Pathogenesis and post-infectious complications in giardiasis

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Pathogenesis and post-infectious complications in giardiasis Thibault Allain, Andre G. Buret∗ University of Calgary, Host-Parasite Interactions Program, Inflammation Research Network, Department of Biological Sciences, Calgary, Canada ∗ Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Pathophysiology and pathogenesis 2.1 Intestinal pathophysiology: Mucosal alterations 3. Interactions between Giardia and the gut microbiota 4. Parasite virulence factors and immune evasion strategies 5. Polymicrobial infections 6. Extraintestinal and post-infectious complications 6.1 Gastrointestinal sequelae of giardiasis and post-infectious IBS 7. Conclusions References

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Abstract Giardia is an important cause of diarrhoea, and results in post-infectious and extraintestinal complications. This chapter presents a state-of-the art of our understanding of how this parasite may cause such abnormalities, which appear to develop at least in part in Assemblage-dependent manner. Findings from prospective longitudinal cohort studies indicate that Giardia is one of the four most prevalent enteropathogens in early life, and represents a risk factor for stunting at 2 years of age. This may occur independently of diarrheal disease, in strong support of the pathophysiological significance of the intestinal abnormalities induced by this parasite. These include epithelial malabsorption and maldigestion, increased transit, mucus depletion, and disruptions of the commensal microbiota. Giardia increases epithelial permeability and facilitates the invasion of gut bacteria. Loss of intestinal barrier function is at the core of the acute and post-infectious complications associated with this infection. Recent findings demonstrate that the majority of the pathophysiological responses triggered by this parasite can be recapitulated by the effects of its membrane-bound and secreted cysteine proteases.

Advances in Parasitology ISSN 0065-308X https://doi.org/10.1016/bs.apar.2019.12.001

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1. Introduction Giardia intestinalis (synonymous G. duodenalis, G. lamblia) is an extracellular protozoan parasite of the gut in a variety of animal species and humans. This binucleated microaerophilic parasite belongs to the phylum Diplomonadida. G. intestinalis is responsible for over 280 human infections each year world-wide, in high-income countries as well as in places with poor sanitation (Ankarklev et al., 2010; Cotton et al., 2011). The organism exists in eight different genotypes, or Assemblages, from A to H, A and B being infectious to humans as well as other animal species (Ankarklev et al., 2010; Bartelt and Sartor, 2015; Cotton et al., 2011). Assemblage E has a predilection for infection in livestock, where it may impair weight gain as it does in children (Geurden et al., 2012; Olson et al., 1995). It has been well established that Giardia can be transmitted between humans and animals (Buret et al., 1990a; Dixon et al., 2011; Geurden et al., 2010, 2012; Olson et al., 1995; Ryan and Caccio, 2013). The infection appears to be endemic in countries with low income and poor sanitation, where it has been implicated in the failure to thrive and growth retardation in children (Bartelt and Sartor, 2015; Muhsen and Levine, 2012). In developing countries, the prevalence of human giardiasis may range from 20% to 30%, with reports of 100% prevalence in some studies; in high-income countries, the prevalence may range from 3% to 10% (Ankarklev et al., 2010; Bartelt and Sartor, 2015; Cotton et al., 2011; Jensen et al., 2009; Muhsen and Levine, 2012). These observations instigated the inclusion of giardiasis in the World Health Organization’s “neglected disease initiative” (Bartelt and Sartor, 2015; Muhsen and Levine, 2012; Savioli et al., 2006). Most often, the infection spreads between species via contaminated water or faecal-oral ingestion of the infective tetranucleated cysts. Giardia trophozoites preferentially colonize the duodenum and jejunum, and hence must survive in an intestinal environment rich in host digestive enzymes and bile. Membrane variant surface proteins coat the parasite and protect it against biochemical and host immune attack (Ankarklev et al., 2010; Singer et al., 2001; Touz et al., 2018). The Giardia genome has a minimal metabolic pathway system which may reflect its obligate parasitic life cycle as well as its early evolutionary position (Morrison et al., 2007). Giardia causes disease without invading the intestinal tissues, although isolated reports suggest that Giardia trophozoites, the motile dividing form of the parasite, may, albeit rarely, be found in subepithelial spaces (ReynosoRobles et al., 2015). Most infections are self-limiting; however, re-infection and chronic infections may occur (Roxstrom-Lindquist et al., 2006).

