Characterization of Secreted Giardia Intestinalis Cysteine Proteases

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1763

Characterization of secreted Giardia intestinalis cysteine proteases

JINGYI LIU

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0551-6 UPPSALA urn:nbn:se:uu:diva-372997 2019 Dissertation presented at Uppsala University to be publicly examined in A1:111a, Uppsala Biomedicinska Centrum BMC, Husarg. 3, Uppsala, Thursday, 28 February 2019 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Andre Buret (University of Calgary).

Abstract Liu, J. 2019. Characterization of secreted Giardia intestinalis cysteine proteases. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1763. 72 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0551-6.

Giardia intestinalis, the causative agent of the diarrheal disease giardiasis, is a protozoan parasite that colonizes the upper small intestine of mammals, including humans. It can be divided into eight genotypes or assemblages (A through H) and only assemblage A and B are infective to humans. Giardiasis is a multi-factorial disease but few giardial virulence factors have been identified and characterized. In this thesis, we used proteomics to identify the major excretory-secretory products (ESPs) released by Giardia trophozoites of the WB and GS isolates during interaction with intestinal epithelial cells (IECs) in vitro (Paper I). To deepen our understanding of the role of ESPs in giardiasis, we focused on three specific secreted Giardia cysteine proteases (CPs; CP14019, CP16160 and CP16779). All the three CPs are capable of opening the apical junction complexes between IECs to degrade chemokines produced in response to Giardia (Paper II). This can partly explain the induction of symptoms and immunosuppression seen during giardiasis. We further studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The preferred sequences were used to search potential human in vivo targets and a number of candidates were identified, including human immunoglobulins as well as defensins, that were subsequently shown to be efficiently cleaved by the CPs (Paper III). To investigate the involvement of CPs in mucus degradation, we tested the CPs on recombinant MUC2 constructs and full-length MUC2. MUC2 is the major component of the mucus layer in the small intestine. It was shown that CP14019 cleave MUC2 in the N-terminal, suggesting a mechanism that the parasite can use to disrupt/release the mucus gel network and get access to the intestinal epithelium of the host (Paper IV). In summary, this thesis has studied secreted Giardia CPs and their roles in Giardia infections, providing significant insights into the molecular pathogenesis of giardiasis.

Keywords: Giardia intestinalis, ESPs, CPs, apical junction, chemokines, phage display, immunoglobulins, defensins, mucin.

Jingyi Liu, Department of Cell and Molecular Biology, Microbiology, Box 596, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-0551-6 urn:nbn:se:uu:diva-372997 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-372997)

To my family

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Ma'ayeh, S.Y., Liu, J., Peirasmaki, D., Hornaeus, K., Bergström Lind, S., Grabherr, M., Bergquist, J., and Svärd, S.G. (2017). Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells. PLoS Negl Trop Dis, 11(12): e0006120.

II Liu, J., Ma'ayeh, S., Peirasmaki, D., Lundström-Stadelmann, B., Hellman, L., and Svärd, S.G. (2018). Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines. Virulence, 9(1): 879-894.

III Liu, J., Fu, Z., Hellman, L., and Svärd, S.G (2018). Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins. Mol Bio- chem Parasitol, 227: 29-38.

IV Arike, L.*, Recktenwald, C.V.*, Liu, J., Trillo-Muyo, S., Svärd, S.G., Hansson, G.C (2019). MUC2 mucin is cleaved by Giardia intestinalis cysteine protease 14019. Manuscript

*These authors contributed equally

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Giardia ...... 11 Life cycle of Giardia ...... 12 The trophozoite ...... 13 Cell differentiation ...... 16 Giardiasis ...... 17 Giardia pathogenesis ...... 17 Microvilli shortening, destruction, malabsorption ...... 19 Intestinal barrier disruption ...... 19 Intestinal microbiota ...... 21 Mucus layer ...... 23 Host cytokine profile ...... 26 Virulence factors ...... 27 Attachment ...... 28 Antigenic variation ...... 29 ADI and OCT ...... 30 Cysteine proteases ...... 31 Other factors ...... 33 Current investigation ...... 35 Secretome study of IECs-parasites interplay (Paper I) ...... 35 Secreted Giardia CPs are key virulence factors (Papers II, III and IV) ... 37 Tight junction disruption and chemokine degradation (Paper II) ...... 37 Degradation of immunoglobulins and defensins (Paper III) ...... 40 Mucin degradation (Paper IV) ...... 41 Conclusion and future perspectives ...... 43 Cleavage specificity of other secreted Giardia CPs ...... 43 Knockout study of Giardia CPs ...... 44 CPs role in vaccines and as drug targets ...... 45 How are the activities of CPs regulated? ...... 45 Assemblage-specific differences of CPs ...... 46 Svensk sammanfattning ...... 47 Acknowledgements ...... 49

References ...... 51

Abbreviations

ADI Arginine deiminase AJ Adherence junction AME Arginine metabolizing enzyme AMP Antimicrobial peptide AP Adaptor protein ATP Adenosine triphosphate CCL (-2, -20) Chemokine with double cysteine (CC) N-terminal motif COP Coat protein CXCL (-1, -2, -3) Chemokine with CC motif separated by 1 amino acid CP Cysteine protease DC Dendritic cell EF-1α Elongation factor 1 alpha ER Endoplasmic reticulum ESP Excretory-secretory product FeS Iron-sulfur HCMP High-cysteine membrane protein IBS Irritable bowel syndrome IEC Intestinal epithelial cell Ig (-A, -G) Immunoglobulin A, G LC-MS Liquid chromatography-mass spectrometry MLCK Myosin light chain kinase MMP Matrix metalloprotease 7 NO Nitric oxide NOS Nitric oxide synthase OCT Ornithine carbamoyl transferase PV Peripheral vacuole SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electro- phoresis TJ Tight junction VSP Variant-specific surface protein ZO Zonula occludens α-HD (-5,-6) α-defensin (-5,-6) β-HD1 β-defensin 1

Introduction

Our understanding of Giardia’s pathogenesis is increasing, partially attributed to the understanding of the effects of excretory-secretory products (ESPs) released by Giardia during host-parasite interactions. However, the understanding is limited to effects induced by culture supernatants in most studies and the specific virulence factors have rarely been identified and characterized. Proteases constitute a large proportion of the ESPs and secreted proteases have been suggested to be key players in the pathogenesis of many protozoan parasitic diseases, including giardiasis.

Giardia Giardia was initially described by van Leeuwenhoek in 1681 when he was examining his own diarrheal stools under his primitive microscope (Dobell, 1920). The organism was described in more detail by Lambl in 1859 who thought it belonged to Cercomonas and named it Cercomonas intestinalis (Lambl, 1859). Thereafter, several names were introduced for the organism. Giardia was for the first time used as genus name by Kunstler in early 1880’s. Lamblia intestinalis was later suggested by Blanchard in 1888 (Blanchard, 1888) and changed to G. duodenalis by Stiles in 1902 (Stiles, 1902). In 1915, Kofoid and Christiansen proposed the name G. lamblia (Kofoid, 1915). It continued to be a controversy about the name and number of different Giardia species in the next 100 years. Based on the morphological differences, Filice described a detailed morphology of Giardia and proposed three species names; G. duodenalis, G. muris, and G. agilis based on the morphology of the median body (Filice, 1952). Thereafter, three additional species, G. psittaci from parakeets (Erlandsen and Bemrick, 1987), G. ardeae from herons (Erlandsen et al., 1990) and G. microti from voles and muskrats (Feely, 1988), were identified by electron microscope. Newly introduced molecular biological tools have later been applied in the Giardia classification. G. intestinalis (syn. G. lamblia, G. duodenalis) human isolates were divided into two major assemblages, assemblage A and B, based on their marked genetic as well as biological differences (Lu et al., 1998, Nash et al., 1987, Karanis and Ey, 1998). More recently, a number of additional assemblages have been proposed for Giardia isolates from a vari-

11 ety of mammals, based on the differences of protein sequences. A few dog Giardia isolates (assemblage C and D) were identified and found to be quite distinct from assemblage A and B (Monis et al., 1998). Separate assemblages (E trough H) have also been proposed for hoofed animals (Ey et al., 1997), cat (Monis et al., 1999), and rat (Monis et al., 1999) isolates (Table 1). In contrast to assemblage A and B parasites, other assemblages display a higher degree of species specificity. In 2009, a revision of the Giardia taxonomy and a new nomenclature at species level was proposed (Table 1, (Monis et al., 2009)). Since this thesis focuses on assemblage A and B, I have chosen to use the assemblage designations to indicate G. intestinalis genetic groups and Giardia will also be used to denote G. intestinalis unless otherwise specified.

Table 1. Taxonomy and host-range of recognized Giardia species, G. intestinalis assemblages. Adapted from (Monis et al., 2009). Species Genotype Proposed species name Host G. intestinalis Assemblage A G. duodenalis Humans and other mammals Assemblage B G. enterica Humans and other mammals Assemblage C,D G. canis Domestic and wild canids Assemblage E G. bovis Hoofed animals Assemblage F G. cati Cats Assemblage G G. simondi Rodents Assemblage H Sea mammals G. muris Rodents G. agilis Amphibians G. microti Rodents G. ardeae Herons G. psittaci Parakeets

Life cycle of Giardia Giardia is a single-celled protozoan parasite. It has a relatively simple life cycle (Figure 1) consisting of two major life-cycle stages; vegetative trophozoites and infectious cysts. The infection is initiated by ingestion of cysts. After exposure to the acidic environment in the stomach and bile and trypsin in the duodenum the cysts undergo excystation (Figure 1) to generate trophozoites. The released motile trophozoites colonize the upper small intestine of the hosts where they divide by binary fission and establish an infection. Thereafter, the trophozoites undergo dramatic biological changes to survive outside the intestine of their host, by differentiating into resistant cysts (encystation, Figure 1). The cyst wall enables the parasite to survive for long periods of time in the environment until the next infection.

Figure 1. The life cycle of Giardia. Giardia has a simple life cycle with two main life cycle stages; infective cysts and vegetative trophozoites. The cyst is resistant to harsh environments. Trophozoites attach to intestinal epithelial cells and replicate in the small intestine. These two life cycle stages are generated by two cell differentiation processes; encystation and excystation.

The trophozoite The Giardia trophozoite is a pear-shaped cell and it is approximately 12-15 µm long and 5-7 µm wide. The trophozoite has two nuclei symmetrically located in the anterior region. The cytoskeleton is extensive, consisting of a median body, four pairs of flagella, and a ventral disc (Elmendorf et al., 2003).

The nuclei Giardia trophozoites possess two nuclei symmetrically located in the anteri- or half of the cell. These nuclei are equivalent in size and in the amount of DNA, and both nuclei are transcriptionally active (Kabnick and Peattie, 1990, Yu et al., 2002). However, differences have been seen between the nuclei. Aneuploidy between the two nuclei seems to occur in certain isolates (Tumova et al., 2007). The two nuclei are distinct in the nuclear envelope and the number and distribution of nuclear pores (Benchimol, 2004). Ex- pression of the small nucleolar RNA (snoRNA) GlsR17 was found to occur primarily in only one nucleolus (Saraiya and Wang, 2008). One nucleus usually starts replication of DNA before the other (Hofstetrova et al., 2010).

13 Cytoskeleton Giardia has an elaborate microtubule cytoskeleton that can be seen in deter- gent extracted cells (Dawson, 2010). The microtubule cytoskeleton consists of structures commonly found in flagellated protists (four pairs of flagella and two mitotic spindles). It also possesses structures such as the ventral disc and the median body (Dawson, 2010). The structures mainly contain alpha- and beta-tubulin but also Giardia-specific protein families such as α- giardins, Nek kinases, ankyrin repeat proteins, striated-fiber assemblin-like proteins (SALP-1, β-,δ-giardin) and the ventral disc protein γ-giardin (Palm et al., 2005, Nohria et al., 1992). The eight flagella (9+2 microtubule arrangement) of the trophozoite are organized as four pairs; the anterior flagella (AF), posteriorlateral flagella (PF), caudal flagella (CF) and ventral flagella (VF). Flagellar beating is important for cellular motility, and they are possibly also involved in the encystation/excystation processes (Ginger et al., 2008). A recent study has shown that the flagellar motility indirectly contributes to giardial attachment and it is important for positioning and orienting trophozoites prior to attachment (House et al., 2011). The flagellar motility has also been found to be essential for cell division by initiating daughter cell separation and providing a source for membrane tension (Hardin et al., 2017). The flagella originate from eight basal bodies located in between the two nuclei. Basal bodies are involved in the regulation of Giardia trophozoite growth, cell division and differentiation (Abel et al., 2001, Gibson et al., 2006, Lauwaet et al., 2007). The ventral disc of Giardia is regarded as a key virulence factor since the establishment of Giardia infection depends on the trophozoites attachment to the epithelium to avoid being eliminated from the small intestine by peristalsis. The ventral disc is a unique structure of Giardia and it covers the anteri- or half of the ventral surface of the trophozoite. The ventral disc is a concave spiral array of microtubules perpendicularly joined by microribbons in the cytoplasm (Dawson, 2010). The spirals are joined at the tips and create a circular area devoid of cytoskeleton proteins. The ventrolateral flange, lateral shields, lateral crest and bare area sequentially make contact with the substrate during attachment (House et al., 2011). The median body is a haystack of disordered microtubules present on the dorsal side of Giardia trophozoites, forming the “crooked smile” of Giardia trophozoites (Dawson, 2010). The structure is unique to Giardia and its morphology has been used to define the morphologic characteristics of Gi- ardia species (Filice, 1952). The functional role of the median body is still unknown.

Mitosome The mitosomes of Giardia are the reduced forms of eukaryotic mitochondria with a size less than 0.2 µm. Each cell possesses 20 to 100 mitosomes. There

14 are two types of mitosomes. The central mitosomes locate between the two nuclei and they are split during the mitosis. The other mitosomes are scat- tered within the cytosol (Tovar et al., 2003, Dolezal et al., 2005, Regoes et al., 2005, Dagley et al., 2009). Giardia mitosomes have lost the ability to generate energy but retained some mitochondrial features such as presence of a double-membranes, assembly of iron-sulfur (FeS) clusters and require- ment of translocation signals for import of mitosomal proteins (Nyindodo- Ogari et al., 2014). The import machinery is much reduced compared to other eukaryotes and the few identified component are outer membrane porin Tom40 and inner membrane translocases Pam18 and Pam16 (Jedelsky et al., 2011, Dagley et al., 2009). Protein folding is mediated by Chaperonin 60 and 10. Although adenosine triphosphate (ATP) is required to provide energy for mitosomal functions, such as import and maturation of mitosomal proteins and transfer FeS clusters to apoproteins, no component involved in ATP synthesis has been identified in giardial mitosomes. How the ATP is transferred to the mitosome remains unknown. “Tail-anchored proteins” such as Sec20, a member of SNARE (soluble N-ethylmalemide-sensitive- factor attachment protein receptors), and VAMP associated protein (VAP) have also been identified in mitosomes (Elias et al., 2008, Jedelsky et al., 2011), suggesting they might have a role in linking the mitosomal membranes with the endomembrane system.

