endoplasmic reticulum (ER), transitional elements, putative tubular-vesicular elements, Golgi-like smooth perinuclear membrane stacks, and lysosome-like peripheral vesicles. Moreover, we observed many small (50-80 nm) vesicles, many of which were coated, that resemble the small transport vesicles that carry proteins between successive ER and Golgi compartments. Importantly, many of these membrane elements appeared to be captured in the process of budding (or fusing). These elements of the endomembrane system are present during both vegetative growth and encystation of Giardia lamblia. In contrast, the encystation-specific vesicles (ESV) are novel large regulated secretory vesicles that transport cyst antigens to the nascent wall. The present studies suggest that ESV may have unusual pathways of formation and traffic. Our findings support the idea that Giardia, a primitive parasite, has complex structures for protein transport. The elements that show similarities to higher cells may have evolved early, while those that differ may represent biologic fossils or specializations for the parasitic life cycle.

Animals↗
PubMed1994

Giardia lamblia: traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching.

Both trophozoites and cysts of Giardia lamblia have unique outer surfaces that protect them from very different hostile environments. However, little is known about the transport of these important molecules to the cell surface. We used monospecific anti-recombinant TSA 417 antibodies and mAb 8C5 in double label immunoelectron microscopy to compare the localization and transport of this major trophozoite surface antigen (TSA) with that of a prominent cyst wall epitope during vegetative growth, encystation, and antigenic switching in vitro. TSA 417 is a marker of the constitutive transport of the major plasma membrane protein, while the 8C5 epitope traces a differentiation-regulated secretory pathway to the cyst wall. Both proteins localized to the nuclear envelope endoplasmic reticulum (ER) cisternae, ER, and cytoplasmic membrane cisternae, reflecting their site of synthesis, but only the differentiation-specific epitope 8C5 localized to the encystation-specific vesicles (ESV). These large secretory vesicles form only during encystation and transport cyst antigens to the nascent wall. In contrast, only TSA 417 was found on the outer surface of the plasmalemma of trophozoites and encysting cells and underlaying the walls of many cysts, while only 8C5 localized to the cyst wall. As encystation progressed, TSA 417 disappeared from the plasmalemma and increased in the lysosome-like PV and other large cytoplasmic vesicles. In contrast to their segregation in the ESV and on the cell surface, both TSA 417 and 8C5 were found in the peripheral vesicles, presumably an endocytic compartment. We propose that this may be the initiation of a stage in differentiation-driven antigenic switching of TSA 417, in which this antigen is no longer synthesized or exported to the plasmalemma, but is taken back inside the cell.

Animals↗
PubMed1994

Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides.

Antimicrobial polypeptides such as the defensins kill a wide range of organisms, including bacteria, fungi, viruses, and tumor cells. Because of the recent finding that intestinal defensins, also known as cryptdins, are synthesized by the Paneth cells of the small intestinal crypts and released into the lumen, we asked whether defensins and other small cationic antimicrobial peptides could kill the trophozoites of Giardia lamblia, which colonize the small intestine. Four mouse cryptdins, two neutrophil defensins (HNP-1 [human] and NP-2 [rabbit]), and the unique tryptophan-rich bovine neutrophil polypeptide indolicidin each had some antigiardial activity against trophozoites in vitro. Cryptdins 2 and 3, indolicidin, and NP-2 each reduced viability by more than 3 log units in 2 h, and killing by all peptides was dose and time dependent. Exposure of trophozoites to peptides frequently resulted in cell aggregation and dramatic changes in morphology. The mechanism of binding and lysis appeared to involve charge interactions, since 150 mM NaCl as well as millimolar levels of Ca2+ and Mg2+ inhibited killing by most of the peptides. Our studies show that G. lamblia is sensitive to defensins and indolicidin and suggest that these small polypeptides could play a role in nonimmune host defenses.

Amino Acid Sequence↗
PubMed1994

GP49, an invariant GPI-anchored antigen of Giardia lamblia.