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Giardiasis causes a broad spectrum of symptoms ranging from no symptom at all, to severe, acute or chronic diarrhoea, bloating, nausea, and abdominal pain. This wide symptom variability has led some to suggest that Giardia may act both as a pathogen and a commensal microorganism (Ankarklev et al., 2010; Bartelt and Platts-Mills, 2016; Bartelt and Sartor, 2015; Cotton et al., 2011; DuPont, 2013). Regardless, the parasite can cause disease during the acute phase of the infection, as well as post-infectiously, where it presents as post-infectious irritable bowel syndrome and chronic fatigue (Hanevik et al., 2014; Robertson et al., 2010). Moreover, giardiasis can lead to a variety of extra-intestinal complications, including lower cognitive function, ocular pathologies, arthritis, allergies, hypokalaemic myopathy, and has even been associated with cancer (Halliez and Buret, 2013). Protein, vitamin A and zinc deficiencies, which are common in malnourished children, may be a predisposition to Giardia infection (Bartelt et al., 2013; Rahman et al., 2001). In turn, Giardia may impair zinc uptake, and make vitamin supplementation ineffective (Abou-Shady et al., 2011; Astiazaran-Garcia et al., 2010). As a result, several investigations have attempted to assess the association of giardiasis with growth impairment and stunting in young children. Findings from prospective longitudinal cohort studies indicate that giardiasis in early life was indeed a risk factor for stunting at 2 years of age (Donowitz et al., 2016; Rogawski et al., 2017). In these large children cohorts, G. intestinalis was the fourth most prevalent enteropathogen in the first 12 months of life, and the second most prevalent in the second year of life (Platts-Mills et al., 2015). During the acute stage of infection, Giardia disrupts the triple intestinal barrier constituted by the microbiome, the mucus, and the epithelial lining, which in turn launches the pathophysiological processes ultimately responsible for the disease (Allain et al., 2017a). Despite the ever-increasing number of research findings on the host-parasite interactions during giardiasis, our understanding of the pathogenesis of giardiasis, and the biological mechanisms explaining its diverse symptomatology remains incomplete. Recent findings suggest that the parasite has potent immunomodulatory effects which may protect against diarrhoea caused by concurrent enteropathogens. These enteropathogens drive diarrhoea via inflammatory events in the gut; this may at least in part explain why Giardia appears to “protect” against diarrheal symptoms in areas of the world where giardiasis, as well as polymicrobial infections, are endemic (Astiazaran-Garcia et al., 2010; Cotton et al., 2015; Muhsen and Levine, 2012; Muhsen et al., 2014; Veenemans et al., 2011). G. intestinalis is also the most commonly reported cause of traveller’s diarrhoea (Ross et al., 2013).

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This chapter offers a state-of-the-art update on our understanding of the pathogenesis of acute and post-infectious disease induced by G. intestinalis.

2. Pathophysiology and pathogenesis Pathophysiology in the acute phase of giardiasis occurs in the absence of invasion of the intestinal tissues by the trophozoites, and the absence of any overt inflammatory cell infiltration with the exception of a modest increase in intraepithelial lymphocytes and mast cells. These cells may be activated via Giardia arginine deiminase or through its metabolic product citrulline (Buret et al., 1992; Cotton et al., 2015; Li et al., 2004; MunozCruz et al., 2018; Tako et al., 2013). Most often, clinical manifestations of giardiasis appear 1–2 weeks after infection. Similar to most infectious diseases, a variety of host and environmental factors modulate disease outcome in giardiasis. These factors include diet, immune factors, age, the gut microbiome, as well as concurrent infections (Ankarklev et al., 2010; Barash et al., 2017; Bartelt and Platts-Mills, 2016; Bartelt and Sartor, 2015; Beatty et al., 2017; Cotton et al., 2011; Halliez and Buret, 2013; Manko et al., 2017a; Roxstrom-Lindquist et al., 2006). Diarrheal symptoms mostly occur during the acute phase of the infection, and at least some of the effects of the infection appear to be isolate-dependent (Allain et al., 2017a; Cotton et al., 2015; Robertson et al., 2010).

2.1 Intestinal pathophysiology: Mucosal alterations The sequence of events leading to diarrhoea in giardiasis includes the induction by the parasite of enterocyte apoptosis (Chin et al., 2002; Panaro et al., 2007), increased intestinal permeability (Scott et al., 2002; Troeger et al., 2007), CD-8+ lymphocyte-dependent brush-border microvillous shortening coinciding or not with villous atrophy and disaccharidase deficiencies (Buret et al., 1990b, 1991, 1992; Cevallos et al., 1995; Chavez et al., 1995; Gorowara et al., 1992; Koot et al., 2009; Scott et al., 2004; SolaymaniMohammadi and Singer, 2013). These effects combine with small intestinal malabsorption of electrolytes, water, and nutrients, anion hypersecretion, and elevated intestinal transit, to produce diarrhoea (Buret et al., 1992; Deselliers et al., 1997; Troeger et al., 2007). Recent observations also indicate that consumption of host arginine by the parasite may further contribute to pathophysiology by impairing enterocyte cell cycle and proliferation (Stadelmann et al., 2013). Together, these processes are implicated at least in part in the growth retardation caused by giardiasis, whether associated

