David Reiner | University of California, San Diego (original) (raw)

Papers by David Reiner

Journal of Biological Chemistry, 2002

Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic di... more Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca 2؉ levels (1,2bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acidtetra(acetyloxymethyl) ester, a Ca 2؉ channel blocker, Ca 2؉ ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca 2؉ levels, and peak Ca 2؉ responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca 2؉ storage compartment in cysts. The ability to sequester ions in membranebounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca 2؉ storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence. Thus, the basal bodies may be a cellular control center to coordinate the resumption of motility and cytokinesis in excystation.

Järn giver skärpa åt järn; så skärper den ena människan den andra.

Genome Biology, 2011

Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryo... more Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton.

Journal of Biological Chemistry, 1991

'Hand 14C-labeled species was released by nitrous acid deamination from ["Clpalmitate-and ['Hlmyo... more 'Hand 14C-labeled species was released by nitrous acid deamination from ["Clpalmitate-and ['Hlmyoinositol-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 [SH]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich 0. 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.

Infection and Immunity, 1992

The variability in duration and severity of infection with Giardia lamblia is likely to be due to... more 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...

Infection and Immunity, 1988

Bile salts and fatty acids stimulated differentiation of cultured Giardia lamblia trophozoites in... more 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.

Infection and Immunity, 1985

We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro ... more We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro and presented evidence that killing requires the bile salt-stimulated lipase of milk. Since this enzyme is activated by bile salts, killing should be dependent on the presence of bile salts. We now show that killing by fresh NHM or NHM stored at -70 degrees C is totally dependent on sodium cholate (a bile salt). With less than 0.4 mM cholate, no parasites were killed, whereas with 1 mM cholate, greater than 99.7% were killed by 5% NHM in 30 min. Moreover, killing activity was completely heat labile. The G. lamblia-killing activity of human milk was greatly altered by storage at -10 or -20 degrees C. In less than 23 days, the 50% lethal dose decreased, cholate dependence was lost, and killing activity became heat stable. In contrast, the activity of milk stored at -70 degrees C remained unchanged. Milk lipase activity, like killing activity, became cholate independent during storage at -1...

Infection and Immunity, 1989

We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are sp... more 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...

It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in... more It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in many areas of biomedical research. To do so, skeletal muscle samples are sectioned transversely to the length of the muscle and labeled for extracellular or membrane proteins to delineate the fiber boundaries and additionally for biomarkers related to function or metabolism. The samples are digitally photographed and the fibers "outlined" for quantification of fiber cross-sectional area (CSA) using pointing devices interfaced to a computer, which is tedious, prone to error, and can be nonobjective. Here, we review methods for characterizing skeletal muscle fibers and describe new automated techniques, which rapidly quantify CSA and biomarkers. We discuss the applications of these methods to the characterization of mitochondrial dysfunctions, which underlie a variety of human afflictions, and we present a novel approach, utilizing images from the online Human Protein Atlas to predict relationships between fiber-specific protein expression, function, and metabolism.

Proceedings of the National Academy of Sciences, 1990

To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we... more 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 ...

PLoS Pathogens, 2009

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genoty... more Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 166 coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus ,0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

PLoS ONE, 2006

Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infecti... more Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, ''HCNCp'' (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many ''CxxC'' tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple ''CxC'' motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp Cterminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing $20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.

Molecular and Biochemical Parasitology, 2011

Proteolytic activity is important in the lifecycles of parasites and their interactions with host... more Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Molecular and Biochemical Parasitology, 2011

Proteolytic activity is important in the lifecycles of parasites and their interactions with host... more Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Molecular and Biochemical Parasitology, 2008

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen i... more Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Molecular and Biochemical Parasitology, 2010

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Seri... more We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia.

Journal of Biological Chemistry, 2003

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection... more 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 leucinerich 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 ϳ50%. 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.

International Journal for Parasitology, 2011

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhea in humans. Its ... more Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterized by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signaling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localization in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localize with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localize to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signaling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.

Current Opinion in Microbiology, 2007

To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoit... more To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoites attach to enterocytes that mature and die. The parasites must 'decide' whether to re-attach or differentiate into cysts that survive in the environment and reactivate when ingested. Other intestinal parasites face similar challenges. Study of these parasites is limited because they do not encyst in vitro. Giardia trophozoites were persuaded to encyst in vitro by mimicking physiological stimuli.Cysts are dormant, yet 'spring-loaded for action' to excyst upon ingestion. Giardial encystation has been studied from morphological, cell biological, biochemical, and molecular viewpoints. Yet important gaps remain and the mechanisms that coordinate responses to external signals remain enigmatic.

Antimicrobial Agents and Chemotherapy, 1982

Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytic... more Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent.

