Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples.

Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.

'Disc-o-Fever': Getting Down with Giardia's Groovy Microtubule Organelle.

Protists have evolved a myriad of highly specialized cytoskeletal organelles that expand known functional capacities of microtubule (MT) polymers. One such innovation - the ventral disc - is a cup-shaped MT organelle that the parasite Giardia uses to attach to the small intestine of its host. The molecular mechanisms underlying the generation of suction-based forces by overall conformational changes of the disc remain unclear. The elaborate disc architecture is defined by novel proteins and complexes that decorate almost all disc MT protofilaments, and vary in composition and conformation along the length of the MTs. Future genetic, biochemical, and functional analyses of disc-associated proteins will be central toward understanding not only disc architecture and assembly, but also the overall disc conformational dynamics that promote attachment.

Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels.

Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n = 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples, 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 µl of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R(2) value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protist taxa.

Giardia intestinalis incorporates heme into cytosolic cytochrome b5.

The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated.

Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome.

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.

A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia.

Cyst walls of Entamoeba and Giardia protect them from environmental insults, stomach acids, and intestinal proteases. Each cyst wall contains a sugar homopolymer: chitin in Entamoeba and a unique N-acetylgalactosamine (GalNAc) homopolymer in Giardia. Entamoeba cyst wall proteins include Jacob lectins (carbohydrate-binding proteins) that crosslink chitin, chitinases that degrade chitin, and Jessie lectins that make walls impermeable. Giardia cyst wall proteins are also lectins that bind fibrils of the GalNAc homopolymer. Although many of the details remain to be determined for the cyst wall of Giardia, current data suggest a relatively simple fibril and lectin model for the Entamoeba cyst wall.

Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690.

Some aaRSs (aminoacyl-tRNA synthetases) develop editing mechanisms to correct mis-charged tRNA. The CP1 (connective peptide 1) domain of LeuRS (leucyl-tRNA synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of GlLeuRS (Giardia lamblia LeuRS) has been identified. Similar substitution with the ESI from HsLeuRS (Homo sapiens LeuRS) impeded the leucine activation, aminoacylation and post-transfer editing of the enzyme, but had no effect on the editing specificity toward non-specific amino acids. Thr341 in GlLeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was crucial for tRNALeu charging and the Asp440 was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg338 and Asp440 of GlLeuRS. Interestingly, GlLeuRS was completely resistant to the AN2690, which is an inhibitor of various LeuRSs. The universally conserved aspartate residue in the LeuRS CP1 domains was responsible for the resistance of GlLeuRS and another recently reported AN2690-resistant AaLeuRS (Aquifex aeolicus LeuRS). Our results indicate the functional divergence of some absolutely conserved sites, improve the understanding of the editing function of eukaryotic/archaeal LeuRSs and shed light on the development of a GlLeuRS-specific inhibitor for the treatment of giardiasis.

Proteomic analysis of Giardia: studies from the pre- and post-genomic era.

The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite. Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is, however, a large amount of future work needed to fully understand the proteome of this parasite.

The exosomes of trypanosomes and other protists.

The archaeal exosome contains three heterodimeric RNase PH subunits, forming a hexamer with RNase activity; on top sits a trimer of two different SI domain proteins. In animals and yeast, six different, but related subunits form the RNase PH-like core, but these lack enzyme activity; there are three different SI-domain proteins and enzyme activity is providedby the endo/exonuclease Rrp44 or--mainly in the nuclear exosome--the Rnase D enzyme Rrp6. Trypanosomes diverged from yeast and mammals very early in eukaryotic evolution. The trypanosome exosome is similar in subunit composition to the human exosome, but instead of being an optional component, trypanosome RRP6 is present in the nucleus and cytoplasm and is required for exosome stability. As in human cells and yeast, the trypanosome exosome has been shown to be required for processing and quality control of rRNA and to be involved in mRNA degradation. Electron microscopy results for a Leishmania exosome suggest that RRP6 is located on the side of the RnasePH ring, interacting with several exosome core proteins. Results of a search for exosome subunits in the genomes of widely diverged protists revealed varied exosome complexity; the Giardia exosome may be as simple as that of Archaea.

Alpha 2 giardin is an assemblage A-specific protein of human infective Giardia duodenalis.

Of the 7 genetic assemblages of the parasite Giardia duodenalis only 2 (A and B) are known to cause infections in humans. These assemblages have been characterized in detail at the genomic level but few studies have examined differences in the proteins expressed. Employing one and two-dimensional PAGE we have identified an assemblage A-specific protein of human infective G. duodenalis; alpha 2 giardin. The protein difference was evident using both electrophoretic techniques. Alpha 2 giardin is known to be a structural protein and associates with the caudal flagella and the plasma membrane; however, its exact function is unknown. Although several proteins unique to assemblage B were also observed, we were unable to identify these proteins due to a lack of genomic data available for assemblage B isolates. Together, these proteins represent distinct phenotypic differences between the human infective assemblages of G. duodenalis and support the need to revise the taxonomy of this parasite.