Protein transport Giardia has a different protein transport system compared to typical eukaryotes. Giardia possesses an endoplasmic reticulum (ER) network that extends from the nuclear envelops through the entire cell body and this is the major secretory system where the proteins mature and sort in the absence of a true Golgi apparatus (Faso and Hehl, 2011). Glycosylation occurs in Giardia without the presence of the Golgi apparatus, but only addition of N-acetyl glucosamine(s) has been described in Giardia ER (Robbins and Samuelson, 2005). A large number of proteins that contribute to protein transport have been identified in the Giardia genome, for example clathrin, SNAREs, coat complexes (COPI) and small GTPases (Murtagh et al., 1992, Marti et al., 2003, Elias et al., 2008). The adaptor proteins (AP1 and AP2) were also shown to be involved in the secretory transport and the endocytic processes (Touz et al., 2004, Rivero et al., 2010b). Giardia has no peroxisomes or lysosomes, but it possesses a large number of vesicles known as peripheral vacuoles (PVs) that are located below the plasma membrane of the dorsal side. PVs have lysosomal properties and contain hydrolases and proteases (McCaffery and Gillin, 1994, Thirion et al., 2003). PVs take up soluble material from the environment, but the exact mechanism needs to be uncovered (Wampfler et al., 2014, Abodeely et al., 2009). Secretory transport is targeted by N-terminal signal peptides of the proteins or combined with signals from a semi-conserved C-terminal region,

15 and transit to plasmamembrane via ER (Faso and Hehl, 2011). Secretion of giardial metabolic enzymes lacking signal peptides via a non-conventional pathway has also been determined (Ringqvist et al., 2008).

Cell differentiation Cell differentiation of Giardia involves two major developmental transitions. Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the parasite. Directly after the ingestion of cysts, the parasites differentiate into the short-lived, flagellated excyzoites in the duodenum, and the excyzoites rapidly undergo two rounds of cytokinesis, generating four fully developed trophozoites.

Encystation Encystation is the process where vegetative trophozoites differentiate to dormant and infectious cysts. Encystation starts in vivo when trophozoites migrate to the lower part of the small intestine. This process can be mim- icked in vitro by exposing the trophozoites to a pH of 7.8 and high concentration of bile salts (Gillin et al., 1996) or starvation of cholesterol (Lujan et al., 1996). The trophozoites quickly become round, detach from the intestinal surface and the ventral disc is fragmented into four crescent-shape structures (Palm et al., 2005). The cyst wall is composed of ~40% protein and ~60% carbohydrate. The transcription of cyst wall proteins 1-3 (CWPs) and genes in the pathway to synthesize carbohydrate monomers is regulated by transcription factor Myb2 (Sun et al., 2002). The synthesized carbohydrate monomers are assembled into giardan (β(1-3)-N-Acetyl-D-galactosamine polymer) by the “cyst wall synthase” (Karr and Jarroll, 2004). During encystation, the two nuclei divide, without intervening cytokinesis, to form four cyst nuclei with four copies of each chromosome (4x4N) in the mature cyst (Bernander et al., 2001, Poxleitner et al., 2008).

The cyst The cyst has an oval shape with a size of 8-12 µm x 7-10 µm. It is covered by a cyst wall that is 0.3-0.5 µm thick. It contains four prominent nuclei, flagellar axonemes and fragments of the ventral disc. The cyst stage enables survival of the parasite in harsh environments outside of the host. Giardia cysts are shed in the feces into the environment until transmission to a new host can occur.

Excystation Excystation of Giardia entails a rapid differentiation of cysts to parasitic trophozoites and it can be induced in vitro. Excystation is induced by exposing cysts to acidic pH like in the stomach, followed by a slightly alkaline solution containing pancreatic proteases, mimicking the environment of the

16 small intestine (Bingham and Meyer, 1979, Boucher and Gillin, 1990). Excystation is initiated by the opening of cyst wall at one pole, the cytoplasm is retracted from the wall and the peritrophic space become progres- sively larger. Subsequently, the flagella emerge through the open pole, followed by release of the entire cell that is a short-lived parasite stage (excyzoite). The excyzoite rapidly goes through cytokinesis and divides twice to form four trophozoites. The generation of four trophozoites from one cyst partly explains the low infection dose of Giardia (Rendtorff, 1954).

Giardiasis Giardia is one of the most common waterborne pathogens distributed worldwide (Lane and Lloyd, 2002). Human giardiasis is caused by assemblage A and B Giardia parasites, both of which are potentially zoonotic. Individuals infected with Giardia can be both asymptomatic and symptomatic. The infection is triggered by ingestion of cysts through contaminated water or food and the colonization of trophozoites in the upper small intestine causes the infection. Symptoms of Giardia infection vary in severity on individual basis and duration of the infection. The outcomes of the disease range from asymptomatic carriage to severe and chronic diarrhea. Asymptomatic individuals can be reservoirs for spreading of the disease by passing cysts in their feces. Symptoms, aside from diarrhea, typically include abdominal pain, vomiting, nausea, dehydration, weight loss (Farthing, 1996, Gascon, 2006, Panaro et al., 2007). “Continuous passage of loose stool for more than two weeks” is defined as severe diarrhea, and chronic diarrhea is used if the diarrheal state continues for more than four weeks (Pawlowski et al., 2009). The majority of Giardia infected individuals does not develop overt intestinal inflammation. The symptoms of giardiasis typically self-resolve. The host’s clinical status such as nutritional state, immune status and age are factors that have effects on the disease outcomes (Faubert, 2000) since elderly and immuno- compromised individuals are more affected (Stark et al., 2009, Rogawski et al., 2017). Giardia infections may also induce post-infectious syndromes such as irritable bowel syndrome (IBS) and chronic fatigue (D'Anchino et al., 2002, Wensaas et al., 2012, Hanevik et al., 2014, Halliez et al., 2016, Nakao et al., 2017).

Giardia pathogenesis The pathogenesis of giardiasis is incompletely understood and both host and parasite factors contribute to the multifactorial process (Figure 2). To estab-

17 lish an infection in the host’s small intestine, Giardia must overcome the intraluminal fluid flow, peristaltic waves, and a series of host defense mechanisms to replicate and make contact with the intestinal epithelium. Giardia trophozoites strongly adhere to the epithelial surface of the small intestine via a ventral adhesive disc (Sousa et al., 2001, Chavez and Martinez-Palomo, 1995, Chavez et al., 1995, Erlandsen et al., 2004). In most cases, Giardia causes infection without invading the host cells. At the height of infection, millions of Giardia trophozoites closely associate with the intestinal epithelial surface. The physical attachment of trophozoites to epithelial cells acti- vates downstream events that impair the host cells and cause associated signs and symptoms of giardiasis via targeting specific signaling pathways.

Figure 2. Giardiasis is a multi-factorial disease. (1) Diffuse shortening of microvilli, disaccharidase impairment and increased crypt/villus ratio. (2) Upregulation of chemokines expression and recruitment of immune cells. (3) Disruption of tight junction. (4) Induction of apoptosis. (5) Increase in intestinal epithelial permeability. (6) Arginine starvation and reduced NO production. (7) Increased chloride secretion. (8) Alteration of intestinal microbiota. (9) Disruption of mucin integrity. Adapted from (Allain et al., 2017a, Einarsson et al., 2016a).

18 Microvilli shortening, destruction, malabsorption The brush border surface provides the major interface for nutrient absorption, and reduction of the microvilli surface area is linked to a reduced activity of enzymes that are necessary for proper digestion and absorption of nutrients (Farthing, 1997). The disruption of the brush border could damage the digestive enzymes leading to malabsorption. Loss of brush border surface area was observed in gerbils infected with Giardia which was associated with the inhibition of disaccharidase activities and fluid malabsorption (Buret et al., 1992, Buret et al., 1991). This is consistent with the observation of decreased total duodenal surface area in patients with chronic giardiasis (Troeger et al., 2007). It has been shown that microvillus shortening, disaccharidase impairment and increased crypt/villus ratios are regulated by CD8+ T cells (Scott et al., 2004) but there is also a direct, physical decrease of microvilli due to tight binding of the disc to the IECs (Sousa et al., 2001, Chavez et al., 1995). Villin is the main microvillus actin-bundling protein. Breakdown of villin results in the destruction of the microvillus architecture. Remodeling of the brush border microvillus protein villin was observed via CD4+ and CD8+ immune responses during Giardia infection in vivo (Solaymani-Mohammadi and Singer, 2013). Co-incubation of Giardia with Caco-2 cells has also induced cleavage of villin in a cysteine protease dependent manner, partly via myosin light chain kinase (MLCK) activation, as MLCK inhibition did not prevent villin degradation at early time points (2 h) whereas it prevented villin degradation at later time points (24 h) (Bhargava et al., 2015). Taken together, Giardia-induced CD8+ T cell dependent shortening of microvilli surface area, increased epithelial cell replication and mi- gration contribute to the reduced levels of disaccharidase activity, resulting in malabsorption and further diarrhea.

Intestinal barrier disruption The intestinal epithelial barrier, which is maintained by epithelial junctional complexes, functions as a physical barrier between the lumen and the sub- epithelial tissue. This barrier prevents the passage of microorganisms and luminal antigen to the underlying tissue, playing an essential role in the maintenance of intestinal homeostasis (Schumann et al., 2017).

Epithelial apoptosis One of the most striking outcomes of parasite attachment is the activation of cell death mechanisms such as apoptosis. Apoptosis is characterized as a regulated and controlled autonomous cell death process that plays an important role in cell turnover and maintain cellular homeostasis (Elmore, 2007). Apoptosis can be activated extrinsically by receptor-mediated signaling (activation of procaspase-8) or intrinsically by disruption of mitochon-

19 dria (activation of procaspase-9), both of which activate downstream caspase-3 (Thornberry and Lazebnik, 1998) resulting in the cell apoptosis. A number of enteric pathogens including Cryptosporidium parvum, en- terohemorrhagic Escherichia coli (EHEC) and Clostridium difficile have the capability to induce enterocyte apoptosis and negatively affect the intestinal homeostasis (McCole et al., 2000, Keenan et al., 1986, Fiorentini et al., 1998). Giardia infections have been shown to induce enterocyte apoptosis. An increased rate of enterocyte apoptosis has been observed in biopsy specimens from patients with chronic giardiasis (Troeger et al., 2007). Genes associated with apoptosis in human Caco-2 cells were found to be upregulated when exposed to Giardia products (Roxstrom-Lindquist et al., 2005). Co- incubation of human HTC-8 cells with Giardia also results in host cell apoptosis. In this case, apoptosis was effectively inhibited by a caspase-3 inhibitor, indicating Giardia-induced apoptosis is activated through caspase-3 signaling pathway (Koh et al., 2013). Giardia-induced caspase-3 dependent apoptosis also occurs in human SCBN cells exposed to Giardia and is strain- dependent (Chin et al., 2002). This is consistent with the observation of caspase-3 activation in human HTC cells exposed to Giardia (Panaro et al., 2007). Furthermore, Giardia infection induces activation of both intrisic and extrisinc apoptotic pathways, increased expression of proaopotic Bax and decreased expression of antiapoptotic Bcl-2 (Panaro et al., 2007). Arginine starvation may contribute to enterocyte apoptosis (Potoka et al., 2003). It is likely to be the case also during Giardia infection, as enterocyte proliferation was observed to be inhibited by arginine starvation caused by parasite’s arginine consumption (Stadelmann et al., 2012). Very little is known how the host cells respond to Giardia-induced apoptosis. One study has shown that activation of caspase-3 is linked to the inter- nal glucose level of host epithelial cells, as low glucose level induces apoptosis and high concentration prevents apoptotic phenomenon. In this study, sodium-dependent glucose cotransporter (SGLT)-1-mediated glucose uptake was upregulated by a soluble proteolytic fraction of Giardia in association with increased SGLT-1 expression on epithelial cells (Yu et al., 2008), indicating a possible novel cell rescue mechanism against Giardia-induced apoptosis via SGTL-1 activation and enhanced glucose uptake.

Apical junction disruption The intestinal apical junctional complex is composed of tight junctions (TJs), adherence junctions (AJs) and desmosomes, and they consist of a large number of cytosolic and transmembrane proteins (Schumann et al., 2017, Allain et al., 2017a). TJs localize on the apical surface of the intestine and connect the paracellular space between epithelial cells. They contain transmembrane proteins such as claudins, occludins, junctional adhesion molecules (JAMs), cytosolic zonula occludens (ZO) proteins and cytosolic regulatory proteins F-actin and α-actinin (Schumann et al., 2017). AJs contain the

20 transmembrane proteins cadherins including E-cadherin that has a cytoplasmic tail that bind to β-catenin, together with α-catenin form cadherin-catenin complex (Nishimura and Takeichi, 2009, Pokutta and Weis, 2007). The in- tactness of the apical junction is important in order to maintain intestinal homeostasis. Giardia trophozoites induce alterations in tight junctions by breakdown and/or rearrangement of ZO-1, transmembrane tight junction proteins and cytoskeleton proteins. Alteration of these key junctional and cytoskeletal proteins results in increased intestinal permeability and intestinal barrier dysfunction. Most studies were done in vitro using human intestinal cell lines. Co-incubation of human cell lines (Caco-2, SCBN, HTC-8) with trophozoites induces reorganization of cytoskeleton proteins F-actin and α- actin, disruption of tight junction protein ZO-1, in some cases, resulting in increased intestinal permeability (Chin et al., 2002, Buret et al., 2002, Teoh et al., 2000, Koh et al., 2013, Humen et al., 2011). A study using electron microscopy also observed that F-actin was retracted and concentrated near cellular contacts resulting in microvillus atrophy (Maia-Brigagao et al., 2012). The expression level of claudin-1, the transmembrane protein responsible for the sealing properties of tight junctions, was reduced both in vitro and in biopsy specimens from patients with chronic giardiasis (Troeger et al., 2007, Humen et al., 2011). Alteration of small intestinal permeability has also been seen in G. muris infected mice in vivo (Scott et al., 2002). The mechanisms responsible for the intestinal barrier abnormalities have also been investigated. One study demonstrated that loss of intestinal barrier function is caspase-3 dependent, as caspase-3 inhibitors can abolish the disruption of ZO-1 and increase epithelial permeability (Chin et al., 2002). An- other study has shown that the reorganization of ZO-1, F-actin and increased permeability were regulated by MLCK via the observation of the increased epithelial myosin light chain (MLC) phosphorylation in SCNB cells exposure to Giardia sonicates and the inhibition of phosphorylation by a MLCK inhibitor (Scott et al., 2002).

Giardia-induced enterocyte apoptosis together with apical junctional disruption have disruptive effects on the intestinal barrier function. The breakdown of the epithelial barrier allows normal flora bacteria, macromolecules, elec- trolytes and other molecules to pass into sub-underlying tissue, by-passing the normal uptake by epithelial cells. The paracellular flow of nutrients, elec- trolytes and other molecules can not only contribute to malabsorption but may also induce host immune responses.

