Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis.

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 um in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened crescent or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Disc-associated proteins mediate the unusual hyperstability of the ventral disc in Giardia lamblia.

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The 'cup-like' disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains - DAP5188KD and DAP6751KD -with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

Microtubule organelles in Giardia.

Giardia lamblia is a widespread parasitic protist with a complex MT cytoskeleton that is critical for motility, attachment, mitosis and cell division, and transitions between its two life cycle stages-the infectious cyst and flagellated trophozoite. Giardia trophozoites have both highly dynamic and highly stable MT organelles, including the ventral disc, eight flagella, the median body and the funis. The ventral disc, an elaborate MT organelle, is essential for the parasite's attachment to the intestinal villi to avoid peristalsis. Giardia's four flagellar pairs enable swimming motility and may also promote attachment. They are maintained at different equilibrium lengths and are distinguished by their long cytoplasmic regions and novel extra-axonemal structures. The functions of the median body and funis, MT organelles unique to Giardia, remain less understood. In addition to conserved MT-associated proteins, the genome is enriched in ankyrins, NEKs, and novel hypothetical proteins that also associate with the MT cytoskeleton. High-resolution ultrastructural imaging and a current inventory of more than 300 proteins associated with Giardia's MT cytoskeleton lay the groundwork for future mechanistic analyses of parasite attachment to the host, motility, cell division, and encystation/excystation. Giardia's unique MT organelles exemplify the capacity of MT polymers to generate intricate structures that are diverse in both form and function. Thus, beyond its relevance to pathogenesis, the study of Giardia's MT cytoskeleton informs basic cytoskeletal biology and cellular evolution. With the availability of new molecular genetic tools to disrupt gene function, we anticipate a new era of cytoskeletal discovery in Giardia.

Recent advances in functional research in Giardia intestinalis.

This review considers current advances in tools to investigate the functional biology of Giardia, it's coding and non-coding genes, features and cellular and molecular biology. We consider major gaps in current knowledge of the parasite and discuss the present state-of-the-art in its in vivo and in vitro cultivation. Advances in in silico tools, including for the modelling non-coding RNAs and genomic elements, as well as detailed exploration of coding genes through inferred homology to model organisms, have provided significant, primary level insight. Improved methods to model the three-dimensional structure of proteins offer new insights into their function, and binding interactions with ligands, other proteins or precursor drugs, and offer substantial opportunities to prioritise proteins for further study and experimentation. These approaches can be supplemented by the growing and highly accessible arsenal of systems-based methods now being applied to Giardia, led by genomic, transcriptomic and proteomic methods, but rapidly incorporating advanced tools for detection of real-time transcription, evaluation of chromatin states and direct measurement of macromolecular complexes. Methods to directly interrogate and perturb gene function have made major leaps in recent years, with CRISPr-interference now available. These approaches, coupled with protein over-expression, fluorescent labelling and in vitro and in vivo imaging, are set to revolutionize the field and herald an exciting time during which the field may finally realise Giardia's long proposed potential as a model parasite and eukaryote.

'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.

Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine.

Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology.

Genetic analysis reveals the identity of the photoreceptor for phototaxis in hormogonium filaments of Nostoc punctiforme.

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.

Novel structural components of the ventral disc and lateral crest in Giardia intestinalis.

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.

Mapping the protistan 'rare biosphere'.

The use of cultivation-independent approaches to map microbial diversity, including recent work published in BMC Biology, has now shown that protists, like bacteria/archaea, are much more diverse than had been realized. Uncovering eukaryotic diversity may now be limited not by access to samples or cost but rather by the availability of full-length reference sequence data.