Characterization of the N-linked glycans of Giardia intestinalis.

This article reports the first rigorous evidence for the existence of N-glycans in Giardia intestinalis, a parasite that is a widespread human pathogen, being a major cause of enteric disease in the world. Excreted/secreted molecules of G. intestinalis are known to stimulate the immune system. Structural strategies based on MALDI and electrospray mass spectrometry were employed to examine the excreted/secreted molecules for their N-glycan content. These revealed that the major oligosaccharides released by peptide N-glycosidase F are complex-type structures and correspond to bi-, and triantennary structures without core (alpha1,6) fucosylation. The major nonreducing epitopes in these complex-type glycans are: Galbeta1-4GlcNAc (LacNAc) and NeuAc alpha2-6Galbeta1-4GlcNAc (sialylated LacNAc).

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains.

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Primary structure and biochemical properties of a variant-specific surface protein of Giardia.

Trophozoites of Giardia duodenalis express at their cell surface variant-specific proteins (VSPs) that are believed to contribute to the protection of the parasite from immunological and other host defense mechanisms. In the present study, we have cloned and characterized the gene encoding a VSP (VSP4A1, originally designated CRISP-90) that is expressed by the sheep-derived Giardia variant clone O2-4A1. The gene was isolated by probing a genomic library with a near-full-length gene-specific polymerase chain reaction (PCR) product. The VSP4A1 gene specifies a 70729 Da protein with features common to all previously reported VSPs, including a high cysteine and threonine content, a highly conserved hydrophobic carboxy-terminal domain and little similarity in the remaining polypeptide sequence. Comparison of the predicted sequence with the amino-terminal sequence of purified VSP4A1 revealed the absence of an amino-terminal hydrophobic extension from the mature protein. VSP4A1 purified from the O2-4A1 variant clone was found to undergo conformational changes resulting in the formation of two additional electrophoretic species. Free thiol groups were not detected in purified VSP4A1, indicating that all cysteine residues may be involved in disulphide crosslinking. Possibly as a consequence of this VSP4A1 was found to be fairly resistant to proteolytic digestion. Although VSP4A1 is able to bind zinc following blotting to a nitrocellulose membrane, other analyses with both the purified and cell associated VSP have failed to confirm significant zinc ion binding to this protein. The latter result questions the assumption previously made by other authors that zinc binding to VSPs constitutes an important structural and functional aspect of these proteins.

Presence and localization of vinculin in Giardia.

A requisite element of pathogenicity in Giardia infections is the parasites' ability to adhere to the intestinal epithelial brush border. The presence of vinculin in Giardia was studied because this protein is known to link the cytoskeleton to the plasma membrane and is localized at adhesion foci in many cell-cell and cell-substrate contact sites. Actin, alpha-actinin, and vinculin were identified in Giardia by western blot analysis. Giardia trophozoites attached to glass substrates were examined by interference reflection microscopy (IRM) and immunofluorescence. The IRM defined the lateral crest, bare area, and overlap region of the ventral disk, as well as the ventrolateral flange and lateral shields as close contact areas between parasite and substrate. These close contact regions were then correlated with immunofluorescence localization of actin, alpha-actinin, and vinculin. Actin was seen in the lateral crest, while alpha-actinin was observed in the ventral disc periphery and lateral shields. Vinculin was viewed at the bare and overlap areas of the ventral disc and portions of the lateral crest, as well as the ventrolateral flange and lateral shields. The correspondence of close contact sites with vinculin localization suggests a role for vinculin in Giardia attachment and adherence.

The 3' terminal region of a gene encoding a cysteine-rich surface protein in Giardia duodenalis.

DNA derived from chromosome band 3 of the cloned Giardia duodenalis line, WB-1B was used to construct a cloned library in E. coli. One of these clones, C3/23, has been identified as the 3' coding region of a G. duodenalis cysteine-rich variable surface protein (CRVSP) gene by homology with other published CRVSPs and also contains 720 bp of the 3' flanking region. The sequence of C3/23, was derived from genomic DNA independently of cDNA, or expression copies of the CRVSP genes. The 3' flanking region is not homologous to the 3' untranslated regions of published CRVSPs which probably reflects its genomic origin. Subclones of C3/23 were used to show that the 3' flanking region was conserved in all strains examined in this study and was repeated many times in the genome. The 3' flanking repeats were located on three chromosome bands and were not always associated with the coding sequence of C3/23 which was represented, although not equally, on all chromosome bands. The highly conserved nature of the 3' flanking region and its multiple representation in the genome emphasize the probable role of this sequence in the localization or regulation of expression of the CRVSPs in G. duodenalis.