Intestinal microbiota The interactions between Giardia and intestinal microbiota play an important role in giardiasis. It has been reported that the colonization of Giar-

21 dia trophozoites in mice is affected by the composition of intestinal microbiota (Singer and Nash, 2000). Intestinal microbiota has also been reported to stimulate the pathogenicity of Giardia (Torres et al., 2000) and the intestinal microbiota may contribute to CD8 T cell-mediated brush border enzyme impairment during Giardia infection in mice (Keselman et al., 2016). The interactions between Giardia and intestinal microbes have been studied experimentally. A recent report has shown that Giardia can synergize with commensal bacteria to disrupt homeostatic host-microbiota interactions by the observation that Giardia-induced functional changes in commensal E. coli can kill Caenorhabditis elegans in the absence of Giardia trophozoites (Gerbaba et al., 2015). Giardia-induced alteration of microbiota biofilms has also been demonstrated, partly mediated by its cysteine proteases, resulting in epithelial apoptosis, tight junctional ZO-1 disruption, bacterial translocation, increased epithelial TLR4 expression and CXCL-8 secretion (Beatty et al., 2017). Moreover, a recent report has investigated the effect of protein malnutrition on mice infected with Giardia. It was shown that Giardia disrupts microbiota-mediated pathogen clearance, causing alteration in the composition of resident microbiota, and impaired host growth (Bartelt et al., 2017). The alteration of intestinal immune responses to enteroaggregative E. coli (EAEC) has also been seen in persistently Giardia infected mice (Bartelt et al., 2017). Furthermore, mice infected with Giardia exhibited systemic dysbiosis of gut commensal bacteria with expansions in aerobic and decreas- es in anaerobic bacteria (Barash et al., 2017). Together, these observations suggest that the interactions between Giardia and intestinal microbiota have an important role in affecting the outcomes of the disease. Probiotic therapies have been proposed as a possible way to control Giar- dia infections. Lactobacilli were shown to have an inhibitory effect on trophozoite proliferation in vitro (Perez et al., 2001) and have anti-giardial probiotic effects in animal models (Humen et al., 2005, Goyal et al., 2011, Shukla and Sidhu, 2011). The toxicity to Giardia trophozoites was associated with bile salts. Conjugated bile salts have been shown to have promoting effects on Giardia growth in vivo (Halliday et al., 1988), whereas unconju- gated bile salts generated by secreted Bile-Salt Hydrolases (BSH) from the probiotic strain L. johnsonii La1 may have a inhibitory effect on trophozoite growth (Perez et al., 2001, Travers et al., 2016). The anti-giardial mechanism by L. johnsonii La1 was further studied in more detail and it has shown that activities of two BSHs derived from L. johnsonii La1 contribute to anti- giardial effects in vitro and in vivo (Allain et al., 2017b). In addition, probiotic oral feeding of Enterococcus faecium and Lactobacillus casei separately both lessened the parasite burden, improved specific immune responses (Benyacoub et al., 2005) and reduced mucosal damage (Shukla et al., 2008). Most studies were done using mouse models or biopsy samples from patient with gastrointestinal diseases. Little is known about the composition of microbiota at specific regions of the human intestine in the presence and ab-

22 sence of Giardia, which also limits our understanding of the interplay between Giardia and human intestinal microbiota.

Mucus layer The mucus layer that lays on the surface of the intestinal epithelium acts as both a physical and immunological barrier, providing protection of the luminal surface of the gastrointestinal tract (Johansson and Hansson, 2016). It is important to prevent normal flora bacteria and enteric pathogens to gain access to underlying tissue. The thickness of the mucus layer in the intestinal tract increases in a proximal-distal direction. There are two types of mucus organization in the intestinal tract. The colon has two mucus layers with an outer loose mucus layer and an inner, thick mucus layer. The outer layer is the habitat for commensal bacteria, while the inner thick mucus layer firmly attached to the epithelial surface and is impermeable to bacteria (Johansson et al., 2008). In contrast to the colon, there is only one single mucus layer in the small intestine that usually covers the villi tips (Atuma et al., 2001, Ermund et al., 2013). The mucus layer in the small intestine is not anchored to the epithelial surface and it moves with the peristaltic waves in a distal direction. Fresh mucus is constantly secreted from the goblet cells, especial- ly in the crypt openings (Ermund et al., 2013). The mucus layer acts as a protective barrier that microorganisms must traverse in order to reach the epithelial surface. There are numerous other antimicrobial proteins such as defensins, lysozyme and secretory antibodies (sIgA) embedded in the mucus layer (Johansson and Hansson, 2016).

MUC2 The main structural component of the small intestinal mucus layer is MUC2 which is a gel-forming mucin secreted by goblet cells in the small intestine (Carlstedt et al., 1993, Herrmann et al., 1999). MUC2 consists of two large highly O-glycosylated mucin domains. The less O-glycosylated N- and C- terminal cysteine-rich domains are more likely to be targeted by parasitic proteases. To reach and make contact with the intestinal epithelium, Giardia trophozoites must acquire the ability to cross the protective mucus barrier. Giardia flagella generate high motility and they position and orient trophozoites prior to attachment (House et al., 2011), but it has been shown that the flagellar motility may not be sufficient to overcome the mucus barrier (Paget and James, 1994). Mucus degradation is well-studied in Entamoeba histolytica, a diarrhea-causing parasite in the colon that induces mucin secretion, depletes mucin stores and ultimately facilitates invading by the depletion of the mucus layer (Chadee and Meerovitch, 1985, Tse and Chadee, 1992). This process is completed by cysteine proteases (CPs) that degrade colonic mucin (Moncada et al., 2000). A later study found that E. histolytica CPs degrade

23 the MUC2 C-terminal domain at two distinct sites resulting in the mucus depolymerization (Lidell et al., 2006). Very little is known how Giardia interacts with the intestinal mucus layer. It was suggested that mucins have a protective role against Giardia via their inhibitory effect on trophozoites attachment (Roskens and Erlandsen, 2002). A recent report demonstrated that Giardia trophozoites disrupt the intestinal mucus layer by degrading MUC2 in a CP-dependent manner. Infection of MUC2 knock-out mice resulted high levels of trophozoites in the small intestine and impaired weight gain. CPs activities are also responsible for depletion of mucin stores and induction of differential mucin gene expression (Amat et al., 2017). Howev- er, the specific giardial CP causing this and the mechanism of how giardial CP disassembles the mucin polymer has yet to be determined.

Antimicrobial peptides There is a large number of molecules imbedded in the mucus layer that play critical roles in the intestinal homeostasis and protect against enteropatho- gens (Bevins and Salzman, 2011). Induction of antimicrobial peptides (AMP) was reported in human and mice infected with a wide range of en- teropathogens (Muniz et al., 2012, Tumanov et al., 2011, O'Neil et al., 1999). AMPs are produced by a variety of host cells including Paneth cells (Ayabe et al., 2004) and can be classified as magainins, cathelicidins and defensins. Defensins are stored in the secretory granules in the Paneth cells located in the intestinal crypts (Lasserre et al., 1999, Ouellette et al., 1999, Porter et al., 1997a). Paneth cells continuously secret these molecules at a baseline level and higher amounts of secretion was observed when IECs were exposed to bacteria (Ayabe et al., 2000). Humans produce both α-defensins and β- defensins. α-defensin 5 and 6 (α-HD5,-6) are enteric, continuously expressed (Chairatana et al., 2016, Jones and Bevins, 1992) and released into the mucus layer and lumen following degranulation. α-HD5 has been reported to kill a wide range of microbes under laboratory conditions (Porter et al., 1997b, Molau et al., 2005, Furci et al., 2015, Wommack et al., 2014). During Giardia infections, a number of anti-parasitic factors are produced in order to clear Giardia. It has been shown that defensins were cytotoxic for both trophozoites and cysts in vitro (Aley et al., 1994). In vivo studies have suggested that matrix metalloprotease 7 (MMP7), which is a mediator to activate α-defensins, play an important role in the control of G. muris (Eckmann, 2003) and G. intestinalis infections (Tako et al., 2013). Blocking of MMP7 and inducible nitric oxide synthase (iNOS) in mice resulted in significant defects in infection control (Tako et al., 2013). It has also been shown that there is an increase in expression and production of AMP in human IECs in a CP-dependent manner when co-incubated with Giardia and enteropathogenic E. coli (Manko et al., 2017).

24 Immunoglobulins It is known that Giardia-infected humans generate serum antibodies (IgG) and intestinal secretory antibodies (sIgA) in response to Giardia infections (Smith et al., 1981, Nash et al., 1987) and patients with immunoglobulin deficiencies develop chronic giardiasis (Olmez et al., 2014). Anti-Giardia IgG was detected in colostrum from Giardia-infected calves (O'Handley et al., 2003). Anti-Giardia IgA was also detected in human saliva and breast milk and associated with protection against disease (Walterspiel et al., 1994, Tellez et al., 2005). The measurements of anti-Giardia IgA and IgG were used as a diagnostic tool to monitor the exposure of children to Giardia infections (Rodriguez et al., 2004, El-Gebaly et al., 2012). IgA is the most abundant antibody in the mucosal secretions (Cerutti and Rescigno, 2008) and transported to mucosal surface by a polymeric immunoglobulin receptor (pIgR). IgG is also synthesized and transported to intestinal lumen by human neonatal Fc receptor (Yoshida et al., 2004). Studies conducted in mice have shown that IgA is required for effective clearance of both G. muris and G. intestinalis (Langford et al., 2002). However, the sensitivity of the two Giar- dia species to IgA seems to be different, as G. muris infected pIgR-deficient mice developed chronic infections (Langford et al., 2002), while IgA level in the mucosal surface was sufficient to clear G. intestinalis and no difference in parasite clearance was detected in pIgR-deficient mice and the control mice (Davids et al., 2006). Many intestinal bacteria and parasites, colonizing the human intestinal tract, secrete proteases that degrade IgA (Mistry and Stockley, 2006, Garcia-Nieto et al., 2008, Lee et al., 2014b). An early report showed that Giardia extracts have a proteolytic activity on IgA1 but the protease was never identified (Parenti, 1989).

Nitric oxide Intestinal epithelial cells produce nitric oxide (NO) that functions as antimicrobial substance. NO is produced from L-arginine by three types of NOSs, neuronal NOS (nNOS, NOS1), iNOS or NOS2 and endothelial NOS (eNOS, NOS3). In IECs, NO is largely produced by iNOS. Patients infected with Giardia exhibited reduced expression of iNOS and increased levels of NO (Mokrzycka et al., 2010, Matowicka-Karna et al., 2011). An early study has shown that NO is cytostatic to Giardia trophozoites growth, prevents both encystation and excystation induced in vitro and thus could maintain infection in the small intestine and interfere with the parasite transmission (Eckmann et al., 2000). Studies conducted in vivo did also show that NO is involved in the clearance of Giardia in mice models (Li et al., 2006, Andersen et al., 2006, Tako et al., 2013).

25 Host cytokine profile The transcription of genes coding for components of intestinal mucosal immunity using in vitro and in vivo models are of great importance to understand host cellular responses during Giardia infections. In response to pathogens, intestinal enterocytes produce proinflammatory chemokines and cytokines that regulate the immune responses (Maaser and Kagnoff, 2002). Co- incubation of human Caco-2 cells with Giardia resulted in upregulation of genes coding for chemokines, including CCL2, CCL20, CXCL1, CXCL2 and CXCL3 (Roxstrom-Lindquist et al., 2005). This unique chemokine profile targets different populations of immune cells, like dendritic cells (DC), T cells and B cells by CCL20, neutrophils by CXCL1 to CXCL3, and macrophages and T-cells by CCL2 to the site of infection (Roxstrom-Lindquist et al., 2005). Assemblage B Giardia trophozoites, isolate GS, induce a stronger inflammatory signal than parasites of the assemblage A isolate WB (Ma'ayeh et al., 2018). Cell cycle inhibitory genes were upregulated after co- incubation of host cells and trophozoites, leading to a reduced cellular proliferation (Stadelmann et al., 2012). Host gene expression of Giardia infected mice has shown that genes associated with antibodies, mast cell proteases and MMP7 were upregulated (Tako et al., 2013). Pro-inflammatory cytokines (CXCL1, CXCL2, CCL2 and IL-8) were induced by co-incubation of Caco-2 cells with macrophages and the secretion was abolished by the addition of Giardia, indicating immune-modulatory of Giardia (Fisher et al., 2013). This is in line with the observations that Giardia can alter the host’s immune responses (Kamda and Singer, 2009, Cotton et al., 2014b). It has been shown that cysteine proteases released by Giardia have a role in degrading IL-8 induced by other pro-inflammatory stimuli (Cotton et al., 2014a), but the involvement of cysteine proteases in immune-modulatory of Giardia requires further investigations. There are a few cytokines, such as IL-6, IL-17A and TNFα, that have been shown to be implicated in giardiasis in vivo. IL-6-deficient mice have a diminished ability to clear the infection caused by Giardia (Bienz et al., 2003, Zhou et al., 2003). DC-derived IL-6 is essential to clear Giardia infection as transferring DC into IL-6-deficient mice promotes clearance of the parasite (Kamda et al., 2012). Activation of DC maturation by Giardia was also shown to increase expression of pro-inflammatory cytokines such as IL- 6, TNF-α, IL-12 and surface molecules such as CD80, CD86, MHC class II molecules (Lee et al., 2014a, Grit et al., 2014). Mice lacking TNFα was found to be delayed in the clearance of parasites (Zhou et al., 2007) and this is consistent with increased TNFα levels in sera of infected children (Bayraktar et al., 2005). IL-17 has six members (A-F), of which IL-17A is best studied. IL-17A produced by CD4+ T cells has been shown to contribute to clearance of infections caused by G. muris and G. intestinalis (Dreesen et al., 2014, Dann et al., 2015, Saghaug et al., 2016).

26 Virulence factors Giardiasis is a multifactorial disease. Factors that are involved in the attachment, the flagellar motility and antigenic variation are considered as virulence factors (Ankarklev et al., 2010, Emery et al., 2016). Additionally, ESPs have also been suggested to have an essential role in pathogenesis of Giardia. To survive in the harsh environment in the hosts’ small intestine, Giardia must develop the ability to avoid the attacks from the host. Upon the interaction, Giardia releases large numbers of ESPs. An early study of Giar- dia ESPs showed an effect on intestinal absorption and secretion (Samra et al., 1988). Caco-2 and SCBN monolayers exposed to sonicated parasites and culture supernatants containing no live trophozoites have shown rearrange- ments of F-actin (Teoh et al., 2000). Oral administration of ESPs from Gi- ardia to pathogen-free mice induces Th2 immune responses and eosinophil infiltration (Jimenez et al., 2004). Glycoproteins in the Giardia ESPs may have a role in inducing a human antibody response (Jimenez et al., 2007). The heat-insensitive ESPs from Giardia have been shown to be sufficient to synergize with commensal bacteria to kill C. elegans (Gerbaba et al., 2015). Identification and characterization of ESPs will be of great importance to understand the molecular pathogenesis of Giardia. But very few specific Giardia virulence factors have been identified and characterized (Table 2).