Journal of Biological Chemistry, 2002

Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic di... more Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca 2؉ levels (1,2bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acidtetra(acetyloxymethyl) ester, a Ca 2؉ channel blocker, Ca 2؉ ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca 2؉ levels, and peak Ca 2؉ responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca 2؉ storage compartment in cysts. The ability to sequester ions in membranebounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca 2؉ storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence. Thus, the basal bodies may be a cellular control center to coordinate the resumption of motility and cytokinesis in excystation.

Järn giver skärpa åt järn; så skärper den ena människan den andra.

Genome Biology, 2011

Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryo... more Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton.

Journal of Biological Chemistry, 1991

'Hand 14C-labeled species was released by nitrous acid deamination from ["Clpalmitate-and ['Hlmyo... more 'Hand 14C-labeled species was released by nitrous acid deamination from ["Clpalmitate-and ['Hlmyoinositol-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 [SH]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich 0. 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.

Infection and Immunity, 1992

The variability in duration and severity of infection with Giardia lamblia is likely to be due to... more 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...

Infection and Immunity, 1988

Bile salts and fatty acids stimulated differentiation of cultured Giardia lamblia trophozoites in... more 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.

Infection and Immunity, 1985

We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro ... more We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro and presented evidence that killing requires the bile salt-stimulated lipase of milk. Since this enzyme is activated by bile salts, killing should be dependent on the presence of bile salts. We now show that killing by fresh NHM or NHM stored at -70 degrees C is totally dependent on sodium cholate (a bile salt). With less than 0.4 mM cholate, no parasites were killed, whereas with 1 mM cholate, greater than 99.7% were killed by 5% NHM in 30 min. Moreover, killing activity was completely heat labile. The G. lamblia-killing activity of human milk was greatly altered by storage at -10 or -20 degrees C. In less than 23 days, the 50% lethal dose decreased, cholate dependence was lost, and killing activity became heat stable. In contrast, the activity of milk stored at -70 degrees C remained unchanged. Milk lipase activity, like killing activity, became cholate independent during storage at -1...

Infection and Immunity, 1989

We induced Giardia lamblia trophozoites to encyst in vitro by exposure to conditions which are sp... more 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...

It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in... more It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in many areas of biomedical research. To do so, skeletal muscle samples are sectioned transversely to the length of the muscle and labeled for extracellular or membrane proteins to delineate the fiber boundaries and additionally for biomarkers related to function or metabolism. The samples are digitally photographed and the fibers "outlined" for quantification of fiber cross-sectional area (CSA) using pointing devices interfaced to a computer, which is tedious, prone to error, and can be nonobjective. Here, we review methods for characterizing skeletal muscle fibers and describe new automated techniques, which rapidly quantify CSA and biomarkers. We discuss the applications of these methods to the characterization of mitochondrial dysfunctions, which underlie a variety of human afflictions, and we present a novel approach, utilizing images from the online Human Protein Atlas to predict relationships between fiber-specific protein expression, function, and metabolism.

Proceedings of the National Academy of Sciences, 1990

To study the interactions between the parasitic protozoan Giardia lamblia and its environment, we... more 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 ...

PLoS Pathogens, 2009

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genoty... more Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 166 coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus ,0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

PLoS ONE, 2006

Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infecti... more Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, ''HCNCp'' (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many ''CxxC'' tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple ''CxC'' motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp Cterminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing $20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.

Molecular and Biochemical Parasitology, 2011

Proteolytic activity is important in the lifecycles of parasites and their interactions with host... more Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Molecular and Biochemical Parasitology, 2011

Proteolytic activity is important in the lifecycles of parasites and their interactions with host... more Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Molecular and Biochemical Parasitology, 2008

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen i... more Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Molecular and Biochemical Parasitology, 2010

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Seri... more We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia.

Journal of Biological Chemistry, 2003

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection... more 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 leucinerich 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 ϳ50%. 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.

International Journal for Parasitology, 2011

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhea in humans. Its ... more Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterized by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signaling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localization in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localize with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localize to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signaling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.

Current Opinion in Microbiology, 2007

To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoit... more To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoites attach to enterocytes that mature and die. The parasites must 'decide' whether to re-attach or differentiate into cysts that survive in the environment and reactivate when ingested. Other intestinal parasites face similar challenges. Study of these parasites is limited because they do not encyst in vitro. Giardia trophozoites were persuaded to encyst in vitro by mimicking physiological stimuli.Cysts are dormant, yet 'spring-loaded for action' to excyst upon ingestion. Giardial encystation has been studied from morphological, cell biological, biochemical, and molecular viewpoints. Yet important gaps remain and the mechanisms that coordinate responses to external signals remain enigmatic.

Antimicrobial Agents and Chemotherapy, 1982

Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytic... more Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent.