Chapter Three - Recent advances in functional research in Giardia intestinalis

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Abstract

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.

Introduction

Undertaking comprehensive functional studies has remained a persistent obstacle in parasitological research, outside of a small number of apicomplexans, such as species of Plasmodium and Toxoplasma gondii (Limenitakis and Soldati-Favre, 2011; Meissner et al., 2007). Primary obstacles to genetically tractable parasites include an inability to readily culture them outside of a host, a lack of knowledge of the genetic and regulatory systems of parasites or the unavailability of tools to apply to them. Giardia intestinalis has been in vitro culturable for several decades in complex media, principally Keister's modified TYI-S-33 (Davids and Gillin, 2011) and its regulatory genetics and gene composition have been explored through the publication of reference genomes (Franzen et al., 2009; Jerlstrom-Hultqvist et al., 2010; Morrison et al., 2007) and other “omics” driven research over the past decade (e.g., Ansell et al., 2015a, Ansell et al., 2017; Emery et al., 2018; Franzén et al., 2013; Ma'ayeh et al., 2018; Spycher et al., 2013), albeit with much still to be done. The complex binucleate and multiploidal (2N, 4N and 8N at various stages of the life cycle; Bernander et al., 2001) cellular biology of Giardia has proven a persistent obstacle to functional research, and for much of its post-genomic period efforts to develop functional tools for Giardia have met with limited success (Luján and Svärd, 2011). Because of its importance as a parasite, its scalable culturability in cell-free media, and its deep-branching position within the eukaryotic tree of life (Morrison et al., 2007), Giardia has long been proposed as an intriguing and potentially impactful model organism (Luján and Svärd, 2011), but its recalcitrance to genetic manipulation has proven a persistent road-block to realising this potential.

Here, we review advances in functional research of Giardia, drawing on recent publications in the field, as well as novel advances in other fields that may be applicable for Giardia. Our hope is that this review will act not only as a summary of the progress in research in this field over the last few years, but also as stimulus for renewed thinking on functional research in Giardia and on the potential to exploit these technologies for novel anti-giardial therapies, but to harness this fascinating protist as a model for eukaryotic biology.

Section snippets

Advances in in vitro cultivation

The inability to culture parasites in a laboratory setting, either in vitro or in vivo, presents the primary obstacle to advancing functional research. Fortunately, this obstacle has been overcome for Giardia for several decades. In vitro cultivation of G. intestinalis is readily undertaken in TYI-S-33 media (Davids and Gillin, 2011), with or without nitrogen-sparging for culture-adapted strains isolates. Giardia intestinalis trophozoites (at least of the assemblage A) can be triggered into in

Major knowledge gaps in functional biology in Giardia

Draft genomes have been available for Giardia intestinalis assemblage A (WB: Morrison et al., 2007), B (GS: Franzen et al., 2009) and E (P15: Jerlstrom-Hultqvist et al., 2010) for a number of years. These assemblies have provided the basis for predicting Giardia's ~ 5500 coding gene models. Recent efforts (see current release in GiardiaDB) to curate these models have significantly improved their functional annotation; yet, ~ 60% of these are defined as “conserved” or “hypothetical” proteins.

Computational and “omic” approaches to functional biology research in Giardia

It is clear that the next major breakthroughs in our understanding of the molecular biology of Giardia must make full use of advances in functional research. Conceptually, these advances can be categorized into three major and complimentary themes: (i) improved in silico inference, (ii) direct empirical assessment of function through “omics”, labelling and other approaches and (iii) direct empirical assessment of function through targeted genetic manipulation.

Direct, targeted-based assessment of gene function

As most parasites are unculturable and genetically intractable, much of the functional biology is understood through in-direct inference by in silico modelling and, more recently, “omics” and systems-based research. These are powerful tools, but are most valuable when they are used to guide empirical interrogation of gene function through direct, target-based studies. For many years, Giardia was proposed as a model parasite because of its ready in vitro cultivation, but significant challenges

Concluding remarks

Giardia has proven a tantalizingly close but frustratingly distant model eukaryote and genetically tractable parasitic species. Its ready culturability and early discoveries in its regulatory biology, as well as its status as an early diverging branch of the eukaryotic tree and cause of significant disease in humans and animals, has piqued interest in the species for many years. However, its binucleate cell, tetraploid genome and limited acceptance of seemingly attractive genetic tools, such as

Acknowledgements

A.J. acknowledges funding support from the Australian National Health and Medical Research Council (Career Development Fellowship APP1126395), Melbourne Water and the Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme.

References (204)

  • L. Favennec et al.

    Cytopathogenic effect of Giardia intestinalis, in vitro

    Parasitol. Today

    (1991)
  • M.Y. Fink et al.

    The intersection of immune responses, microbiota, and pathogenesis in giardiasis

    Trends Parasitol.

    (2017)
  • P.R. Gargantini et al.

    Antigenic variation in the intestinal parasite Giardia lamblia

    Curr. Opin. Microbiol.

