High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Similarity in cyst wall protein (CWP) trafficking between encysting Giardia duodenalis trophozoites and CWP-expressing human embryonic kidney-293 cells

Cyst wall proteins 1 and 2 (CWP1 and CWP2) are major constituents of the giardial cyst wall and are expressed with similar kinetics by encysting trophozoites. In the present study, we were interested to determine if the expression of giardial CWPs as heterologous proteins in a higher eukaryotic cell would result in their trafficking across the secretory pathway, as is the case in encysting trophozoites. Recombinant (r)CWP1 and rPro-CWP2 were detected in the lysate and culture media of transfected HEK-293 cells. We then conducted intracellular localization experiments using confocal microscopy and found that the proteins were trafficked in membrane enclosed vesicles across the secretory pathway and released to the culture medium by transfected HEK-293 cells. We then dissected the rCWP1 and rPro-CWP2 molecules to identify the portion(s) responsible for their secretion and found that the putative N-terminal signal peptide was sufficient for directing the secretion of rCWP1, while both the putative N-terminal signal peptide and the 13kDa C-terminal regions were necessary for the secretion of rPro-CWP2 by transfected HEK-293 cells. Taken together, these results demonstrate the degree of conservation of signal peptide recognition between lower and higher eukaryotes.     

Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation

To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.     

Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.     

Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote,Giardia lamblia. Stage-specific activation and intracellular localization

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.     

Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis

14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.     

Identification of antigens of Colombian Giardia duodenalis isolates recognized by total IgG and subclasses

Antigen profiles were described for Giardia duodenalis cysts and trophozoites that are recognized by IgG and its anti-G. dudodenalis subclasses (IgG1, IgG2, IgG3, IgG4). Antigens were identified by Western blot from G. duodenalis cyst and trophozoite isolates. Cysts and trophozoites were each subjected to protein separation by SDS-PAGE. The proteins were then transferred to nitrocellulose membranes by electroimmunoblot, and their antigenicity was determined by exposing them to sera from patients with confirmed diagnosis of G. duodenalis infection. The antigen-antibody reaction was revealed by specific alkaline phosphatase antibody conjugates against IgG, IgG2, IgG3, IgG4: bands were visualized by addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate and the stain nitro blue tetrazolium. The bands were read and analyzed by linear regression using Quantity One software. Thirty two antigens were simultaneously recognized by total IgG anti-G. duodenalis in the cyst and trophozoite stages. The antigens varied in molecular weight from 22 to 185 kDa. Nineteen antigens were identified by both IgG, and IgG3 anti-G duodenalis, with molecular weights ranging from 42 to 180 kDa. IgG2 and IgG4 did not identify any antigen in either stage. The antigens of molecular weights 27, 30, 31, 33, 45, 49, 57, 78, 89 and 170 kDa are shared with G. duodenalis isolates from other geographical regions of Colombia. The recognition of cyst and trophozoite antigens of Colombian G. duodenalis isolates by IgG, IgG1 and IgG3 anti-G. duodenalis suggested that they are involved in the induction of the host immune response.     

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.     

Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc₂), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.     

Respiration in the cysts and trophozoites of Giardia muris

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp

Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

Background  

Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.     

Mining the Giardia lamblia genome for new cyst wall proteins

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 leucine-rich 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 approximately 50%. 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.     

Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.