Table 2. Identified and characterized specific virulence factors in Giardia intestinalis. “A thiol proteinase” and “A 58 kDa ESP” are from the P1 strain and all other factors are from the WB strain. Giardia factor Function Localization References A thiol proteinase IgA hydrolysis - (Parenti, 1989) (Kaur et al., 2001, Shant Fluid accumulation in et al., 2002, Shant et al., 58 kDa ESP host intesine Cell membrane 2005) (Weiland et al., 2003, Attachment in vitro, Jenikova et al., 2011, α-1 giardin vaccine candidate Plasma membrane Weeratunga et al., 2012) δ-giardin Attachment in vitro Ventral disc (Jenkins et al., 2009) Punctuate pattern (Ringqvist et al., 2008, ADI Arginine consumption within cytoplasm Stadelmann et al., 2012) Cytoplasm with accumulation near OCT Arginine consumption plasma membrane (Ringqvist et al., 2008) ER, cytoplasmic (Ward et al., 1997, CP1 or CP10217 Excystation vesicles DuBois et al., 2006) Excystation, encystation, (DuBois et al., 2008, CP2 occludin and claudin-1 Abodeely et al., 2009, or CP14019 degradation, apoptotic ER, cytoplasmic Ortega-Pierres et al., or Giardiapain-1 damage vesicles 2018, Ward et al., 1997) CP3 or CP16779 Excystation - (Ward et al., 1997) Cell monolayers disrupt- (Cabrera-Licona et al., VSP9B10A ion Cell membrane 2017)

27 Attachment The first step to establish a Giardia infection is parasite attachment to host intestinal villi, in order to proliferate and avoid peristalsis. The attachment of trophozoites to host cells is an important virulence factor; without the attachment they would be swept away by the peristaltic wave (Crouch et al., 1991). It has also been shown that adhesion-deficient clones isolated from Giardia have a reduced ability to establish infections in Mongolian gerbils (Hernandez-Sanchez et al., 2008). Attachment is accomplished by the ventral disc, flagella and a number of giardial surface proteins. Giardial attachment occurs as a stepwise process. Trophozoite’s ventrolateral flange, lateral crest, lateral shield and bare area sequentially make contact with the substrate during the attachment procedure and the detachment occurs in reverse order to the stages of attachment (House et al., 2011). The flagellar motility is critical for cellular orientation and positioning prior to attachment, but it is not directly required to maintain attachment (House et al., 2011). A number of parasitic factors have been implicated in the process, e.g. surface proteins with lectin activity. Attachment can be impaired by binding the entire surface of live trophozoites with anti-surface antibodies or by pre- incubating parasite and IECs with monosaccharides (Gillin et al., 1990, Magne et al., 1991, Inge et al., 1988, Sousa et al., 2001, Katelaris et al., 1995). Giardins are Giardia-specific proteins that include α-, β-, γ- and δ- giardin that are associated with microtubules in the ventral disc and plasma membrane of trophozoites (Faubert, 2000). Antibodies against α-1 giardin were detected in serum samples of patients recently infected with Giardia (Palm et al., 2003) and recombinant giardial α-1 giardin binds to human intestinal epithelial cells in vitro (Weiland et al., 2003). The crystal structure determination of this protein also demonstrated its direct role in binding to host glycosaminoglycan and thus provides a mechanism of attachment of the adhesive-disc free excyzoite implicated in the establishment of Giardia infections in the host (Weeratunga et al., 2012). Immunization of mice using α-1 giardin generated a marked protection against Giardia infection, making it a suitable vaccine candidate against giardiasis (Jenikova et al., 2011). Pretreatment of Giardia trophozoites with anti-δ-giardin sera induced morphological changes in the parasites and inhibited trohpozoites binding to the surface of cell culture slides (Jenkins et al., 2009). Membrane lipid rafts of the trophozoites are also implicated in the trophozoites attachment to human enterocyte-like cells, as diminished attachment was observed by the treatment with methyl‐β‐cyclodextrin, a lipid raft disorganizing agent (Humen et al., 2011). Moreover, it was suggested that unknown giardial cysteine proteases aid in attachment during host-parasite interaction (Rodriguez-Fuentes et al., 2006).

28 Antigenic variation Surface antigenic variation occurs in a variety of organisms, including protists, bacteria and viruses (reviewed in (Deitsch et al., 1997)). Giardia trophzoite undergoes surface antigenic variation by changing the expression of its variant-specific surface proteins (VSPs) periodically. Giardia possesses approximately 200 VSP genes (Adam et al., 2010, Morrison et al., 2007). VSPs are the major surface protein and densely coat the entire surface of this parasite, including ventral disc and flagella, provide a dense interface between the parasite and host (Pimenta et al., 1991), are thought to be a resistance mechanism protecting the parasites from host intestinal proteases (Nash, 2002). VSPs are characterized by the presence of N-terminal variable regions containing a high frequency of CXXC motifs, a conserved C-terminal transmembrane domain and a conserved cytosolic CRGKA tail (Adam, 2001, Mowatt et al., 1991). The structures of VSPs suggest that they may have essential roles in contributing to Giardia infections by interacting with the host cells. Each parasite expresses only one VSP at a time and change surface VSPs spontaneously in the absence of stimuli or anti-VSP antibodies (Nash et al., 1988, Nash and Mowatt, 1992). Changes in VSP expression also occur during encystation and excystation (Svard et al., 1998, Carranza et al., 2002, Einarsson et al., 2016b). The frequency of VSP switching is isolate-dependent with switching every 12-13 generations in WB isolate and 6.5 generations in the GS isolate (Smith et al., 1982, Nash and Keister, 1985, Nash et al., 1985). VSPs have been linked to the evasion of host’s immune system (Prucca and Lujan, 2009), the generation of protection against Giar- dia in the gerbil model of giardiasis (Rivero et al., 2010a), trophozoites attachment to epithelial cells (Bermudez-Cruz et al., 2004) and in protection of the parasite from intestinal proteases (Nash et al., 1991). Most in vivo studies of antigenic variation have been conducted in mice using the Giardia GS isolate. Early studies showed that humans infected with the GS isolate expressing a particular VSP (VSPH7) had parasites expressing other VSPs than in the initial clone after 17 days post-infection and the initial VSP was completely replaced 21 days post-infection (Nash et al., 1990). Similar outcomes were observed in mice infected with the same isolate (Bienz et al., 2001, Singer et al., 2001, Stager et al., 1998). These results indicate that immune pressure is one factor contributing to VSPs switching. However, a study conducted in immunodeficiency mice revealed that other signals for switching exist apart from pressure of the immune system (Singer et al., 2001). Soluble host factors may have a role in driving antigen switching events (Emery et al., 2016). Differential expression in VSPs was observed when Giardia trophozoites were co-incubated with soluble host factors generated from HT-29 cells. A quarter of all expressed VSP variants were upregulated after exposure to soluble host factors, some of which con-

29 tain additional functional domains including metalloprotease domain homologue to Leishmania virulence factor leishmanolysin and BmKX domain found in scorpion toxins, suggesting VSPs might also act as independent virulence factors (Emery et al., 2016). A recent study of high molecular weight proteins secreted by Giardia during interaction with a IEC-6 cell monolayer identified the VSP9B10A protein. Further characterization using transfected trophozoites that constitutively express the VSP9B10A protein demonstrated a proteolytic activity of the VSP9B10A protein and it caused cytotoxic damage in these cell monolayers (Cabrera-Licona et al., 2017), indicating the relevance of the specific VSP with trophozoite’s virulence. VSPs switching is essential for the survival of Giardia, but the mechanism of how the switch is triggered and regulated is unknown.

ADI and OCT A wide variety of pathogens are known to interfere with the host arginine metabolism by competing with host NOSs for arginine via up-regulation of host arginases or via competition from parasitic arginases (Das et al., 2010, Morris, 2010). NO produced by the conversion of L-arginine via NOSs is critical for the clearance of Giardia infection as mentioned above. However, Giardia has also evolved strategies to evade NO-mediated host defenses. Arginine is the primary energy source of Giardia (Schofield et al., 1992). Earlier secretome studies using two-dimensional gel analyses identified four proteins, including arginine deiminase (ADI), ornithine carbamoyl transferase (OCT), α-enolase and elongation factor 1-α (EF-1α)) from the culture supernatant of trophozoites following co-incubation with human intestinal epithelial cells (Ringqvist et al., 2008). ADI and OCT belong to arginine metabolizing enzymes (AMEs) and they are involved in arginine dihydrolase pathway. Within the pathway, arginine is converted into citrulline by ADI, then into ornithine and carbamoyl phosphate by OCT and finally into ammonia and CO2 by carbamate kinase (CK), resulting in the generation of ATP through substrate level phosphorylation. The competition between ADI and iNOS for arginine uptake affects NO production and further affects the outcome of the disease. It has been shown that arginine consumption by Giardia trophozoites prevents NO production by IECs. During infection, Giardia consumes arginine as energy source and arginine starvation was observed after 4 h of co-incubation with Caco-2 cells (Eckmann et al., 2000). Supplementation with recombinant giardial ADI protein to IECs in vitro induced a reduced NO production from IECs, indicating a cross talk between arginine consumption and NO reduction (Ringqvist et al., 2008). In addition to NO reduction, arginine consumption by Giardia ADI decreased the amount of arginine in the growth medium during in vitro growth, leading to reduced IEC proliferation. Thereby, it was proposed that the reduced host NO response and slower IEC proliferation

30 could favor the proliferation of the trophozoites and maintain a more stable niche for colonization (Stadelmann et al., 2012). Additionally, Giardia ADI has a role in antigenic variation by modifying the cytoplasmic C-terminal tail of VSPs proteins (Touz et al., 2008). Arginine depletion is known to induce apoptosis in another system (Potoka et al., 2003) and it is likely that arginine depletion by ADI also could cause enterocyte apoptosis during giardiasis. Giardia-induced arginine depletion has also been shown to inhibit cytokine production by human dendritic cells (Banik et al., 2013).

Cysteine proteases Cysteine proteases (CPs) are characterized by an active site with one cysteine and one histidine residue, and they are categorized as clan CA cysteine proteases. They have been subdivided into three categories: cathepsin B, cathepsin C and cathepsin K/L. Cathepsin B contains a 20 amino acid (aa) peptide loop insertion referred to as “occluding loop” that enables cathepsin B to function as an endo- and exopeptidase (Sajid and McKerrow, 2002). The additional exopeptidase activity of human cathepsin B is attributed to two histidine residues (Musil et al., 1991). CP is synthesized as a proenzyme containing a signal peptide, a pro-domain and a catalytic domain or mature enzyme (Figure 3). The pro-domain maintains the protease in an inactive state. The protease is processed to an active mature enzyme by losing the pro-domain via cleavage by another protease or exposure to acidic environment that induces self-cleavage (Turk et al., 2000). The Giardia assemblage A WB genome contains 26 cysteine protease genes with 9 cathepsin B-like, 4 cathepsin C-like and 13 cathepsin K/L-like genes (Morrison et al., 2007, DuBois et al., 2008). In contrast to human cathepsin B, Giardia cathepsin Bs lack the occluding loop, suggesting that the Giardia CPs only function as endopeptidases (Figure 3) (Sajid and McKerrow, 2002).

31 Figure 3. Structure of Giardia intestinalis cathepsin B cysteine proteases. Giardial CP is synthesized as proenzyme with a signal peptide, a pro-domain and a mature enzyme. The protease can be auto-cleaved during purification. “CEHHVNGS- RPPCTGEGDTPKC” is the occluding loop in human cathepsin B protein and “HH” in the occluding loop enables the protease to have exopeptidase activity in addition to its endopeptidase activity. Adapted from (Sajid and McKerrow, 2002, DuBois et al., 2008).

CPs have been described as virulence factors in many pathogenic microbes and are involved in the pathogenesis of other protozoan parasites, including E. histolytica, Trichomonas vaginalis, Blastocystis spp. and T. cruzi (Lidell et al., 2006, Ndao et al., 2014, Hernandez et al., 2014, Nourrisson et al., 2016). Accumulating studies in Giardia have shown that CPs released upon contact with host cells possess proteolytic activities that degrade various substrates. The presence of CP activities in the ESPs fraction was reported in the Giardia P1 strain (Jimenez et al., 2000) and these CPs were found to have proteolytic activity on substrate proteins such as gelatin, collagen, albumin (Coradi and Guimaraes, 2006). CP activities have also been detected in the ESPs fractions of another Giardia strain (BTU-11) and they are the main proteolytic activities that responsible for the degradation of gelatin and collagen type I (de Carvalho et al., 2008). CP activities were also detected in the WB strain during the co-incubation with rat IEC-6 monolayers (Rodriguez-Fuentes et al., 2006). Activities of CPs have also been acknowledged to be essential to pathogenesis of giardiasis. An increase in CP secretion has been seen during host- parasite interactions in vitro and CPs have been shown to be associated with trophozoite attachment (Rodriguez-Fuentes et al., 2006). CP activities have been reported to contribute to mucosal injury and humoral immune responses in BALB/c mice that were orally administrated with ESPs (Jimenez et al., 2009). CPs are also important in differentiation since they play a critical role in excystation and encystation of Giardia (Ward et al., 1997, DuBois et al., 2008). A more recent report demonstrated that Giardia CPs degrade CXCL8 induced by pro-inflammatory interlukin-1β or Salmonella typhimurium, resulting in the attenuation of CXCL8-induced polymorphonuclear leukocytes or neutrophils (PMN) chemotaxis (Cotton et al., 2014a). CP activities are also responsible for disruption of cellular junctions (Chin et al., 2002), cleavage and redistribution of microvillus protein villin (Bhargava et al., 2015), affecting the bacterial normal flora and microbiota biofilm formation (Gerbaba et al., 2015, Beatty et al., 2017), inhibiting the growth of the intestinal bacterial pathogens (Manko et al., 2017), degrading the intestinal mucus layer (Amat et al., 2017). However, most studies of CPs role in the pathogenesis of giardiasis are limited to use of culture supernatants and/or inhibitors of CPs. Very little is known about the involvement of specific CPs in the disease mechanism.

32 Cathepsin B-like proteases are the most highly expressed cathepsins in Giardia and many are upregulated during differentiation and IEC-parasite interactions in vitro (Table 3) (Ringqvist et al., 2011, Ma'ayeh and Brook- Carter, 2012, Ferella et al., 2014, Emery et al., 2014, Emery et al., 2016, Einarsson et al., 2016b, Dubourg et al., 2018). Specific CPs have been suggested to play an important role in excystation (CP1 or CP10217, CP2 or CP14019 and CP3 or CP16779) (Ward et al., 1997), encystation (CP14019) (DuBois et al., 2008), and degradation of endocytosed proteins (Abodeely et al., 2009). A recent study using mAb1G3 antibody purified a cysteine protease CP14019, was given the name giardiapain-1 due to its papain-like features, from Giardia trophozoites. Characterization of this protease has shown that giardiapain-1 is capable of degrading and delocalizing tight junction proteins occludin and claudin-1, inducing apoptotic damage (Ortega-Pierres et al., 2018). Otherwise, no more study regarding specific CPs has been doc- umented. The roles of specific CPs in the molecular pathogenesis of giardiasis remain unclear.