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with diarrhoea or not (Buret et al., 1990b; Kosek and MAL-ED Network Investigators, 2017; Muhsen and Levine, 2012; Rogawski et al., 2017). Caspase-3 dependent disruptions of tight junctional proteins and loss of epithelial barrier function appear to represent a shared pathophysiological mechanism among a variety of gut disorders, as they may be triggered by other enteropathogens, by bacterial lipopolysaccharide, via proteinaseactivated receptors, or by anoxia (Buret and Bhargava, 2014; Chin et al., 2003, 2006; O’Hara and Buret, 2008; Zehendner et al., 2011). The effects are sensitive to myosin light-chain kinase inhibitors and may be blocked by SGLT1-mediated glucose uptake through an innate cellular rescue process, or via luminal epidermal growth factor (Buret et al., 2002; Chen et al., 2013; Scott et al., 2002; Yu et al., 2005, 2006, 2008). Moreover, mixed infections with different Giardia Assemblages may worsen enterocyte apoptosis and tight junctional disruptions (Koh et al., 2013). Cysteine protease-dependent degradation of villin, a key epithelial cytoskeletal actin-binding protein, also occurs upon attachment to intestinal epithelial cells, concomitantly with activation of myosin light-chain kinases in the structure of brush-border microvilli (Bhargava et al., 2015). Villin breakdown contributes, at least in part, to a loss of epithelial brush-border microvilli integrity. Intestinal permeability plays a critical role in the maintenance of homeostasis and health. Epithelial junctional complexes (EJC) control the transepithelial passage of luminal toxins, microorganisms, nutrient, and antigens. These complexes are arranged as transmembrane proteins such as claudins (e.g. claudin-1, -4) and occludins as cytosolic anchor proteins like the Zonula Occludens family (e.g. ZO-1), or regulatory proteins like F-actin and alpha-actinin. EJC are in turn functionally regulated by a myosin light-chain kinase-controlled cellular actomyosin ring. Therefore, any disruption to these epithelial junctional complexes will affect intestinal barrier function and homeostasis. Disruptions of epithelial integrity play a key role in the pathophysiology of giardiasis (Allain et al., 2017a; Kraft et al., 2017; Maia-Brigagao et al., 2012). Altered structure and functions of epithelial junctions have been reported in human patients, in animal model systems in vivo, as well as in co-culture experiments in vitro (Buret et al., 1992; Chin et al., 2002; Fisher et al., 2013; Kraft et al., 2017; Scott et al., 2002; Teoh et al., 2000; Troeger et al., 2007; Tysnes and Robertson, 2015). Disruptions of epithelial junctional complexes in giardiasis are, at least in part, mediated by the parasite’s secreted cysteine proteases (see Section 4) (Allain et al., 2019a; Liu et al., 2018; OrtegaPierres et al., 2018).

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To access host tissues, intestinal microbes must be able to cross the mucus barrier lining the gut mucosa. The mucus barrier is composed mostly of water and MUC2 mucin, a large glycoprotein polymer, which forms a thick protective layer at the surface of the epithelium. When this layer is disrupted, it can lead to an impairment of host innate immunity and gastrointestinal disorders (Hansson, 2012). In the context of giardiasis, the mucus layer provides a barrier against highly motile trophozoites, as well as the pathobionts bacteria created upon exposure to the parasite. Recent studies using human intestinal biopsy tissues, mouse models, and human goblet cell lines have shown that this barrier could be circumvented in giardiasis. In particular, Giardia cysteine proteases can cleave human mucin-2 and trigger mucus hypersecretion from goblet cells, hence leading to depletion of mucin stores, both in the small and large intestine in an Assemblage-dependent manner (Amat et al., 2017). Moreover, Giardia trophozoites trigger a CP-dependent increase in MUC2 gene expression in human goblet cells, due to a potential compensatory response to intracellular mucin depletion (Amat et al., 2017). Those discoveries are in line with previous observations with other intestinal parasites showing that the proteolytic activity of Entamoeba histolytica and Trichuris muris is responsible for intestinal mucus degradation (Hasnain et al., 2012; Lidell et al., 2006). Recent experiments performed in our lab are starting to shed new light on how Giardia may regulate mucin expression and hypersecretion. Indeed, Giardia cysteine proteases can cleave and activate protease-activated receptor-2 on intestinal goblet cells, leading to intracellular calcium release and ultimately altered mucin expression and secretion (Fekete et al., 2019). Thus, those results suggest that goblet cell mucin production may be regulated upon activation of PAR2. In our attempts to better understand the pathophysiology of giardiasis, these observations on altered gut permeability in giardiasis, via the disruption of the epithelium and the mucus layer, are key. This importance is due to the fact that loss of intestinal barrier function in giardiasis may cause stunting in children, in the absence of diarrheal disease (Garzon et al., 2017; Kosek and MAL-ED Network Investigators, 2017; Rogawski et al., 2017).

