Human milk kills parasitic intestinal protozoa.

Giardia lamblia, a common pathogenic intestinal parasite of humans, was rapidly killed by exposure to normal human milk in vitro. The killing did not depend on secretory immunoglobulin A. Entamoeba histolytica, the dysentery amoeba, was also killed by normal human milk. Giardia-cidal activity cochromatographed with an unusual lipase that is present in the milk of humans but not of lower mammals. Human milk may play a protective role in infants exposed to this parasite.

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

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

Mechanism of killing of Giardia lamblia trophozoites by complement.

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

Attachment of the flagellate Giardia lamblia: role of reducing agents, serum, temperature, and ionic composition.

The flagellated protozoan Giardia lamblia has been grown only in highly complex media under reduced oxygen tension. Therefore, the organic and physiological requirements for in vitro attachment and short-term (12-h) survival of this organism were determined. In defined maintenance media, a thiol reducing agent (e.g., cysteine) was absolutely required for attachment and survival of this aerotolerant anaerobe. The crude bovine serum Cohn III fraction greatly stimulated attachment and survival. Attachment was decreased at a reduced temperature (24 degrees C as compared with 35.5 degrees C) and absent at 12 degrees C or below. Attachment and survival were strongly dependent upon pH and ionic strength, with optima at pH 6.85 to 7.0 and 200 to 300 mosmol/kg. Sodium chloride was better tolerated than KC1. Reduction of Ca2+ and Mg2+ to below 10(-8) M did not significantly affect attachment.

8-Azaguanine resistance in mammalian cells. I. Hypoxanthine-guanine phosphoribosyltransferase.

Chinese hamster cells were treated with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, and mutants resistant to 8-azaguanine were selected and characterized. Hypoxanthine-guanine phosphoribosyltransferase activity of sixteen mutants is extremely negative, making them suitable for reversion to HGPRTase(+). Ten of the extremely negative mutants revert at a frequency higher than 10(-7) suggesting their point mutational character. The remaining mutants have demonstrable HGPRTase activity and are not useful for reversion analysis. Five of these mutants have < 2% HGPRTase and are presumably also HGPRTase point mutants. The remaining 14 mutants utilize exogenous hypoxanthine for nucleic acid synthesis poorly, and possess 20-150% of wild-type HGPRTase activity in in vitro. Their mechanism of 8-azaguanine resistance is not yet defined.

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

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

Dependence of Giardia lamblia encystation on novel transglutaminase activity.

Earlier, we found that three protein disulfide isomerases (PDI) from Giardia lamblia (gPDI) also have transglutaminase (TGase) activity in vitro. We now show that differentiating Giardia cells contain isopeptide bonds (epsilon(gamma-glutamyl)lysine), the biological product of TGase activity that results in irreversible crosslinking of proteins in vivo. HPLC analyses showed the highest isopeptide bond content in cells encysting for 21 h, indicating an important role for TGase early in encystation. We were not able to detect isopeptide bonds in water-resistant cysts, possibly because they could not be extracted. One of the hallmarks of early encystation is the formation of encystation secretory vesicles (ESV) that transport nascent cyst wall proteins (CWPs) to the outer cell surface. ImmunoEM and live-cell immunofluorescence assays of encysting parasites revealed that gPDIs 1-3 are located in ESV and that gPDI-2 is also novel in that it is localized on the cell surface. Cystamine, a widely used TGase inhibitor, caused a dose-dependent inhibition of ESV formation by 21 h, thereby preventing development of trophozoites into cysts. Since cystamine (0.5-1 mM) inhibited the TGase activity of recombinant gPDIs 1-3 in vitro, PDIs appear to be the physiologic targets of cystamine. We found that when parasites were treated with cystamine, CWPs were not processed normally. These data suggest that TGase-catalyzed reactions may be needed for either the machinery that processes CWP precursors or their recruitment to ESV.

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.

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.

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.

A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia.

The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Effects of oxygen tension and reducing agents on sensitivity of Giardia lamblia to metronidazole in vitro.

The effects of oxygen tension and reducing agents upon the sensitivity of Giardia lamblia to metronidazole in vitro were determined using two different assays. First, the parasites were exposed to drug under high or low oxygen tension in liquid medium with or without fresh cysteine and ascorbic acid. Survival was determined by colony assay. Second, the effect of thiol reducing agents was measured directly by colony assay in semi-solid media (low pO2 conditions). Trophozoites were strikingly (greater than 100-fold) less sensitive to metronidazole under the aerobic condition of the first assay. In contrast, the reducing agents had much lesser effects (2- to 5-fold) depending upon the assay. Finally, G. lamblia isolated from a patient treated with metronidazole unsuccessfully four times showed similar metronidazole sensitivity to a standard strain under both aerobic and reduced O2 tension conditions.

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.

A new method for purification of Giardia lamblia cysts.

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

Tolerance of axenically cultured Entamoeba histolytica and Giardia lamblia to a variety of antimicrobial agents.

Twelve antimicrobial agents were tested for use in axenic cultures of Entamoeba histolytica and Giardia lamblia. Ten did not inhibit parasite growth at concentrations of at least two-fold above the effective antibacterial or antifungal levels. We have found the combination of piperacillin (500 micrograms/ml) plus amikacin (125 micrograms/ml) to be useful in axenization of a new strain of E. histolytica, eradication of multiply drug-resistant contaminants in cultures and prevention of bacterial growth in experiments with non-sterile components.

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.

An atypical proprotein convertase in Giardia lamblia differentiation.

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 homologs 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 1mM 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.