Giardia lamblia is a primitive protozoan and a major cause of waterborne enteric disease throughout tropical and temperate zones. The ability to grow the infective trophozoites in culture as well as the discovery of the method of in vitro encystation made it possible to study the biology of this primitive protozoan and to characterize the surface antigens. Giardia trophozoites are exposed to high concentrations of fatty acids in the human small intestine. This raises the possibility that intestinal fatty acids may become incorporated into Giardia. Therefore, we determined the pattern of fatty acylation of Giardia surface molecules. By metabolic labeling with radiolabeled fatty acids we identified a single glycosylphosphatidylinositol (GPI)-anchored surface protein in Giardia. GP49 differs from the cysteine-rich variable surface antigens described previously. The presence of a GPI anchor in GP49 was supported by the metabolic incorporation of [14C]-ethanolamine, [3H]-myoinositol and fatty acids into the protein. This was confirmed by chemical and enzymatic cleavage experiments. Most interestingly, GP49 was found to be present in different isolates of Giardia and thus can be considered as an invariant surface antigen. Although the biological function of GP49 is not known, recently we have found that intact and soluble GP49 altered the electrolyte fluxes which regulate fluid secretion in the cultured human intestinal epithelial cell line, T84. These studies indicate that the GPI-anchored invariant antigen of Giardia may play an important role in the pathophysiology of giardiasis.

Animals↗
PubMed1993

Giardia lamblia: post-translational processing and status of exposed cysteine residues in TSA 417, a variable surface antigen.

Giardia lamblia develops and thrives within the harsh and variable environment of the human small intestine. To survive this environment, Giardia has evolved a unique family of antigenically variable, extremely cysteine-rich surface proteins. We have characterized the expression of one of these antigens, TSA 417, at the gene and protein levels. TSA 417 mRNA and protein were expressed at constant levels during both logarithmic growth and encystation. On the trophozoite surface, TSA 417 exists in two forms, an 85-kDa protein and a derived 66-kDa form. Both species have identical amino-terminal sequences that match the translated sequence of TSA 417 after removal of a predicted 17 amino acid signal peptide. Since TSA 417 is highly cysteine rich (ca. 12 mole%), we asked whether it was complexed with itself or with other molecules. Although we found no interchain disulfide bonds, there was substantial intrachain bonding that helped retain the gross structure of isolated TSA 417 after partial trypsin digestion. Because the hydropathy profile of TSA 417 suggested that most of the cysteine and all 29 of its conserved CXXC motifs are on the cell surface, we investigated whether TSA 417 might contain some of the free thiols previously shown to be on the trophozoite surface (Gillin et al., Molecular and Biochemical Parasitology, 13, 1-12, 1984). However, no free thiols were detected, either exposed on native TSA 417 or in the unfolded protein. The absence of free thiols and resistance to proteolytic digestion suggest that most of the cysteine residues are in intrachain bridges, probably either disulfide bonds or cysteine-metal complexes. This internal crosslinking may help explain the resistance of Giardia to proteases and other degradative enzymes in the intestinal fluid.

Amino Acid Sequence↗
PubMed1993

Giardia lamblia: absence of cyst antigens and reduced secretory vesicle formation and bile salt uptake in an encystation-deficient subline.

Encystation of Giardia lamblia entails the appearance of a number of new antigens, as well as formation of a novel class of large encystation-specific secretory vesicles (ESV) that transport stage-specific proteins to the nascent cyst wall. The monoclonal antibody GCSA-1, which was raised against purified cyst walls, recognizes protein species of approximately 26-46 kDa that are regulated by exposure to bile (plus lactic acid) and alkaline pH, the factors that induce encystation. The GCSA-1 epitope is maximally expressed after approximately 14 hr of encystation and localizes to the interior, but not the membrane of the ESV as shown by frozen section immunoelectron microscopy. To further understand the process of encystation, we compared two sublines of strain WB that differ in their ability to encyst in vitro. Water-resistant cysts were not detected in subline A6 under conditions in which subline C6 formed approximately 2 x 10(5) cysts/ml. Moreover, subline A6 did not form ESV efficiently or detectably express antigens recognized by mAb GCSA-1 or by polyclonal anti-cyst sera. Finally, uptake of the bile salt taurocholate by A6 was reduced 4- to 20-fold, compared with that of C6, although transport by both strains was sodium-dependent and regulated by bile salt starvation. The decrease in bile salt uptake by A6 may be related to its defect in encystation.

