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S B Aley

Publications and source records attributed to S B Aley.

At least 19 recordsLinked to original sources
PubMed2000

The Giardia genome project database.

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

Animals↗
PubMed1999

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Amino Acid Sequence↗
PubMed1998

Sequence survey of the Giardia lamblia genome.

The parasitic protozoan Giardia lamblia represents one of the earliest diverging lineages in the evolutionary history of eukaryotic organisms as well as an important human pathogen. A representative sampling of gene sequences from this early diverging protozoan could provide insights into genotypic and phenotypic innovations associated with the origin of eukaryotes. Currently, known giardial gene sequences are heavily biased toward a few gene families, including variant surface proteins (VSPs), structural proteins, and ribosomal RNA genes. One-pass sequences of Giardia genomic DNA were obtained using vector flanking priming sequences on the ends of cosmids in two independent libraries. Comparisons of 2304 of these sequences against the GenBank database identified 205 potential giardial genes with BLAST scores P(n) < 10(9). These coding regions encompass a wide range of metabolic, repair, and signaling enzymes, and include some genes not predicted by our current understanding of Giardia biochemistry. The efficiency of identification of putative genes is consistent with earlier findings that coding regions in the Giardia genome are densely packed and do not appear to contain introns. Our current results suggest that direct genome sequencing is an efficient method for identifying giardial genes for evolutionary and biochemical studies.

Animals↗
PubMed1997

Giardia lamblia: evidence for carrier-mediated uptake and release of conjugated bile acids.

Giardia lamblia trophozoites colonize the human small intestine, where they are exposed to high concentrations of conjugated bile acids. Previous work has shown that bile acids enhance trophozoite survival, multiplication, and differentiation into the cyst stage. Therefore, experiments were performed to test whether carrier-mediated uptake of conjugated bile acids is present in this primitive parasite. Uptake of both cholyltaurine (C-tau) and cholylglycine (C-gly) was increased manyfold after culturing trophozoites in medium lacking bile acids. Absence of uptake at 4 degrees C and inhibition by other conjugated bile acids provided additional evidence for carrier-mediated uptake. Uptake of C-tau was greater than that of C-gly under all experimental conditions and appeared to be mediated by a different carrier. The major evidence for different carriers is that C-tau uptake was Na(+)-dependent, while C-gly uptake was not. In addition, C-tau uptake was more strongly inhibited by DTNB and several organic anions than C-gly uptake. Radiolabeled C-tau and C-gly were each released rapidly from trophozoites at 37 degrees C but not at 4 degrees C, suggesting that release of conjugated bile acids was also carrier-mediated. These findings are consistent with the notion that multiple transporters for conjugated bile acids are present in a lower eukaryote. We speculate that intracellular bile acids may facilitate lipid trafficking and membrane biosynthesis.

Animals↗
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↗
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

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

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↗
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↗
PubMed1989

Expression of Plasmodium berghei circumsporozoite antigen on the surface of exoerythrocytic schizonts and merozoites.

The intracellular distribution of circumsporozoite (CS) antigen was traced by immunoelectron microscopy in cultures of Plasmodium berghei exoerythrocytic (EE) schizonts with monoclonal antibody (Mab) 3D11 to the immunodominant repeat region of the P. berghei CS protein. CS antigen was localized on the parasitophorous vacuole (PV) membrane and pellicular complex of recently invaded sporozoites and on electron-dense masses of sloughed CS antigen in the host cell cytoplasm. CS antigen persisted throughout the complete EE cycle of P. berghei on the surface of EE schizonts and was incorporated into the plasma membrane of budding EE merozoites. Erythrocytic merozoites were not labeled by Mab 3D11, indicating that these 2 populations of merozoites differ in antigenic composition. Significant internal labeling occurred in 50 hr EE schizonts in association with the limiting membranes of peripheral vesicles and short, tube-like structures attached to their outer surfaces. These vesicles contained an electron-dense flocculent material also present in the PV space. Association of CS antigen with the limiting membranes of these vesicles suggests that they either develop as endocytotic invaginations of the schizont plasma membrane or transport newly synthesized CS antigen from the endoplasmic reticulum and Golgi of developing EE schizonts to the parasite surface.

Animals↗
PubMed1987

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts.

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.

Animals↗
PubMed1987

Plasmodium falciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells.

The immunodominant epitope region of the circumsporozoite protein of Plasmodium falciparum sporozoites contains 37 tandem repeats of the tetrapeptide Asn-Ala-Asn-Pro and 4 repeats of Asn-Val-Asp-Pro. Synthetic peptides and recombinant proteins of the repeat region were used to immunize mice using different doses and adjuvants. Antisera were tested for inhibition of sporozoite invasion of cultured human hepatoma cells. Synthetic peptides and recombinant proteins elicited high levels of antibodies that inhibited sporozoite invasion when emulsified with complete Freund's adjuvant. Since recombinant proteins with alum elicited a better antibody response to sporozoite invasion than they did without adjuvant, it may be that a recombinant protein containing 32 tandem copies of the tetrapeptide repeat combined with alum could be a candidate malarial vaccine suitable for human trials.

Animals↗
PubMed1987

Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs.

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K+ phenotype) synthesize a 90 kDa histidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submembranous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunofluorescence and immunoelectron microscopy of intact, non-fixed K+ infected erythrocytes using mAb 89 and a rabbit antiserum raised against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.

Animals↗
PubMed1987

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Animals↗