Table 3. Giardia WB CP transcriptional changes and secretion during host parasite interactions in vitro. Gene ID Observed References GL50803_10217 Protein was detected in supernatant (Dubourg et al., 2018) GL50803_14019 Gene expression was upregulated (Ferella et al., 2014, during host-parasite interaction and Ma'ayeh and Brook-Carter, protein was detected in supernatant 2012, Dubourg et al., 2018) GL50803_15564 Protein was detected in supernatant (Dubourg et al., 2018) GL50803_16468 Protein was detected in supernatant (Dubourg et al., 2018) GL50803_16779 Gene expression was upregulated (Ringqvist et al., 2011, during host-parasite interaction and Emery et al., 2016, Dubourg protein was detected in supernatant or et al., 2018) when exposed to host soluble factors GL50803_17516 Gene expression was upregulated and (Ringqvist et al., 2011, protein was detected in supernatant Ferella et al., 2014, Dubourg et al., 2018)

Other factors There are several semi-identified but uncharacterized proteins that may have a role in the pathogenicity of Giardia. Investigation of Giardia assemblage A P1 strain sonicates revealed a thiol protease that can cleave human IgA (Parenti, 1989). A 58 kDa active glycoprotein purified from ESPs of assemblage A P1 strain was found to induce fluid accumulation in ligated rabbit ileal loops and in the intestine of sealed adult mice and it caused morphological changes in HEp-2 cells (Kaur et al., 2001, Shant et al., 2002). A later study of the specific ESP found that it could bind to a 41 kDa glycoprotein on the enterocytes and increase the intracellular Ca2+ concentration (Shant et

33 al., 2004). It has also been shown that the cause of ion secretion and fluid accumulation in mice intestine was via diverse signal transduction pathways (Shant et al., 2005). Additionally, earlier immunization of mice with this specific protein led to failure of colonization (Kaur et al., 1999). However, the identified partial amino acid sequence from the 58 kDa protein (AD- FVPQVST) (Kaur et al., 2001) does not resemble any sequences in the WB genome but partly with bovine serum albumin (BSA) (Ringqvist et al., 2008). Later work in serum free medium with non-interacting assemblage A WB trophozoites has confirmed a 58 kDa fragment to be ADI (Skarin et al., 2011). Whether Giardia WB ADI contains the toxin-like features of the described 58 kDa protein, or if the 58 kDa protein is a P1 strain-specific protein requires further investigation. An early secretome study identified two more proteins, enolase and EF- 1α, in addition to ADI and OCT (Ringqvist et al., 2008). Our understanding of enolase is limited to its cytoplasmic localization in trophozoites (Ringqvist et al., 2008). Molecular modelling suggested that Giardia enolase can bind plasminogen but it has not been verified experimentally (Aguayo- Ortiz et al., 2017). Little is known about EF-1α. EF-1α localizes to ER and closer to cell membrane upon contact with human cells. It is an immunoreactive protein recognized by antibodies from patients who had previously giardiasis (Skarin et al., 2011). EF-1α secreted by Leishmania has been shown to downregulate host inflammatory cell signaling (Nandan and Reiner, 2005). A recent proteomic study has shown that EF-1α is only detected in assemblage A and raises the possibility of its relevance to the differences in pathogenicity or host ranges between assemblage A and B (Dubourg et al., 2018). However, the role of those two proteins as putatively virulence factor in giardiasis remains unknown. High-cysteine membrane proteins (HCMPs) and Tenascins may also have important roles in the pathogenesis of Giardia. HCMPs have a similar structure as VSPs but lack the C-terminal tail. HCMPs have been reported to be transcriptionally upregulated during host cell interaction (Ma'ayeh and Brook-Carter, 2012, Roxstrom-Lindquist et al., 2005, Ferella et al., 2014). Tenascins are another group of proteins highly secreted not only in axenic culture but also induced by soluble host factors and involved in the regulation of attachment of trophozoites (Emery et al., 2016, Dubourg et al., 2018), indicating their may have a role as virulence factors in pathogenesis of giardiasis.

34 Current investigation

It is known that Giardia infection induces symptoms via several mechanisms involving both host and parasitic factors. However, little is known about the specific virulence factors involved. The aim of this thesis is to identify putative virulence factors in Giardia and characterize their roles in disease induction. To do this, proteomics was used to study the interplay between host and parasites (Paper I). Three major secreted CPs identified from a secretome study were further characterized (Papers II, III and VI). The three CPs are capable of opening the apical junctional complexes and degrading chemokines (Paper II). Phage display was applied to study the substrate cleavage specificity of these CPs in order to search for human potential in vivo substrates (Paper III). Studies later on were conducted to verify these identified substrates, including immunoglobulins, defensins and mucus (Papers III and VI).

Secretome study of IECs-parasites interplay (Paper I) In this study, we applied proteomics to identify the major ESPs when Giar- dia trophozoites of the WB or GS isolates interact with IECs in vitro in order to identify putative virulence factors and elucidate mechanistic pathways leading to disease. This study provides new insights into host-parasite interactions and provides the basis for further studies of specific putative virulence factors.

Secretome of Giardia in the absence and presence of host cells We collected the ESPs from Giardia grown axenically or in the presence of IECs in order to identify proteins in the Giardia secretome. 196 WB proteins and 155 GS proteins were identified when were maintained in modified serum-free medium. In the co-culture from Giardia incubated with IECs, 248 WB proteins and 152 GS proteins were identified. The top 50 proteins identified from the co-culture overlaps with those in axenic culture. Comparison between the Giardia secretome incubated with or without IECs identified 87 WB interaction-specific proteins and 41 GS interaction-specific proteins. Only 13 proteins overlap between the two isolates amongst the interaction- specific proteins, indicating the assemblage-specific difference during IEC- Giardia interaction. The majority of the identified proteins have functions

35 associated with metabolism. Proteins involved in anti-oxidation, proteolysis and encystation responses were identified. Immunodominant and glycosylated proteins were also seen in the secretome of axenic cultures.

Secretome of differentiated Caco-2 cells during parasite interaction We also analyzed the released proteins of differentiated Caco-2 cells incubated with Giardia trophozoites. 384 Caco-2 cell proteins were identified in the medium of incubation with WB trophozoites and 355 proteins were identified in co-culture incubated with GS trophozoites. Comparisons between the secretomes of co-culture and control identified 76 proteins released specifically in response to WB and 45 proteins specifically in response to GS. Amongst the specific proteins, 31 proteins overlapped in the responses to both isolates. The secretome profile of parasitized IECs showed that the identified proteins are associated with metabolism, cell cytoskeleton and immunological functions.

Transcriptional responses of IECs to Giardia ESPs To investigate the effect of Giardia ESPs on the induction of chemokines in IECs, differentially expressed genes were analyzed in differentiated Caco-2 cells exposed to Giardia ESPs at different time points. We identified 120 and 87 genes differentially expressed in IECs when incubated with ESPs of WB and GS trophozoites, respectively. The majority of these genes were upregulated at 6 h compared to 2 h and they were associated with inducing inflammatory responses, glucose homeostasis and cell cycle.

The effect of ESPs on cell signaling Next, we collected Giardia ESPs from axenic culture, labelled the ESP with fluorescent dyes and incubated them with IECs in order to examine the interactions with IECs. Confocal microscopy demonstrated that the labelled ESPs bind to the surface, cellular borders or junctions and some are even internalized into the Caco-2 cells. The MAPK signaling pathway in IECs was examined when exposed to Giardia ESPs. A slight decrease in the phosphorylation levels of ERK1/2 and P38 at 2 h but an increase in the level of NFκB nuclear translocation were seen, suggesting other factors such as stress, growth factors, hypoxia contributing to NFκB activation. A marked decrease in ERK1/2 and P38 phosphorylation was seen at 6 h and this is coincided with a decrease in NFκB nuclear translocation. Incubation of Gi- ardia ESPs with inflamed IECs exhibited reduced phosphorylation of ERK1/2 and P38 as well as nuclear recruitment of NFκB compared to the control, indicating Giardia ESPs can attenuate inflammatory signaling mediated by MAPK phosphorylation and NFκB recruitment. These results suggest that specific parasitic factors are released to subvert such effect.

36 Secreted Giardia CPs are key virulence factors (Papers II, III and IV) CP activities play a significant role during Giardia infections. However, most studies were conducted using Giardia cell extracts and/or CP inhibitors, but the function of specific CPs in Giardia’s pathogenicity requires further investigation. In this thesis, we focused on three identified secreted Giardia CPs (CP14019, CP16160 and CP16779), characterized them and identified their potential roles in the molecular pathogenesis of Giardia infections.

Tight junction disruption and chemokine degradation (Paper II)

Identification of secreted Giardia CPs To assess the secretion of CPs from Giardia WB trophozoites, we collected supernatants from both axenic cultures and co-culture with IEC monolayers and examined their proteolytic activity in the presence or absence of E64 on gelatin zymogram gel. Subsequently, we cut the bands from the gels and analyzed them by mass spectrometry. Two CPs, CP14019 and CP16779, were identified in the gel bands, which is consistent with previous reports that the two CPs are upregulated at transcriptional level during host-Giardia interactions and are released into the medium (Ringqvist et al., 2011, Ma'ayeh and Brook-Carter, 2012, Ferella et al., 2014, Emery et al., 2014, Emery et al., 2016, Dubourg et al., 2018). CP16160 could also be released into the medium as it shows high similarity with the other two CPs and this was verified by our recent secretome study that the three CPs are the main CPs released during IECs-Giardia interactions (Paper I).

Localization of Giardia CPs Three CPs with epitope tags were constructed, transfected into Giardia WB trophozoites, and imaged by immunofluorescence microscopy. The results showed that all three CPs have similar localization pattern and they localized to ER and vesicles-like structures within the cytoplasm, similar to what has been seen earlier with CP14019 and CP10217 (Abodeely et al., 2009, DuBois et al., 2006). Therefore, together with CP10217 all four CPs have similar localization.

Characterization of recombinant CP14019, CP16160 and CP16779 To study the role of specific CPs, we decided to use P. pastoris to express recombinant proteins for later studies. The three CPs were expressed with a polyhistidine-tag at C terminal and purified by Ni-column. The recombinant CP14019 and CP16779 were purified as mature enzyme (28 kDa), whereas the recombinant CP16160 was purified as two forms, proenzyme (37 kDa)

37 and mature enzyme (28 kDa). But the proenzyme can be auto-cleaved to mature enzyme by a 2 h incubation in an acidic environment. Then, we tested the activity of the mature enzymes and calculated their kinetic parameters by measuring their ability to cleave artificial substrates N-carbobenzoxy- phenylalanyl-arginyl-7-amido-4-methylcoumarin (Z-FR-AMC) and N- carbobenzoxy-arginyl-arginyl-7-amido-4-methylcoumarin (Z-RR-AMC). The results showed that all three CPs were more active under acidic condi- tion and they cleaved the substrates with different reaction dynamics.

Breakdown of epithelial barrier by Giardia ESPs and recombinant CPs Giardia causes intestinal barrier dysfunction by the rearrangement of tight junction proteins, ZO-1, occludin, caludin-1 and cytoskeleton proteins, α- actinin and F-actin. This has been found to be associated with an increase in epithelial permeability (see above). We decided to investigate the effect of Giardia ESPs and recombinant CPs on the integrity of the intercellular junctions. First, we collected ESPs from axenic culture, concentrated the supernatant and added to IECs. The changes in intracellular junctional structures were determined by immunofluorescence. The flocculation and reorganization of ZO-1, occludin and claudin-1 in IECs was observed after exposure to Giardia concentrated ESPs. Second, we assessed the effect of CPs on the apical junctional complex by using Western blot and immunofluorescence to examine the degradation of junctional proteins and distribution in IECs after treatment with recombinant CPs. Recombinant CP14019 and CP16779 showed a similar cleavage pattern that they were able to degrade claudin-1 and -4 more pronounced at lower enzyme concentrations, whereas recombinant CP16160 degraded claudin-1 and 4, occludin in a dose-dependent manner. We further showed the degradation of recombinant human occludin and recombinant human E-cadherin by all three CPs. Furthermore, the reduced levels and rearrangement of occludin, caludin-4, E-cadherin and β-catenin were also observed in IECs exposed to recombinant CPs. The reduced disruption could be seen with E64 pretreated CPs. These findings indicate that the three CPs released by Giardia might play a role in compromising the integrity of the intestinal epithelial barrier.

Chemokine degradation by Giardia ESPs and recombinant CPs The activity of CPs has been shown to cleave IL-8 and reduce inflammation (Cotton et al., 2014a). Giardia has also been reported to induce chemokine expression during IECs-Giardia interactions (Roxstrom-Lindquist et al., 2005). To follow up these studies, we tested the effects of Giardia ESPs and recombinant CPs on cytokines/chemokines induced during the interaction between host cells and Giardia (Roxstrom-Lindquist et al., 2005). Concen- trated ESPs and recombinant CPs was incubated with different cytokines/chemokines and potential degradation was visualized by gel electro- phoresis. Giardia ESPs could clearly degrade IL-8, CCL20, CXCL1 and

38 TNF-α. Complete degradation of CXCL3 was seen with ESPs. Recombinant CP14019 and CP16779 cleaved CXCL2, CCL20, CCL2 and IL-8. No CXCL3 degradation could be seen with CP14019 and CP16779, indicating substrate specificity of the two CPs. All tested chemokines were either degraded (e.g. CXCL1-3) or cleaved (e.g. CCL2, CCL20 and IL-8) by CP16160. For all the CPs tested, the degradation of chemokines was abolished by CP inhibitor, E64. These results indicate that Giardia ESPs, specifically CPs, might play a role in modulating inflammatory responses via their ability to degrade cytokines/chemokines produced by IECs.

In this study, we characterized three secreted Giardia CPs (CP14019, CP16160 and CP16779). Their ability to cleave and reorganize apical junctional proteins makes it possible to propose a model (Figure 4) that can explain how Giardia CPs interact to reduce chemokines effects produced by IECs during host-parasite interactions. This model can also partly explain the disruption of intestinal barrier observed in other studies and low levels of inflammation seen during giardiasis. This also highlights that the identified CPs are key virulence factors in Giardia.

Figure 4. Model of cathepsin B cysteine proteases function during Giardia-host cells interactions. Giardia cathepsin B cysteine proteases released during host- parasite interaction have proteolytic activity and are capable of destroying the junctional complexes (TJs and AJs). The CPs can pass the epithelial barrier and degrade chemokines that are induced by intestinal epithelial cells and released on the baso- lateral side in response to Giardia infection.

39 Degradation of immunoglobulins and defensins (Paper III) The role of specific CPs is still incompletely understood. To follow up our study described in Paper II, we decided to investigate the other roles of the three CPs in giardiasis by characterization of their substrate cleavage specificity. Combining the results from substrate phage display and two- thioredoxin approach we generated consensus peptides for potential targets screening against human proteome. A number of host potential in vivo substrates were identified and tested.

Substrate phage display A nonapeptide library containing 5 x 107 unique phage-displayed peptides was used in this study. These phages were modified to have a nine aa long random peptide followed by a polyhistidine-tag displayed on the surface of T7 phages. First, the phage library was bound to Ni-nitriloacetic acid (Ni- NTA) beads and unbound phages were removed by washing. Then, recombinant CPs were added separately to the Ni-NTA bounded phages and phages with susceptible peptide were cleaved and amplified in bacteria as phage sub-library for next round of selection. After a few rounds of selection, the phage plaques were isolated, amplified and sequenced. In our study, we tried all three CPs on phage display but only succeed with CP16160. The reason why we did not succeed in the other two CPs is unknown. We aligned the phage peptide sequences of CP16160, from which we generated a standard peptide “VVSSFSGGV” for further analyses.