3. Interactions between Giardia and the gut microbiota In healthy individuals, the intestinal commensal microbiota is composed of five major phyla, respectively, Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia (Lozupone et al., 2012). The relative

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abundance and prevalence of these phyla dependent upon environmental, physical (pH, oxygen), and nutritional (diet) factors, as well as other factors such as exposure to enteropathogens (Lozupone et al., 2012). Several findings suggest that the crosstalk between Giardia parasite and the gut microbiota is a two-way interaction. On the one hand, commensal microbiota control the colonization and establishment of the parasite in the host (Singer and Nash, 2000). This protective anti-Giardia microbiota can be effectively transferred to different mice and prevents the establishment of the parasite (Singer and Nash, 2000). The mechanisms of such protective effects as well as the identity of commensal bacteria displaying anti-Giardia activities have yet to be uncovered. Furthermore, a recent study found that the CD8+ T cell-dependent disaccharidase deficiencies observed in giardiasis (Scott et al., 2004) develop in a microbiota-dependent manner (Keselman et al., 2016). On the other hand, the gut microbiota composition is altered in the small intestine and the colon during the acute phase of giardiasis in conventional mice (Barash et al., 2017). The abundance of major phyla such as Firmicutes was decreased in infected mice while several minor taxa were enriched (Barash et al., 2017). These changes in microbiota composition may subsequently be responsible for symptoms observed during giardiasis both at the site of infection and beyond, in the colon (Allain et al., 2017a; Amat et al., 2017). In humans, overgrowth of duodenal commensals has been reported during symptomatic giardiasis (Chen et al., 2013; Torres et al., 2000). Moreover, the composition of commensal microbiota can modulate the pathogenesis of giardiasis (Bar et al., 2015). As discussed above, Giardia disrupts human gut commensal biofilms, and promotes the formation of pathobionts in these communities, which in turn causes intestinal abnormalities in germ-free mice (Beatty et al., 2017). When transferred to germ-free mice infected with Giardia and given microbiota isolated from patients with giardiasis, more severe pathology develops than in mice infected with Giardia alone or given the microbiota alone, in further support of a role for host’s microbiota in the pathogenesis of Giardia infection (Torres et al., 2000). Ever-increasing evidence also indicates that the host’s nutritional status plays a role in Giardia-induced microbiota alterations. In mice fed with a protein-deficient diet, the Giardia-induced shift of duodenal microbiota composition combines with persistent parasite colonization (Bartelt et al., 2017). Recent experiments performed in our laboratory indicate that short-term consumption of western diet (high-fat) increases trophozoite burden in Giardia-infected mice, and that microbiota abundance and diversity is altered in infected mice in a diet-dependent manner

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(Allain et al., 2019b). More research is required to determine whether diet interventions may modulate disease outcome and subsequent microbiota alterations. Most of the anti-Giardia drugs used today carry significant risks of side effects. In view of the key role played by tight junctional disruptions in the pathophysiology of giardiasis and other protozoan parasites as well as in a variety of other intestinal disorders, these processes have become hot topics of research in our attempts at discovering novel therapies. Alternative treatments and probiotics potential developments include the use of natural products or probiotics. As defined in 2001 by the World Health Organization, probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” Over the last decade, probiotic strains of lactic acid bacteria, including Lactobacillus sp., yeast and complex multispecies fermented products have shown promising results by exhibiting direct trophozoite killing effects as well as reducing parasite burden, cyst shedding and duration of infection (Allain et al., 2017a,b, 2018; Benyacoub et al., 2005; Chen et al., 2013; Coelho and Singer, 2018; Franco et al., 2013; Goyal et al., 2013; Goyal and Shukla, 2013; Humen et al., 2005; Lalle and Hanevik, 2018; Perrucci et al., 2019; Shukla and Sidhu, 2011; Shukla et al., 2013; Travers et al., 2016). The underlying mechanisms of these anti-Giardia effects remain unclear; however, enhancement of the host innate and adaptive immune responses, competition for ecological niches and metabolites, and the accumulation of toxic deconjugated bile via probiotic bile-salt hydrolase activities are key contributing factors (Allain et al., 2017a).

4. Parasite virulence factors and immune evasion strategies Our understanding of the role of Giardia excretory-secretory products (ESPs) in disease pathogenesis remains incomplete. Exposure to host enterocytes activates Giardia trophozoites to modify the expression of a broad range of their genes and to release a variety of secretory compounds (Ma’ayeh et al., 2017; Ringqvist et al., 2008, 2011; RodriguezFuentes et al., 2006). Adhesion of trophozoites to the host’s enterocytes involves surface lectins, and parasite proteins such as giardin, variantspecific surface proteins (VSPs), tenascins, and secreted cysteine proteases (Cabrera-Licona et al., 2017; DuBois et al., 2006; Dubourg et al., 2018; Jenkins et al., 2009; Ortega-Pierres and Arguello-Garcia, 2019;