Animals↗
PubMed1993

Antigenic switching of TSA 417, a trophozoite variable surface protein, following completion of the life cycle of Giardia lamblia.

Expression of TSA 417, the predominant cysteine-rich variable surface protein of Giardia lamblia WB clone C6 trophozoites, did not change during encystation in vitro. However, in vitro excystation of cysts derived in vitro or in vivo consistently produced TSA 417 nonexpressing trophozoite populations, suggesting that completion of the life cycle leads to antigenic switching.

Animals↗
PubMed1993

Cysteine-dependent zinc binding by membrane proteins of Giardia lamblia.

The abundant, highly variable surface proteins (VSPs) which cover the surface of Giardia lamblia trophozoites compose a group of extremely cysteine (C)-rich proteins in which more than half of the cysteines are in the motif CXXC. Because of the constancy of these features among the known VSPs and the prominence of cysteine and particularly CXXC in proteins that bind zinc and other metals, we asked whether G. lamblia VSPs bind zinc in vitro. VSPs are the major protein component of Triton X-114 detergent-phase extracts of G. lamblia trophozoites and can be readily identified by surface iodination of intact cells. The partitioning of 65Zn binding into the Triton X-114 detergent phase and the correspondence between surface iodination and zinc binding patterns of four G. lamblia strains or sublines with different VSPs support the idea that VSPs bind zinc. The requirement for renaturation of blots with a reducing agent indicates that Zn2+ is coordinated by cysteine residues, rather than by other amino acids. Binding did not appear to be specific to zinc since it was inhibited by competition with other divalent metal ions. The abundance of the VSPs and the prevalence of metal binding motifs among all known variants suggest that they may play an important role in trophozoite survival and colonization in the host.

Animals↗
PubMed1992

Encystation of Giardia lamblia leads to expression of antigens recognized by antibodies against conserved heat shock proteins.

During in vitro encystation, Giardia lamblia expresses several stage-specific proteins which are recognized in immunoblots by antisera raised against antigens from three different pathogens. The antigens belong to two different families of conserved stress proteins: (i) HSP60 purified from Legionella pneumophila and recombinant HSP60 from Mycobacterium bovis BCG and (ii) recombinant HSP70 from Plasmodium falciparum.

Animals↗
PubMed1992

Human secretory and serum antibodies recognize environmentally induced antigens of Giardia lamblia.

The variability in duration and severity of infection with Giardia lamblia is likely to be due to trophozoite interactions with immune and nonimmune components of the small intestinal milieu. Despite its potential importance, nothing is known of the isotype or the specificity of the secretory antibody response to G. lamblia. In the present study, we show that serum and secretory antibodies recognize many Giardia antigens whose expression is induced by exposure to selected intestinal conditions. Isotype-specific immunoblots of antigens from trophozoites grown at pH 7.0 without bile or at the intestinal pH of 7.8 with bile were reacted with milk or serum antibodies from subjects with or without histories of giardiasis. While the results were complex, several key observations emerged. Serum and secretory immunoglobulin A (IgA), IgM, and IgG antibodies reacted with many regulated antigens. Antigen recognition patterns varied with isotype and between milk and serum antibodies of the same isotype. Antigen recognition also differed among subjects. Antibodies from virtually every patient recognized some G. lamblia antigens. Furthermore, milk and/or serum samples from putative controls without histories of giardiasis were positive more frequently than would be predicted from published prevalence studies, suggesting either that these antibodies may be cross-reactive or that undiagnosed infections with G. lamblia may be more common than previously thought. Thus, recognition of neoantigens induced by host conditions may be due to conserved or cross-reactive epitopes which could constitute a form of immune evasion by G. lamblia.

Animals↗
PubMed1991

Chitin synthase in encysting Entamoeba invadens.

Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [3H]GlcNAc ([3H]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.

Animals↗