    (2016)
  • L.A. Gilbert et al.

    CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes

    Cell

    (2013)
  • K. Hanevik et al.

    Whole genome sequencing of clinical isolates of Giardia lamblia

    Clin. Microbiol. Infect.

    (2015)
  • L.M. Iyer et al.

    Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes

    Int. J. Parasitol.

    (2008)
  • G. Jeelani et al.

    Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cysteine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica

    J. Biol. Chem.

    (2010)
  • D. Kaczmarzyk et al.

    Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX

    Metab. Eng.

    (2018)
  • R.D. Adam

    Biology of Giardia lamblia

    Clin. Microbiol. Rev.

    (2001)
  • R.D. Adam et al.

    Genome sequencing of Giardia lamblia genotypes A2 and B isolates (DH and GS) and comparative analysis with the genomes of genotypes A1 and E (WB and Pig)

    Genome Biol. Evol.

    (2013)
  • A. Aggarwal et al.

    Comparison of two antigenically distinct Giardia lamblia isolates in gerbils

    Am. J. Trop. Med. Hyg.

    (1987)
  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • N. Andreu et al.

    Noninvasive biophotonic imaging for studies of infectious disease

    FEMS Microbiol. Rev.

    (2011)
  • J. Ankarklev et al.

    Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage a isolates

    BMC Genomics

    (2015)
  • B.R. Ansell et al.

    Time-dependent transcriptional changes in axenic Giardia duodenalis trophozoites

    PLoS Negl. Trop. Dis.

    (2015)
  • B.R. Ansell et al.

    Divergent transcriptional responses to physiological and xenobiotic stress in Giardia duodenalis

    Antimicrob. Agents Chemother.

    (2016)
  • B.R. Ansell et al.

    Transcriptomics indicates active and passive metronidazole resistance mechanisms in three seminal Giardia lines

    Front. Microbiol.

    (2017)
  • B.R.E. Ansell et al.

    Annotation of the Giardia proteome through structure-based homology and machine learning

    Gigascience

    (2019)
  • T.L. Bailey

    DREME: motif discovery in transcription factor ChIP-seq data

    Bioinformatics

    (2011)
  • N. Barash et al.

    Giardia colonizes and encysts in high density foci in the murine small intestine

    mSphere

    (2017)
  • L.A. Bartelt et al.

    Advances in understanding Giardia: determinants and mechanisms of chronic sequelae

    F1000Prime Rep.

    (2015)
  • L.A. Bartelt et al.

    Persistent G. lamblia impairs growth in a murine malnutrition model

    J. Clin. Invest.

    (2013)
  • V.M. Bedell et al.

    In vivo genome editing using a high-efficiency TALEN system

    Nature

    (2012)
  • Benchling

    Biology Software

  • G. Benson

    Tandem repeats finder: a program to analyze DNA sequences

    Nucleic Acids Res.

    (1999)
  • H.M. Berman et al.

    The protein data bank

    Nucleic Acids Res.

    (2000)
  • R. Bernander et al.

    Genome ploidy in different stages of the Giardia lamblia life cycle

    Cell. Microbiol.

    (2001)
  • A. Buret et al.

    Genotypic characterization of an epithelial cell line for the study of parasite-epithelial interactions

    J. Parasitol.

    (2008)
  • A.G. Buret et al.

    Giardia lamblia disrupts tight junctional ZO-1 and increases permeability in non-transformed human small intestinal epithelial monolayers: effects of epidermal growth factor

    Parasitology

    (2002)
  • L.G. Byrd et al.

    Giardia lamblia infections in adult mice

    Infect. Immun.

    (1994)
  • L. Cevenini et al.

    Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites

    Anal. Chem.

    (2014)
  • X.S. Chen et al.

    High throughput genome-wide survey of small RNAs from the parasitic protists Giardia intestinalis and Trichomonas vaginalis

    Genome Biol. Evol.

    (2009)
  • Y. Chen

    In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses

    PLoS One

    (2017)
  • K.J. Clark et al.

    A TALE of two nucleases: gene targeting for the masses?

    Zebrafish

    (2011)
  • C.H. Contag et al.

    Photonic detection of bacterial pathogens in living hosts

    Mol. Microbiol.

    (1995)
  • J.A. Cotton et al.

    Disruptions of host immunity and inflammation by Giardia duodenalis: potential consequences for co-infections in the gastro-intestinal tract

    Pathogens

    (2015)
  • M. Dan et al.

    Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme

    Mol. Microbiol.

    (2000)
  • S.M. Dann et al.

    Giardia infection of the small intestine induces chronic colitis in genetically susceptible hosts

    J. Immunol.

    (2018)
  • S. D'Archivio et al.

    Non-invasive in vivo study of the Trypanosoma vivax infectious process consolidates the brain commitment in late infections

    PLoS Negl. Trop. Dis.

    (2013)
  • B.J. Davids et al.

    Methods for Giardia culture, cryopreservation, encystation, and excystation in vitro

  • Cited by (0)

    Equal contribution.

    View full text