Verification of phage display sequence using two-thioredoxin approach Recombinant two-thioredoxin (2-trx) substrates described in (Thorpe et al., 2016) were used to verify the phage display results. We generated eleven peptides by changing a single aa in the standard peptide to address small variation of aa in the aligned phage sequences. The selected peptide sequences were inserted in the linker region of two adjacent thioredoxin proteins and purified from E. coli. Recombinant CP16160 was incubated with each recombinant 2-trx protein at different time pointes and the degradation was visualized by SDS-PAGE gel. CP16160 showed broad substrate specificity as it cleaves Phe, Trp, Tyr, Leu, Arg at P1 position efficiently and it can also tolerate Leu or Tyr at P2 position as well as a Leu or Arg in P1´ position. Although phage display selected a number of negative charged aa, the cleavage of 2-trx proteins containing Glu was much reduced. Since CP14109 and CP16779 are closely related to CP16160, we tested their ability to cleave the recombinant 2-trx proteins. The cleavage pattern of CP14019 was similar to that of CP16160. Interestingly, CP16779 exhibited a different cleavage pattern. It preferred the 2-trx proteins in the trx structure instead of the inserted peptides between the two trx structures, indicating

40 CP16779 has markedly different cleavage specificity than CP14019 and CP16160.

Screening of potential in vivo substrates Combining the data from substrate phage display and 2-trx approach, we set up three aa sequences with P2 (L/S/Y), P1 (F/W/Y/R/L) and P1´ (S/L/R) for screening of human intestinal targets. The most favorable targets are junctional proteins, chemokines, immunoglobulins, defensins and mucins. We have demonstrated in Paper II that the three CPs are capable of degrading apical junctional proteins and chemokines. In this study, we focused on the interactions of CPs with immunoglobulins and defensins.

Cleavage of human immunoglobulins and defensins To study the effects of the three CPs on immunoglobulins and defensins that are involved in the mucosal immune defense, we incubated recombinant CPs separately with human immunoglobulins and defensins. The reaction mix- ture was analyzed by SDS-PAGE gels to study the degradation. All the three CPs degraded the heavy chain of the IgG, IgA1 and IgA2 in a dose- dependent manner. IgG light chain was resistant to CP14109 and CP16160 but slight degradation of light chain can be seen with CP16779. Slight degradation of both IgA1 and IgA2 light chain can be also seen with all three CPs. All three CPs can degrade β-HD1. No clear α-HD5 degradation can be seen with all three CPs. The α-HD6 was degraded by CP14019 and CP16779 in a dose-dependent manner. These results show different substrate cleavage specificity of the three secreted Giardia CPs, suggesting that they might play an important role in the interference with mucosal immune responses during Giardia infection by cleaving anti-Giardia antibodies as well as defensins.

Mucin degradation (Paper IV) To follow up the findings in Paper III that mucins are potential in vivo substrate for Giardia CPs, we decided to assess the roles of recombinant CPs in mucus degradation. In this study, we focus on the MUC2 mucin that is the main structure component of the mucus layer in the small intestine.

Giardia CP14019 cleaves MUC2 N-terminus The activities of the three secreted Giardia CPs (CP14019, CP16160 and CP16779) were tested on MUC2 N- and C-terminal recombinant proteins produced in CHO cells. Activated CPs were incubated with MUC2 N- and C-terminal recombinant proteins at 1:50 ratio under pH 6 at 37 °C for 60 min. The reactions were analyzed under reducing conditions. No C-terminal degradation was observed with all the three CPs, while CP141019 was seen to degrade N-terminus into 150, 100 and 60 kDa products. The cleavage products with molecular masses of 150 and 100 kDa spanned from D’ to the

41 GFP tag. In both products, the N-terminal cleavage site was the same and it is located to the D’ domain (N-I768GQS). The cleavage site is the same as observed for meprin β (Schutte et al., 2014), that has been shown to release anchored small intestinal mucus.

Giardia CP14019 cleaves reduced full length MUC2 We tested CP14109 activity on the reduced full length MUC2 mucin. The MUC2 mucin was purified and reduced-alkylated from human LS174T cells, human jejunum and mouse proximal small intestine. We found that CP14019 was able to cleave the reduced and alkylated MUC2 at multiple sites, leading to a complete loss of MUC2 oligomers and partial degradation of the mono- mer. Mass spectrometric analysis of the human MUC2 cleavage products identified two new N-terminal sites in the CysD1 (N-L1378CYD) and CysD2 (N-D1843VSV) domains.

Giardia CP14019 does not cleave MUC2 in fresh mucus The cleavage in the D’ domain will not disrupt the mucin polymeric nature, while cleavages in CysD domains will dissolve the polymers. Mucin has to have reduced disulfide bonds to observe these CysD cleavage sites, which is mostly not the case in native mucus in vivo. To confirm that CP14019 is capable of disrupting the mucus gel, CP14019 was incubated with freshly collected mucus from a human jejunum and the proximal small intestine of mouse. No degradation was observed, suggesting that Giardia CP14019 is not able to dissolve fresh normal mucus by itself.

In this study, we have studied CPs activity on MUC2 mucin. It was shown that Giardia CP14019 is capable of cleaving MUC2 N-terminal recombinant protein. However, CP14019 does not cleave MUC2 in fresh mucus. The optimal conditions for efficient cleavage by the Giardia CPs are substrate- dependent and several other conditions need to be further tested before we can determine the efficiency of cleavage of MUC2 by these three CPs. It is also possible that some of the other major secreted CPs (e.g. CP10217 and CP16468) actually cut other sites in MUC2 and disrupt the mucus layer and that they work in concert with CP14019 that currently seems to be involved in release of anchored small intestinal mucus.

42 Conclusion and future perspectives

We have studied the secretome of Giardia and a number of potential virulence factors were identified (Paper I). Three major secreted Giardia CPs (CP14019, CP16160 and CP16779) were characterized and their roles in disruption of apical junctional proteins, degradation of chemokines, immunoglobulins, defensins and MUC2 mucin were studied (Paper II-IV). Given the results described in this thesis and together with other studies, CPs can be considered key virulence factors involved in the pathogenesis of Giardia. However, a number of unanswered questions remain.

Cleavage specificity of other secreted Giardia CPs In this thesis, we characterized three closely related CPs (CP14019, CP16160 and CP16779) and they are capable of opening the apical junctional complex and degrading chemokines (Paper II). They have similar substrates but different cleavage specificity (Paper II, III, IV). We applied phage display technology to study the substrate cleavage specificity of all three CPs, but only succeed in obtaining information from CP16160 (Paper III). The reason why phage display failed for CP14019 and CP16779 is still unknown. Although we tested CP14019 and CP16779 on the recombinant 2-trx substrates, the substrate cleavage of the two CPs remains unclear. In paper III, we conclude that CP14019 and CP16160 have similar cleavage specificity whereas CP16779 has distinctly different cleavage specificity. However, the cleavage of CPs is also dependent on the structure of the substrate protein and this can be seen in Paper IV. Only CP14019 is able to degrade MUC2 N-terminal recombinant protein. CP14019 can also cleave the reduced full length MUC2 when the disulfide bond is exposed, but no degradation can be seen with native MUC2 mucin. Interestingly, the two CPs (CP14019 and CP16779) that have remarked different cleavage specificity are capable of cleaving MUC17, a transmembrane mucin that Giardia needs to overcome prior to colonization (data not shown). In addition to the three CPs characterized in this thesis, other CPs, including CP12017, CP15564, CP16468 and CP17516 were identified in secretome study (Paper I) and they are upregulated during IEC-parasite interactions in vitro in earlier studies (Table 3) (Dubourg et al., 2018, Ringqvist et al., 2011, Ferella et al., 2014). But their functions remain unclear. CP10217

43 is one of the earliest studied Giardia CP and it is involved in excystation (Ward et al., 1997). We expressed the recombinant CP10217 protein in P. pastoris and the protein was purified as one form with a molecular size of 28 kDa. However, the protein did not show a similar activity and specificity as the three tested CPs (data not shown). We suspect that CP10217 functions differently as proteolytic protease than the three CPs tested during Giardia infections and it will be interesting to study this CP further. We have also tried to characterize CP16468 but the expression of recombinant CP16468 protein in P. pastoris was unsuccessful. CP17516 is yet another very interesting CP that is secreted during IEC- parasite interactions. In the protein sequence of CP17516, the conserved CP active site residues, cysteine and histidine, are replaced by serine and leucine, respectively. We expressed the protein in P. pastoris and the recombinant protein was purified as two forms, 35 kDa and 28 kDa. However, no activity of CP17516 was detected. We suspect that CP17516 is processed by proteases expressed from P. pastoris to form “active form” (28 kDa) and the protein may have other functions instead of typical proteolytic CP. Our hypoth- esis is that CP17516 might be a binding protein that interacts with host receptor triggering downstream signaling pathways or it functions as a chaper- one aiding the folding of CPs. Given the fact that Giardia possesses only one mutated CP and it is upregulated and secreted during IEC-parasite interactions, the involvement of this protein in Giardia infections is of importance to address. CPs are known to be essential to Giardia infections, but the specific virulence factors are rarely characterized. Studies of the cleavage specificity of specific CP will give a better view of the molecular pathogenesis of Giardia. Different cleavage specificity of three CPs tested and other CPs makes biological sense. In addition, phage display will be a great tool to uncover the functions of the uncharacterized CPs, including CP10217, CP15564 and CP16468.

Knockout study of Giardia CPs Although some secreted Giardia CPs have been identified and characterized, the role of many CPs in the disease mechanism remains unknown. In addition to study the cleavage specificity of CPs, knockout or knockdown studies will be helpful to elucidate the role of specific virulence factors in Giardia. Knockout or knockdown studies of CPs have been used for many protozoan parasites such as Leishmanial species, Plasmodium falciparum, Toxoplasma gondii (Mundodi et al., 2005, Denise et al., 2006, Mottram et al., 1996, Sijwali and Rosenthal, 2004, Sijwali et al., 2006, Sijwali et al., 2004, Dou et al., 2014, Que et al., 2007). However, it is hard to knockout genes in Giardia as it has two diploid nuclei. Since the CPs tested here have similar sub-

44 strates, the effect of knockout or knockdown of one CP might be compen- sated by other CPs as observed in P. falciparum that the abnormality caused by knockout of falcipain-2 was resolved later in the life cycle probably due to falcipain-3 expression (Sijwali and Rosenthal, 2004). A similar effect was seen in T. gondii that inhibition of both TgCPC1 and TgCPC2 is able to reduce parasite intracellular growth and proliferation, but the same phenotype was not seen with a TgCPC1 knockout as TgCPC2 expression was upregulated (Que et al., 2007). However, genetic tools that can be used to knockout or knockdown genes should be developed in Giardia and applied to characterize putative virulence factors or to verify characterized virulence factors.

CPs role in vaccines and as drug targets CPs of Trypanosoma brucei have been validated as drug targets using RNA interference or small-molecular inhibitors in vitro and in animal models (O'Brien et al., 2008, Steverding et al., 2012, Caffrey et al., 2000). In Giar- dia, oral administration of ESPs increased the antibody levels and prior immunization of ESPs leads to failure of trophozoite colonization, and CPs were found to play a major role. This is consistent with the study in vitro that CPs have a role in aiding the trophozoite attachment to rat enterocytes (Rodriguez-Fuentes et al., 2006). Strategies that inhibit Giardia CPs expression as well as block activities of CPs could be a path to prevent and control Giardia infections. Moreover, many studies were carried out in vitro, animal studies in vivo will also be necessary to validate the putative virulence factors.

How are the activities of CPs regulated? Although CPs are essential to the pathogenesis of Giardia, regulation of CP activities is also required to protect the parasite from intracellular damage by the endogenous CPs. The production of CP inhibitors has been described in other parasitic organisms. They play important roles in endogenous process, such as regulation of haemoglobin degradation in Schistosoma species (Morales et al., 2004), protection of endogenous proteolysis by CPs in Clonorchis sinensis (Kang et al., 2014, Kang et al., 2011), Trichomonas vaginalis (Puente-Rivera et al., 2014). The association between a CP inhibitor and CPs has also been seen in T. vaginalis (Puente-Rivera et al., 2014) and E. histolytica (Riekenberg et al., 2005). E. histolytica CP inhibitors are also involved in the regulation of CP secretion, thus in the virulence of this parasite (Sato et al., 2006). However, how the activities of Giardia CPs are regulated is unknown.

45 The Giardia WB isolate possesses one gene (GL50803_27918) that en- codes a CP inhibitor, cystatin. The protein sequence of Giardia cystatin shows that it has a conserved amino acid motif (QVVXG) that is typically seen in cystatins. The lack of a N-terminal signal peptide and disulfide bonds suggests that Giardia cystatin is a member of type I cystatins (stefins) and it is an intracellular protein. However, this protein was detected in the supernatant of Giardia trophozoites co-incubated with host soluble factors (Emery et al., 2016), demonstrating the secretion of cystatin by Giardia. Our hypothe- sis is that Giardia cystatin plays a critical role in regulation of CP activity endogenously by the inhibition of Giardia CPs and exogenously by the inhibition of host secreted CPs during Giardia infections. Our preliminary data has shown that Giardia cystatin can inhibit CPs, including papain, recombinant human cathepsin B, recombinant Giardia CP14019, CP16160 and CP16779 efficiently (data not shown). However, the association between Giardia CPs and cystatin and if cystatin affects CPs secretion require further investigation.

Assemblage-specific differences of CPs Giardia assemblage A and B are infective to humans but they are different in many aspects. For example, only assemblage B is infective to adult mice (Byrd et al., 1994), and they both have strain-specific proteins. In addition, assemblage B is reported to cause more severe symptoms than assemblage A in certain studies and antigenic variation has different frequencies of switching in the two assemblages. Regarding the secretion of CPs, we can find homologues of CPs identified in assemblage A (WB) in assemblage B (GS) (Paper I). It was suggested that assemblage A and B secrete CPs with similar activity, but it has been shown that assemblage A CPs degrade IL-8 and attenuate inflammatory responses, whereas assemblage B CPs failed to degrade CXCL8, indicating an isolate-specific effect (Bartelt et al., 2013, Cotton et al., 2014a). A recent report has also shown that assemblage A isolate NF deplete MUC2 in a CP-dependent manner, but assemblage B isolate could not (Amat et al., 2017). CPs of assemblage A are critical to Giardia’s pathogenicity but CPs of assemblage B requires further investigation.

In conclusion, the secretome study described here provides new insights into the ESPs released by Giardia during IEC-parasite interactions and provides the basis for studies of putative secreted virulence factors. The characterization of three major CPs partly explains the disease-causing mechanism of giardiasis. Hopefully, the work conducted in this thesis will lead to more investigations that contribute to understanding of the molecular pathogenesis of Giardia.