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Ortega-Pierres et al., 2018; Parenti, 1989; Prucca and Lujan, 2009; Ringqvist et al., 2011; Singer et al., 2001). In addition, Giardia helps evade host immune responses via antigenic variation (VSPs) and arginine deiminase, which uses luminal arginine, a metabolite that is required to synthetize anti-Giardia nitric oxide (NO) by intestinal epithelial cells (Eckmann et al., 2000). Among ESPs, Giardia cysteine proteases are major contributors to the pathogenesis of giardiasis (Allain et al., 2019a). Indeed, secreted as well as membrane-bound Giardia cysteine proteases have been implicated in encystation, intestinal barrier dysfunction through the disruption of apical junctional complexes and villin in intestinal epithelial cells, activation of apoptosis (i.e. caspase-3 activation and PARP-1 cleavage), cleavage of mucin and mucus depletion, breakdown of microbiota biofilms ECM and subsequent pathogenic transformation of commensal microbiota biofilms caused by giardiasis (Allain et al., 2019a; Amat et al., 2017; Beatty et al., 2017; Bhargava et al., 2015; Liu et al., 2018; Ortega-Pierres et al., 2018). Adding to the roles of VPSs and metabolic enzymes in Giardia’s host immune evasion, Giardia CPs also exert potent immunomodulatory properties allowing the parasite to evade the host’s immune system and therefore modulate local inflammatory responses. In particular, recent findings demonstrate that Giardia cysteine proteases, including thiol proteinases and cysteine proteases (e.g. cathepsin B and cathepsin L), deployed as secreted proteins, can degrade host immune factors such as immunoglobins (IgG, IgA1, and IgA2), inflammatory mediators (CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL20), and host defensins (α-HD6, β-HD1, and TFF3) (Allain et al., 2019a; Bhargava et al., 2015; Cotton et al., 2014a,b; DuBois et al., 2006; Eckmann et al., 2000; Liu et al., 2018, 2019; Ortega-Pierres et al., 2018; Parenti, 1989). In turn, those effects help prevent the infiltration of anti-microbial neutrophils and coincidentally protect the gut tissues against inflammation triggered by agents such as Clostridium difficile toxins. Indeed, cathepsin B-dependent attenuation of granulocyte chemotaxis has been demonstrated in vivo, with Giardiainfected mice showing significantly lower levels of pro-inflammatory cytokine expression in response to intracolonic administration of C. difficile toxins A and B (Cotton et al., 2014a,b). Similar anti-inflammatory responses of Giardia CPs were also observed in infections with pro-inflammatory bacterial enteropathogens such as Salmonella sp. and attaching effacing strain of Escherichia coli (Allain et al., 2019a). Combined, these immune-evasive effects will facilitate the establishment of infection and hence play a key

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role in pathogenesis. Therefore, a vast majority of the pathophysiological responses triggered by this parasite can be recapitulated by the effects of its proteolytic activity; the effects of Giardia cysteine proteases are illustrated and summarized in this chapter in Figs 1 and 2. The role of these proteases in the pathogenesis of giardiasis was extensively reviewed recently (Allain et al., 2019a). Recent reports also indicate that Giardia secretes small extracellular, membrane-bound vesicles, that play a role in pathogenicity and trophozoite attachment to host epithelial cells (Deolindo et al., 2013; Evans-Osses et al., 2017). Those vesicles, which are for the most part classified in exosomes and microvesicles (MVs), carry proteins, messenger and mi-RNAs, as well as lipids (Deolindo et al., 2013). Recent proteomic analysis of Giardia exosomes has revealed that several aforementioned parasitic factors such as tenascins, VSPs and metabolic enzymes (i.e. arginine deaminase and ornithine carbamoyltransferase) are present, while CPs could not be detected (Evans-Osses et al., 2017). Further research is needed to establish a full proteomic profile of G. intestinalis EVs from Assemblages A and B isolates. Interestingly, host factors such as peptidylarginine deiminase inhibitor and cannabidiol have been shown to significantly decrease EVs shedding from Giardia (Gavinho et al., 2019).

5. Polymicrobial infections Polymicrobial infections are characterized by the presence of microorganisms (bacteria, viruses, protozoa, helminths or fungi), that, under certain circumstances, will generate an ecological niche for the concomitant establishment of one or several other microorganisms in the host (Brogden et al., 2005). Polymicrobial and coinfections are now considered as the norm in developing countries and countries with poor sanitation, and their consequences on child health have led researchers to study microbial-microbial interactions as well as their impact on host physiology (Muhsen and Levine, 2012; Ryan and Caccio, 2013). The presence of G. intestinal in human stools has been associated with concomitant infections with Ascaris sp., Cryptosporidium sp., Cyclospora sp., as well as bacterial enteropathogens such as Helicobacter pylori, C. difficile, Vibrio cholera, enteropathogenic strains of E. coli, Campylobacter sp. and Salmonella sp., and enteroviruses such as norovirus and rotavirus, although most of those microorganisms do not share the same host tropism as Giardia (Ankarklev et al., 2012; Bilenko et al., 2004; Blackwell et al., 2013; Eldash et al., 2013; Fenollar et al., 2003; Grazioli et al., 2006; Hagel et al., 2011; Koru et al., 2006;