46 Svensk sammanfattning

Giardia intestinalis (syn. G. duodenalis, G. lamblia) är en encellig parasit som koloniserar det övre tarmsystemet (duodenum) hos många däggdjur, inklusive människor. Parasiten orsakar diarrésjukdomen Giardiasis som drabbar 200 miljoner människor årligen, framförallt barn i utvecklingslän- der. Den är mycket genetiskt variabel och kan delas in i åtta genotyper (A till H) som har föreslagits vara så olika att de representerar olika arter av Giar- dia. Endast genotyp A och B är infektiösa för människor men de kan också infektera andra däggdjur och är således zoonotiska. Giardia har en relativt enkel livscykel med två huvudsakliga livscykelstadier; infektiösa cystor och replikativa trofozoiter. Cystan är mycket resistent mot olika miljöfaktorer och endast 10 stycken räcker för att starta en infektion i människor. Trofo- zoiterna, som tros orsaka symptomen vid Giardiasis, fäster vid tarmepitelcellerna och replikerar i tunntarmen. Dessa två livsstadier genereras av två celldifferentieringsprocesser; encystering och excystering. Förståelsen av Giardias patogenes har ökat på senare år, delvis på grund av förståelsen av effekterna från exkretoriska och sekretoriska produkter (ESP) som frisätts av Giardia under värd-parasit-interaktioner. Förståelsen är emellertid begränsad till effekter som induceras av odlingssupernatanter i de flesta studierna och de specifika virulensfaktorerna har sällan identifierats och karakteriserats. Proteaser utgör en stor del av ESP och utsöndrade proteaser har tidigare föreslagits vara nyckelaktörer i patogenesen av många para- sitiska sjukdomar, inklusive Giardiasis. I denna avhandling genomförde vi den första storskaliga sekretomstudien i Giardia genom att samla ESP från Giardia i både axenisk kultur och från samkulturer med tarmepitelceller (differentierade Caco-2-celler). Specifika proteiner i ESP identifierades med masspektrometri, LC-MS/MS och totalt identifierades runt 200 Giardia proteiner i ESP. För att fördjupa vår förstå- else för ESP:s roll vid Giardiasis fokuserade vi på tre identifierade utsönd- rade cysteinproteaser (CP14019, CP16160 och CP16779). Dessa tre cysteinproteaserna utgör en stor del av ESP och vi skapade en hypotes där proteaserna är viktiga faktorer vid Giardiasis. För att karakterisera de tre cysteinproteaserna, uttryckte vi först och renade dem från Pichia pastoris, en jäst som ofta används för produktion av rekombinanta proteiner, och testade deras aktiviteter mot artificiella proteas substrat. Vi fann att alla tre rekombinanta cysteinproteaserna är mer aktiva i sur miljö (pH 5,5–6) men med en bred aktivitetsprofil som reflekterar pH variationen i duodenum. Vi testade

47 också om de tre cysteinproteaserna klyver apikala komplexproteiner och kemokiner. Apikala komplexproteiner sammanfogar termepitelceller i tarmen så att tarminnehållet inte läcker in i vävnaden och kemokiner signalerar till immunsystemet. Vi kunde visa att specifika apikala komplexproteiner och kemokiner klyvs a cysteinproteaserna. De utsöndrade proteasernas för- måga att klyva och omorganisera apikala samlingsproteiner gjorde det möj- ligt att föreslå en modell som kan förklara hur utsöndrade cysteinproteaser från Giardia kan inducera vissa av symptomen som man ser under Giardiasis och hur parasiten minskar effekter av kemokiner som produceras av tarmepitelceller under värd-parasit interaktioner. Detta stödjer våran hypotes om att de identifierade cysteinproteaserna är viktiga virulensfaktorer i Giardia. Vi använde oss också av metoden ”phage display” för att bestämma cysteinproteasernas klyvningsspecificitet. De identifierade klyvningssekven- serna användes sedan för att hitta nya potentiella humana målproteiner som uttrycks i tarmen. Ett antal kandidater identifierades och vi visade att immunglobuliner och antimikrobiella peptider klyvs specifikt och effektivt av de rekombinanta cysteinproteaserna. Vidare undersökte vi rollen av cysteinproteaserna vid slemhinnedbrytning. Slemhinnan i duodenum utgörs fram- förallt av proteinet MUC2 och det skyddar tarmepitelcellerna mot mikroorg- anismer och skadliga ämnen i maten. Vi testade om de tre rekombinanta cysteinproteaserna kan klyva rekombinanta MUC2-proteiner och renat MUC2 och resultatet analyserades med masspektrometri. Detta visade att CP14019 klarar av att klyva MUC2 i N-terminalen, vilket gjorde att vi kunde föreslå en mekanism hur parasiten kan lösa upp tarmslemhinnan och få till- gång till värdens tarmepitel. Sammanfattningsvis har denna avhandling studerat tarmparasiten Giar- dias utsöndrade proteiner eller sekretom. Det gav nya insikter i de proteiner som frisläpps av Giardia under interaktioner med tarmepitelceller i tarmen. Karaktäriseringen av de tre mest utsöndrade cysteinproteaserna förklarar delvis den sjukdomsframkallande mekanismen av Giardiasis. Det kan också förklara hur Giardia undviker att elimineras av immunsystemet. Förhopp- ningsvis leder arbetet i denna avhandling till nya forskningsprojekt som yt- terligare bidrar till förståelsen av Giardias molekylära patogenes och till ny diagnostisk och behandlingsmetoder.

48 Acknowledgements

First and foremost, my supervisor Staffan- Thank you for trusting me from the very beginning and applying the CSC funding together with me, that made it possible for me to come to Sweden and spend my wonderful four years of PhD studies here. Thank you for taking good care of me when I arrived in Sweden and being supportive all these four years. Thank you for never say no and always being available for discussion and being enthusias- tic and positive about my work. CSC- Thanks for funding me to do my four- year PhD studies in Sweden. My co-supervisor Lars- Thank you for your help with my projects and be available whenever I want to talk to you. I had great time working in your lab. All other PIs in micro- Thank you all for giving me nice comments and suggestions on my research presentations.

The Giardia group: Ulf, It was nice to have you in the lab. Jon, it is nice meeting you. Elin, I admire your dedication. Thanks for all the protocols. I had great time sharing the office with you, thank you for the nice chats in our tiny office. Showgy, thanks for bringing good energy to the group and teaching me how to grow Giardia and human cell lines. Feifei, happy to have the second Chinese girl in the group, thanks for sharing your pregnancy and parenting experience with me. I am sure I will have more to ask you in the future. Asgeir, thanks for your help with all the random lab stuff I asked for, it was fun to play Pingpong with you and good luck with your dissertation. Dimitra, I am very happy to share the office with you and your decora- tion makes the office so much nicer and cozy. Your laugh makes the lab much happier and good luck with your dissertation. Louise, always nice to chat with you and hope you have a good time working in Rudbeck. Laura, thanks for being a very good friend. You always know how to take good care of me when I get tired or bored. Thanks for feeding me with chocolates and candies. Sascha, thanks for reading my thesis. It was fun to work with you on your master project. I am happy to have you in the group; finally I can find someone to talk about proteins. Jana, the CRISPR and new cysteine protease girl, great to have you in the group and you will have so much fun working with proteases. Anders, good luck with your PhD. Courtney, nice to meet you and have you with the Giardians for dinners. Livia, Viktor, Anna, Karin, Eva, Ines, Adena, all the best to you guys. My collaborators, Gunnar, Liisa, Christian and Sergio, thanks for your contribution to this thesis. Christian K, nice to work with you on cystatin.

49 I also want to thanks the micro program. Thanks for the program meetings, journal clubs, cakes, bubbles. My best friend Zhen, I had so many precious memories with you all these four years not only in the lab but also in my four-year life outside of work in Sweden. We are so similar in many ways. You are like a big sister to me. Thanks for all your concerns and cares, that means a lot to me. The Wagner group, Cedric, thanks for all the chats and dinners. I had great times with you and Zhen and wish you all the best. Mirthe, it was nice to have you around in micro and good luck to you. Bork, thanks for showing me western blot bands quantification. Sofia, Maaike, good luck with everything in the future. The CRISPR girl, Lina, thanks for the nice cakes and deserts. The worm group, Diri, Yani and Ben- jamin, thanks for nice chats. The Dicty group, Jonas and Bart, thanks for nice chats and good luck to you. The CDI group, Disa, thanks for organizing many things in the lad. Katrin, all the best with the little baby. Marcus, thanks for the science talks and good luck with your PhD. Danna, Magnus, Petra, nice talking to you guys and good luck to you too. The Holmqvist group, Yolanda, you are always so happy and energetic and good luck back home. Liis, Alisa, Sofia B, you guys are always working and all the best for future. The Sellin group, Mikael, thanks for being my opponent during my half time and thanks for all the nice suggestions and comments on my projects. Stefan, all the best for your PhD. Maria Letizia, thanks for fikas and dinners, that frees my mind from the thesis. The Hellman group, Zhirong, thanks for teaching me the phage display. I had great time working with you and all the best for the new chapter of your life. Srinivas, thanks for the phage display and protein chats.

There are also other members I would like to thank. Adrian, thanks for introducing me to the Pichia world. Jinfan, Ka-Weng, Lu, thanks for explain- ing me the enzyme kinetics. Wangshu, thanks for suggestions on protein expression. Mikael N, thanks for suggestions on protein expression and purification. Lili, I had great time staying with you. Bingjie, my friend from my bachelor University, thanks for introducing me to so many friends in SLU, good luck to you in UK. Mao, Liang, Chang-Il, thanks for nice chats. Xia, thanks for always cooking me nice Sichuan food. I would also like to thank all the other friends I met in UU and SLU all these four years.

感谢我的老公一直以来对我的爱护和支持，从大学到研究生到博士到找工作，我的每一分收获都有你的参与你的功劳。感谢小坤坤，你的到来让我很幸福，我会努力做个好妈妈。感谢我最亲爱的父母和姐姐，感谢你们一直无条件的爱我支持我。姐姐，谢谢你照顾父母照顾我，有你在父母身边，我可以安心的在外念书。姐夫，谢谢你照顾姐姐和我们的父母。感谢我的公公婆婆对我关爱和支持。博士四年，收获很多，感谢所有关心我的人。

50 References

ABEL, E. S., DAVIDS, B. J., ROBLES, L. D., LOFLIN, C. E., GILLIN, F. D. & CHAKRABARTI, R. 2001. Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia. J Biol Chem, 276, 10320-9. ABODEELY, M., DUBOIS, K. N., HEHL, A., STEFANIC, S., SAJID, M., DESOUZA, W., ATTIAS, M., ENGEL, J. C., HSIEH, I., FETTER, R. D. & MCKERROW, J. H. 2009. A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia. Eukaryot Cell, 8, 1665-76. ADAM, R. D. 2001. Biology of Giardia lamblia. Clin Microbiol Rev, 14, 447-75. ADAM, R. D., NIGAM, A., SESHADRI, V., MARTENS, C. A., FARNETH, G. A., MORRISON, H. G., NASH, T. E., PORCELLA, S. F. & PATEL, R. 2010. The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution. BMC Genomics, 11, 424. AGUAYO-ORTIZ, R., MEZA-CERVANTEZ, P., CASTILLO, R., HERNANDEZ- CAMPOS, A., DOMINGUEZ, L. & YEPEZ-MULIA, L. 2017. Insights into the Giardia intestinalis enolase and human plasminogen interaction. Mol Biosyst, 13, 2015-2023. ALEY, S. B., ZIMMERMAN, M., HETSKO, M., SELSTED, M. E. & GILLIN, F. D. 1994. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. Infect Immun, 62, 5397-403. ALLAIN, T., AMAT, C. B., MOTTA, J. P., MANKO, A. & BURET, A. G. 2017a. Interactions of Giardia sp. with the intestinal barrier: Epithelium, mucus, and microbiota. Tissue Barriers, 5, e1274354. ALLAIN, T., CHAOUCH, S., THOMAS, M., VALLEE, I., BURET, A. G., LANGELLA, P., GRELLIER, P., POLACK, B., BERMUDEZ- HUMARAN, L. G. & FLORENT, I. 2017b. Bile-Salt-Hydrolases from the Probiotic Strain Lactobacillus johnsonii La1 Mediate Anti-giardial Activity in Vitro and in Vivo. Front Microbiol, 8, 2707. AMAT, C. B., MOTTA, J. P., FEKETE, E., MOREAU, F., CHADEE, K. & BURET, A. G. 2017. Cysteine Protease-Dependent Mucous Disruptions and Differential Mucin Gene Expression in Giardia duodenalis Infection. Am J Pathol, 187, 2486-2498. ANDERSEN, Y. S., GILLIN, F. D. & ECKMANN, L. 2006. Adaptive immunity- dependent intestinal hypermotility contributes to host defense against Giardia spp. Infect Immun, 74, 2473-6. ANKARKLEV, J., JERLSTROM-HULTQVIST, J., RINGQVIST, E., TROELL, K. & SVARD, S. G. 2010. Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol, 8, 413-22.

51 ATUMA, C., STRUGALA, V., ALLEN, A. & HOLM, L. 2001. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol, 280, G922-9. AYABE, T., ASHIDA, T., KOHGO, Y. & KONO, T. 2004. The role of Paneth cells and their antimicrobial peptides in innate host defense. Trends Microbiol, 12, 394-8. AYABE, T., SATCHELL, D. P., WILSON, C. L., PARKS, W. C., SELSTED, M. E. & OUELLETTE, A. J. 2000. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol, 1, 113-8. BANIK, S., RENNER VIVEROS, P., SEEBER, F., KLOTZ, C., IGNATIUS, R. & AEBISCHER, T. 2013. Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia. Infect Immun, 81, 2309-17. BARASH, N. R., MALONEY, J. G., SINGER, S. M. & DAWSON, S. C. 2017. Giardia Alters Commensal Microbial Diversity throughout the Murine Gut. Infect Immun, 85. BARTELT, L. A., BOLICK, D. T., MAYNERIS-PERXACHS, J., KOLLING, G. L., MEDLOCK, G. L., ZAENKER, E. I., DONOWITZ, J., THOMAS- BECKETT, R. V., ROGALA, A., CARROLL, I. M., SINGER, S. M., PAPIN, J., SWANN, J. R. & GUERRANT, R. L. 2017. Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co- infection with Giardia lamblia and enteroaggregative Escherichia coli. PLoS Pathog, 13, e1006471. BARTELT, L. A., ROCHE, J., KOLLING, G., BOLICK, D., NORONHA, F., NAYLOR, C., HOFFMAN, P., WARREN, C., SINGER, S. & GUERRANT, R. 2013. Persistent G. lamblia impairs growth in a murine malnutrition model. J Clin Invest, 123, 2672-84. BAYRAKTAR, M. R., MEHMET, N. & DURMAZ, R. 2005. Serum cytokine changes in Turkish children infected with Giardia lamblia with and without allergy: Effect of metronidazole treatment. Acta Trop, 95, 116-22. BEATTY, J. K., AKIERMAN, S. V., MOTTA, J. P., MUISE, S., WORKENTINE, M. L., HARRISON, J. J., BHARGAVA, A., BECK, P. L., RIOUX, K. P., MCKNIGHT, G. W., WALLACE, J. L. & BURET, A. G. 2017. Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms. Int J Parasitol, 47, 311-326. BENCHIMOL, M. 2004. Giardia lamblia: behavior of the nuclear envelope. Parasitol Res, 94, 254-264. BENYACOUB, J., PEREZ, P. F., ROCHAT, F., SAUDAN, K. Y., REUTELER, G., ANTILLE, N., HUMEN, M., DE ANTONI, G. L., CAVADINI, C., BLUM, S. & SCHIFFRIN, E. J. 2005. Enterococcus faecium SF68 enhances the immune response to Giardia intestinalis in mice. J Nutr, 135, 1171-6. BERMUDEZ-CRUZ, R. M., ORTEGA-PIERRES, G., CEJA, V., CORAL- VAZQUEZ, R., FONSECA, R., CERVANTES, L., SANCHEZ, A., DEPARDON, F., NEWPORT, G. & MONTANEZ, C. 2004. A 63 kDa VSP9B10A-like protein expressed in a C-8 Giardia duodenalis Mexican clone. Arch Med Res, 35, 199-208.