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Fig. 1 Examples of direct effects of Giardia cysteine proteases (CP) on the intestinal triple barrier (microbiota, mucus, epithelium). (A) CP-dependent disruption of epithelial villin in Caco-2 cells infected with G. intestinalis. Breakdown of villin is reversed when Giardia is pre-treated with a cysteine protease inhibitor (E-64) (immunofluorescence images). (B) Cleavage of gut microbiota biofilm extracellular matrix (ECM) by Giardia CPs. Cysteine protease inhibitor E-64 pre-treatment prevents the disruption of human microbiota biofilms ECM (wheat germ agglutinin staining; confocal scanning laser microscopy images). (C) Giardia CPs cleave pro-inflammatory mediators. Degradation of interleukin-8 (CXCL8) by G. duodenalis trophozoites is abolished following pretreatment with cysteine protease inhibitor (E-64) (ELISA) (modified). (D) Giardia proteolytic activity can cleave purified human MUC2 mucin cysteine protease-dependent manner. Preincubation of Giardia secreted products with cysteine protease inhibitor E-64 prevents degradation of human MUC2 (Western Blot and relative densitometry). Courtesy of Trends in Parasitology: Allain, T., Fekete, E., Buret, A.G., 2019. Giardia cysteine proteases: the teeth behind the smile. Trends Parasitol. 35, 636–648.

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Fig. 2 Host-parasite interactions during giardiasis. (1) Adhesion of trophozoites to the epithelium via Giardia surface lectins, giardins, variant-specific surface proteins (VSPs), tenascins, and CPs; (2) induction of intestinal epithelial cell apoptosis via caspase-3, 8, 9, BAX and PARP activation; (3) increased permeability and disruption of the epithelial barrier via disruption and alteration of apical junctional complexes, filamentous actin and desmosome; (4) alteration of epithelial brush-border microvilli integrity via MCLKdependent villin breakdown, and diffuse shortening of microvilli; (5) disruption of gut microbiota biofilms and release of pathogenic and invasive bacteria (pathobionts) from dysbiotic microbiota; (6) Giardia exert protective antibacterial effects during polymicrobial infections by promoting host’s antimicrobial peptides (HBD-2 and TFF3) secretion; (7–8) the degrade of host immune factors such as chemokines, immunoglobins and defensins by Giardia cysteine proteases contributes to parasite immune evasion; (9) Giardia trophozoites impairs mucus barrier integrity by depleting goblet cells mucus and degrading host’s mucins, in a CP-dependent manner. Courtesy of Trends in Parasitology: Allain, T., Fekete, E., Buret, A.G., 2019. Giardia cysteine proteases: the teeth behind the smile. Trends Parasitol. 35, 636–648.

Vasco et al., 2016; Wang et al., 2013). The direct interaction between pathogens during polymicrobial infections can either exacerbate the pathogenesis or conversely, elicit a protective response by the host (Rall and Knoll, 2016). Epidemiological evidence indicates that coinfections involving G. intestinalis are associated with a protective effect against enteropathogen driven diarrhoea in developing countries, where diarrheal disease is a major health concern for young individuals (Bilenko et al., 2004; Cotton et al., 2015; Veenemans et al., 2011). For instance, Tanzanian and Bangladeshi

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children infected with Giardia present with reduced diarrheal illness as well as reduced serum inflammatory scores (Bilenko et al., 2004; Haque, 2007; Muhsen et al., 2014; Veenemans et al., 2011). To further understand mechanisms by which Giardia may exert protective effects against attaching and effacing (A/E) pathogens, a murine model of coinfections was designed using Giardia muris and mouse enteropathogen Citrobacter rodentium. Results showed that G. muris can reduce the symptoms of C. rodentium-induced colitis, such as weight loss, intestinal permeability and histopathological damages, by enhancing, at least in part, the production of mucosal antimicrobial peptides (Manko et al., 2017a). Similarly, coinfection with G. intestinalis and enteropathogenic E. coli (EPEC) results in a cathepsin B-dependent increase in Human β-defensin 2 and Trefoil factor 3 gene expression, two major AMPs in intestinal epithelial cells (Manko et al., 2017a). In addition, Giardia trophozoites exert direct anti-EPEC effects. Recent experiments suggest that Giardia coinfection with A/E enteropathogens activates expression and secretion of mucosal AMPs in intestinal epithelial cells through the activation of the NLRP3 inflammasome pathway (Manko et al., 2017b). Research also indicates that disease symptoms during concomitant infection with Giardia and enteropathogens can be exacerbated in young individuals. In a murine model of protein malnutrition, a recent study observed that coinfection with G. intestinalis and enteroaggregative E. coli (EAEC) leads to growth impairment, microbiota-dependent delayed parasite clearance, microbial metabolic perturbations in the gut, as well as an alteration of local host immune responses against EAEC (Bartelt et al., 2017). In addition, in mice co-infected with EAEC, Giardia infection causes extraintestinal manifestations by mediating and attenuating the cytokine response of bone marrow-derived dendritic cells (Burgess et al., 2019). A previous report had demonstrated growth impairment and decreased inflammatory responses in malnourished mice infected with Giardia alone (Bartelt et al., 2013). Together, these results are in accordance with epidemiological data showing that in humans with malnutrition, Giardia infection in humans are more likely to contribute to decreased immune functions and growth impairment (Fink and Singer, 2017).

6. Extraintestinal and post-infectious complications Giardiasis is characterized by a broad range of clinical manifestations ranging from asymptomatic infection to acute and chronic illness. Evidence also indicates that Giardia infection is associated with extraintestinal

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manifestations as well as long-term disorders, even after clearance of the parasite. Extra-intestinal complication, which affects up 30% of infected patients (Cantey et al., 2011), include ocular and retinal pathologies, reactive and post-infectious arthritis, immunoglobulin E (IgE)-mediated cutaneous and intestinal allergies, and diarrhoea associated muscular complication (hypokalaemia) due to an impairment of nutrient absorption (Halliez and Buret, 2013). Post-giardiasis metabolic disorders play an important role in the long-term and post-infectious consequences following symptomatic and asymptomatic giardiasis in low and middle-income countries. Indeed, epidemiological studies conducted on children and adults have reported nutrient deficiencies, failure to thrive, lower height and lower weight, growth retardation, stunting, lower cognitive functions, as well as lower intellectual and social quotients following Giardia infections; those clinical manifestation have been extensively reviewed (Halliez and Buret, 2013). In addition, the host nutritional status plays a pivotal role in the pathogenesis of giardiasis (Fink and Singer, 2017; Halliez and Buret, 2013). It has been well established that G. intestinalis infections disrupt the intestinal barrier and cause malnutrition, micronutrient deficiencies and malabsorption, which may lead to growth retardation, stunting and lower cognitive functions in young individuals (Berkman et al., 2002; Guerrant et al., 1999; Koruk et al., 2010; Simsek et al., 2004). Micronutrient and trace element deficiencies such as zinc, iron and vitamin A deficiencies, have been reported in children with giardiasis, which result from impaired intestinal absorption, parasite-host competition for nutrients, inhibition of micronutrients-related functions by parasitic factors (Astiazaran-Garcia et al., 2010, 2015; Bartelt et al., 2013; Koruk et al., 2010; Rahman et al., 2001; Simsek et al., 2004). In addition, children with micronutrient deficiency exhibit increased susceptibility to Giardia infection (AstiazaranGarcia et al., 2015). Interestingly, zinc and/or vitamin A supplementation have been shown to reduce the incidence and prevalence of Giardiaassociated diarrhoea, improve immune response, and reduce the rate of Giardia infection (Astiazaran-Garcia et al., 2015; Lima et al., 2010). In parallel, malnutrition may lead to inadequate nutrient and electrolyte intake, and impaired parasite-specific immune response in patients infected with Giardia (Bartelt et al., 2013). In Giardia-infected malnourished mice, dietary protein deficiencies induce a decrease of immune mediators and decreased mucosal immune cell recruitment, along with prolonged impairment of intestinal barrier functions and growth faltering (Bartelt et al., 2013).

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Failure to thrive is a major concern in children from developing countries, mostly in the first 2 years of age. It is caused by inadequate food intake, malabsorption and malnutrition, and excessive utilization of metabolic energy to fight infection (Fink and Singer, 2017; Halliez and Buret, 2013; Rogawski et al., 2017). Numerous studies have reported growth retardation in children infected with G. intestinalis, characterized by a lower height and weight, when compared to child growth standards within the same range of age, sex and ethnicity (Halliez and Buret, 2013). Interestingly, no correlation has been established between Giardiaassociated diarrhoea and poor growth in findings from the Malnutrition and the Consequences for Child Health and Development (MAL-ED) birth cohort study (MAL-ED Network Investigators, 2018; Rogawski et al., 2017). This suggests that the high prevalence of asymptomatic Giardia infection is a major contributor to growth retardation in young individuals, and that this may occur independently of diarrheal disease (MAL-ED Network Investigators, 2017).

6.1 Gastrointestinal sequelae of giardiasis and postinfectious IBS Functional gastrointestinal disorders, such as irritable bowel syndrome (IBS) or functional dyspepsia, are characterized by recurring or chronic gastrointestinal symptoms including abdominal discomfort and altered motility, and in certain cases, visceral hypersensitivity (Simsek, 2011). Among the different subtypes of IBS, post-infectious IBS (PI-IBS) is characterized by the manifestation of symptoms of IBS following acute gastroenteritis induced by enteropathogens such as Salmonella sp., Shigella sp., Campylobacter sp. and G. intestinalis (Heitkemper et al., 2011; Rodriguez and Ruigomez, 1999; Spiller, 2007). Indeed, people diagnosed with G. intestinalis have a higher risk of developing PI-IBS symptoms after clearance of the parasite, and 5% to 10% of patients with IBS have been previously infected with G. intestinalis (Dizdar et al., 2010; Grazioli et al., 2006; Hanevik et al., 2009, 2014; Horman et al., 2004). Early treatment of giardiasis may help reduce the risks of chronic Giardia infection and the development of post-infectious gastrointestinal disorders and chronic fatigue (Morch et al., 2009). The mechanisms remain unclear, but abnormalities in intestinal and plasma serotonin (5-HT) and cholecystokinin (CCK) levels, which have been observed in IBS patients, have also been reported in patients with Giardia-associated PI-IBS and functional dyspepsia (Dizdar et al., 2010).

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Hallmarks of IBS such as visceral hypersensitivity and pain were monitored in rats infected with Giardia. The results showed a significant increase in jejunal and rectal hypersensitivity, activation of a nociceptive signalling pathway (c-fos expression) as well as signs of villus atrophy and crypt hyperplasia, 50 days post-challenge, after full clearance of the parasite (Halliez et al., 2016). Studies also found that the loss of gut barrier function caused by giardiasis may persist, at least in part through the formation of a dysbiotic microbiome during the acute stage of the infection, consistent with the known development of post-infectious IBS (Barash et al., 2017; Beatty et al., 2017; Bhargava et al., 2015; Halliez et al., 2016; Kraft et al., 2017; Ma’ayeh et al., 2017; Slapeta et al., 2015; Torres et al., 2000). Recent evidence suggests that gut microbiota dysbiosis is a major cause of the pathophysiology of IBS, especially in patients that have developed PI-IBS (Carroll et al., 2011, 2012; Halvorson et al., 2006; Menees and Chey, 2018). To further decipher the disruption of the microbiome in the development of post-infectious disorders, our group has demonstrated that Giardia is able to break down the extracellular matrix of human mucosal multispecies biofilms and promote the release of pathobionts, in a cysteine protease-dependent manner (Beatty et al., 2017). In turn, these pathobionts can disrupt tight junctions and penetrate the intestinal epithelium, and trigger the release of pro-inflammatory chemokines such as CXCL-8 in a TLR4-dependent fashion. When administered to germ-free mice, dysbiotic biofilms are able to promote colitis through the induction of pro-inflammation signals and the disruption of epithelial barrier integrity (Beatty et al., 2017). Together, these findings indicate that Giardia has the ability to change commensal microbiota bacteria into invasive pathobionts, which in turn can lead to post-infectious visceral hypersensitivity and other complications (Beatty et al., 2017; Buret et al., 2019; Halliez and Buret, 2013). In further support of the key pathogenic role of these alterations, the effects of Giardia on commensals were investigated in a model of Caenorhabditis elegans (Gerbaba et al., 2015). Commensal bacteria, including human isolates of E. coli, were exposed to Giardia and subsequently transferred to C. elegans, causing lethal toxicity to the worms. Bacteria that were not exposed to Giardia showed no signs of toxicity to the nematode, further supporting that Giardia can activate latent virulence factors in commensal bacteria and turn them into pathobionts (Gerbaba et al., 2015). Transcriptomic analyses in this model revealed that Giardia alters the expression of over a hundred genes in E. coli. Consistent with these findings,

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recent studies have reported an increased prevalence of opportunistic pathogen species such as Enterococcus spp. and E. coli in the faecal microbiota of patients infected with Giardia (Allain et al., 2017a; Iebba et al., 2016; Tomkins et al., 1978).

7. Conclusions Giardiasis has been implicated in failure to thrive and growth retardation in children. Because of its detrimental impact on health, giardiasis was included in the World Health Organization’s “neglected disease initiative”. Giardiasis leads to a broad spectrum of symptoms ranging from no symptom at all, to severe, acute or chronic diarrhoea, bloating, nausea, and abdominal pain. Giardia may cause disease during the acute phase of the infection, as well as post-infectiously, where it presents as post-infectious Irritable Bowel Syndrome and chronic fatigue. Moreover, giardiasis can initiate a variety of extra-intestinal complications. The parasite causes epithelial abnormalities in the gut that affect intestinal function and permeability, which in turn are responsible for disease. These are combined with disruptions of the mucus barrier and the commensal gut microbiota biofilm. Recent observations demonstrate that the production of membrane-bound and secreted cysteine proteases by the parasite play a central role in disease pathogenesis. To date, studies have identified a number of pathophysiological events in giardiasis that appear to be shared with other intestinal disorders, and therefore may well become important therapeutic targets. This chapter elaborates in detail on these findings, and paves the road towards future research directions.

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