52 BERNANDER, R., PALM, J. E. & SVARD, S. G. 2001. Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol, 3, 55-62. BEVINS, C. L. & SALZMAN, N. H. 2011. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol, 9, 356-68. BHARGAVA, A., COTTON, J. A., DIXON, B. R., GEDAMU, L., YATES, R. M. & BURET, A. G. 2015. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase. PLoS One, 10, e0136102. BIENZ, M., DAI, W. J., WELLE, M., GOTTSTEIN, B. & MULLER, N. 2003. Interleukin-6-deficient mice are highly susceptible to Giardia lamblia infection but exhibit normal intestinal immunoglobulin A responses against the parasite. Infect Immun, 71, 1569-73. BIENZ, M., SILES-LUCAS, M., WITTWER, P. & MULLER, N. 2001. vsp gene expression by Giardia lamblia clone GS/M-83-H7 during antigenic variation in vivo and in vitro. Infect Immun, 69, 5278-85. BINGHAM, A. K. & MEYER, E. A. 1979. Giardia excystation can be induced in vitro in acidic solutions. Nature, 277, 301-2. BLANCHARD, R. 1888. Remarques sur le megastome intestinal. Bull Soc Zool Fr, 13:18. BOUCHER, S. E. & GILLIN, F. D. 1990. Excystation of in vitro-derived Giardia lamblia cysts. Infect Immun, 58, 3516-22. BURET, A., GALL, D. G. & OLSON, M. E. 1991. Growth, activities of enzymes in the small intestine, and ultrastructure of microvillous border in gerbils infected with Giardia duodenalis. Parasitol Res, 77, 109-14. BURET, A., HARDIN, J. A., OLSON, M. E. & GALL, D. G. 1992. Pathophysiology of small intestinal malabsorption in gerbils infected with Giardia lamblia. Gastroenterology, 103, 506-13. BURET, A. G., MITCHELL, K., MUENCH, D. G. & SCOTT, K. G. 2002. Giardia lamblia disrupts tight junctional ZO-1 and increases permeability in non- transformed human small intestinal epithelial monolayers: effects of epidermal growth factor. Parasitology, 125, 11-9. BYRD, L. G., CONRAD, J. T. & NASH, T. E. 1994. Giardia lamblia infections in adult mice. Infect Immun, 62, 3583-5. CABRERA-LICONA, A., SOLANO-GONZALEZ, E., FONSECA-LINAN, R., BAZAN-TEJEDA, M. L., RAUL, A.-G., BERMUDEZ-CRUZ, R. M. & ORTEGA-PIERRES, G. 2017. Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity. Exp Parasitol, 179, 49-64. CAFFREY, C. R., SCORY, S. & STEVERDING, D. 2000. Cysteine proteinases of trypanosome parasites: novel targets for chemotherapy. Curr Drug Targets, 1, 155-62. CARLSTEDT, I., HERRMANN, A., KARLSSON, H., SHEEHAN, J., FRANSSON, L. A. & HANSSON, G. C. 1993. Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine. J Biol Chem, 268, 18771-81.

53 CARRANZA, P. G., FELTES, G., ROPOLO, A., QUINTANA, S. M., TOUZ, M. C. & LUJAN, H. D. 2002. Simultaneous expression of different variant- specific surface proteins in single Giardia lamblia trophozoites during encystation. Infect Immun, 70, 5265-8. CERUTTI, A. & RESCIGNO, M. 2008. The biology of intestinal immunoglobulin A responses. Immunity, 28, 740-50. CHADEE, K. & MEEROVITCH, E. 1985. Entamoeba histolytica: early progressive pathology in the cecum of the gerbil (Meriones unguiculatus). Am J Trop Med Hyg, 34, 283-91. CHAIRATANA, P., CHU, H., CASTILLO, P. A., SHEN, B., BEVINS, C. L. & NOLAN, E. M. 2016. Proteolysis Triggers Self-Assembly and Unmasks Innate Immune Function of a Human alpha-Defensin Peptide. Chem Sci, 7, 1738-1752. CHAVEZ, B., GONZALEZ-MARISCAL, L., CEDILLO-RIVERA, R. & MARTINEZ-PALOMO, A. 1995. Giardia lamblia: in vitro cytopathic effect of human isolates. Exp Parasitol, 80, 133-8. CHAVEZ, B. & MARTINEZ-PALOMO, A. 1995. Giardia lamblia: freeze-fracture ultrastructure of the ventral disc plasma membrane. J Eukaryot Microbiol, 42, 136-41. CHIN, A. C., TEOH, D. A., SCOTT, K. G., MEDDINGS, J. B., MACNAUGHTON, W. K. & BURET, A. G. 2002. Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase- 3-dependent manner. Infect Immun, 70, 3673-80. CORADI, S. T. & GUIMARAES, S. 2006. Giardia duodenalis: protein substrates degradation by trophozoite proteases. Parasitol Res, 99, 131-6. COTTON, J. A., BHARGAVA, A., FERRAZ, J. G., YATES, R. M., BECK, P. L. & BURET, A. G. 2014a. Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis. Infect Immun, 82, 2772-87. COTTON, J. A., MOTTA, J. P., SCHENCK, L. P., HIROTA, S. A., BECK, P. L. & BURET, A. G. 2014b. Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue. PLoS One, 9, e109087. CROUCH, A. A., SEOW, W. K., WHITMAN, L. M., SMITH, S. E. & THONG, Y. H. 1991. Inhibition of adherence of Giardia intestinalis by human neutrophils and monocytes. Trans R Soc Trop Med Hyg, 85, 375-9. D'ANCHINO, M., ORLANDO, D. & DE FEUDIS, L. 2002. Giardia lamblia infections become clinically evident by eliciting symptoms of irritable bowel syndrome. J Infect, 45, 169-72. DAGLEY, M. J., DOLEZAL, P., LIKIC, V. A., SMID, O., PURCELL, A. W., BUCHANAN, S. K., TACHEZY, J. & LITHGOW, T. 2009. The protein import channel in the outer mitosomal membrane of Giardia intestinalis. Mol Biol Evol, 26, 1941-7.

54 DANN, S. M., MANTHEY, C. F., LE, C., MIYAMOTO, Y., GIMA, L., ABRAHIM, A., CAO, A. T., HANSON, E. M., KOLLS, J. K., RAZ, E., CONG, Y. & ECKMANN, L. 2015. IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen- dwelling enteric parasite Giardia. Exp Parasitol, 156, 68-78. DAS, P., LAHIRI, A. & CHAKRAVORTTY, D. 2010. Modulation of the arginase pathway in the context of microbial pathogenesis: a metabolic enzyme moonlighting as an immune modulator. PLoS Pathog, 6, e1000899. DAVIDS, B. J., PALM, J. E., HOUSLEY, M. P., SMITH, J. R., ANDERSEN, Y. S., MARTIN, M. G., HENDRICKSON, B. A., JOHANSEN, F. E., SVARD, S. G., GILLIN, F. D. & ECKMANN, L. 2006. Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia. J Immunol, 177, 6281-90. DAWSON, S. C. 2010. An insider's guide to the microtubule cytoskeleton of Giardia. Cell Microbiol, 12, 588-98. DE CARVALHO, T. B., DAVID, E. B., CORADI, S. T. & GUIMARAES, S. 2008. Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis. Parasitol Res, 104, 185-90. DEITSCH, K. W., MOXON, E. R. & WELLEMS, T. E. 1997. Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. Microbiol Mol Biol Rev, 61, 281-93. DENISE, H., POOT, J., JIMENEZ, M., AMBIT, A., HERRMANN, D. C., VERMEULEN, A. N., COOMBS, G. H. & MOTTRAM, J. C. 2006. Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5. BMC Mol Biol, 7, 42. DOBELL, C. 1920. The Discovery of the Intestinal Protozoa of Man. Proc R Soc Med, 13, 1-15. DOLEZAL, P., SMID, O., RADA, P., ZUBACOVA, Z., BURSAC, D., SUTAK, R., NEBESAROVA, J., LITHGOW, T. & TACHEZY, J. 2005. Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc Natl Acad Sci U S A, 102, 10924-9. DOU, Z., MCGOVERN, O. L., DI CRISTINA, M. & CARRUTHERS, V. B. 2014. Toxoplasma gondii ingests and digests host cytosolic proteins. MBio, 5, e01188-14. DREESEN, L., DE BOSSCHER, K., GRIT, G., STAELS, B., LUBBERTS, E., BAUGE, E. & GELDHOF, P. 2014. Giardia muris infection in mice is associated with a protective interleukin 17A response and induction of peroxisome proliferator-activated receptor alpha. Infect Immun, 82, 3333- 40. DUBOIS, K. N., ABODEELY, M., SAJID, M., ENGEL, J. C. & MCKERROW, J. H. 2006. Giardia lamblia cysteine proteases. Parasitol Res, 99, 313-6. DUBOIS, K. N., ABODEELY, M., SAKANARI, J., CRAIK, C. S., LEE, M., MCKERROW, J. H. & SAJID, M. 2008. Identification of the major cysteine protease of Giardia and its role in encystation. J Biol Chem, 283, 18024-31.

55 DUBOURG, A., XIA, D., WINPENNY, J. P., AL NAIMI, S., BOUZID, M., SEXTON, D. W., WASTLING, J. M., HUNTER, P. R. & TYLER, K. M. 2018. Giardia secretome highlights secreted tenascins as a key component of pathogenesis. Gigascience, 7, 1-13. ECKMANN, L. 2003. Mucosal defences against Giardia. Parasite Immunol, 25, 259-70. ECKMANN, L., LAURENT, F., LANGFORD, T. D., HETSKO, M. L., SMITH, J. R., KAGNOFF, M. F. & GILLIN, F. D. 2000. Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia. J Immunol, 164, 1478-87. EINARSSON, E., MA'AYEH, S. & SVARD, S. G. 2016a. An up-date on Giardia and giardiasis. Curr Opin Microbiol, 34, 47-52. EINARSSON, E., TROELL, K., HOEPPNER, M. P., GRABHERR, M., RIBACKE, U. & SVARD, S. G. 2016b. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis. PLoS Negl Trop Dis, 10, e0004571. EL-GEBALY, N. S., HALAWA, E. F., MOUSSA, H. M., RABIA, I. & ABU- ZEKRY, M. 2012. Saliva and sera IgA and IgG in Egyptian Giardia- infected children. Parasitol Res, 111, 571-5. ELIAS, E. V., QUIROGA, R., GOTTIG, N., NAKANISHI, H., NASH, T. E., NEIMAN, A. & LUJAN, H. D. 2008. Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia. J Biol Chem, 283, 35996-6010. ELMENDORF, H. G., DAWSON, S. C. & MCCAFFERY, J. M. 2003. The cytoskeleton of Giardia lamblia. Int J Parasitol, 33, 3-28. ELMORE, S. 2007. Apoptosis: a review of programmed cell death. Toxicol Pathol, 35, 495-516. EMERY, S. J., MIRZAEI, M., VUONG, D., PASCOVICI, D., CHICK, J. M., LACEY, E. & HAYNES, P. A. 2016. Induction of virulence factors in Giardia duodenalis independent of host attachment. Sci Rep, 6, 20765. EMERY, S. J., VAN SLUYTER, S. & HAYNES, P. A. 2014. Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation. Proteomics, 14, 2523-34. ERLANDSEN, S. L. & BEMRICK, W. J. 1987. SEM evidence for a new species, Giardia psittaci. J Parasitol, 73, 623-9. ERLANDSEN, S. L., BEMRICK, W. J., WELLS, C. L., FEELY, D. E., KNUDSON, L., CAMPBELL, S. R., VAN KEULEN, H. & JARROLL, E. L. 1990. Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias). J Parasitol, 76, 717-24. ERLANDSEN, S. L., RUSSO, A. P. & TURNER, J. N. 2004. Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia. J Eukaryot Microbiol, 51, 73-80.

56 ERMUND, A., SCHUTTE, A., JOHANSSON, M. E., GUSTAFSSON, J. K. & HANSSON, G. C. 2013. Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches. Am J Physiol Gastrointest Liver Physiol, 305, G341-7. EY, P. L., MANSOURI, M., KULDA, J., NOHYNKOVA, E., MONIS, P. T., ANDREWS, R. H. & MAYRHOFER, G. 1997. Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes. J Eukaryot Microbiol, 44, 626-35. FARTHING, M. J. 1996. Giardiasis. Gastroenterol Clin North Am, 25, 493-515. FARTHING, M. J. 1997. The molecular pathogenesis of giardiasis. J Pediatr Gastroenterol Nutr, 24, 79-88. FASO, C. & HEHL, A. B. 2011. Membrane trafficking and organelle biogenesis in Giardia lamblia: use it or lose it. Int J Parasitol, 41, 471-80. FAUBERT, G. 2000. Immune response to Giardia duodenalis. Clin Microbiol Rev, 13, 35-54, table of contents. FEELY, D. E. 1988. Morphology of the cyst of Giardia microti by light and electron microscopy. J Protozool, 35, 52-4. FERELLA, M., DAVIDS, B. J., CIPRIANO, M. J., BIRKELAND, S. R., PALM, D., GILLIN, F. D., MCARTHUR, A. G. & SVARD, S. 2014. Gene expression changes during Giardia-host cell interactions in serum-free medium. Mol Biochem Parasitol, 197, 21-3. FILICE, F. P. 1952. Studies on the cytology and life history of a Giardia from the laboratory rat. Univ Calif Publ Zool, 57, 53-146. FIORENTINI, C., FABBRI, A., FALZANO, L., FATTOROSSI, A., MATARRESE, P., RIVABENE, R. & DONELLI, G. 1998. Clostridium difficile toxin B induces apoptosis in intestinal cultured cells. Infect Immun, 66, 2660-5. FISHER, B. S., ESTRANO, C. E. & COLE, J. A. 2013. Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system. PLoS One, 8, e81104. FURCI, L., BALDAN, R., BIANCHINI, V., TROVATO, A., OSSI, C., CICHERO, P. & CIRILLO, D. M. 2015. New role for human alpha-defensin 5 in the fight against hypervirulent Clostridium difficile strains. Infect Immun, 83, 986-95. GARCIA-NIETO, R. M., RICO-MATA, R., ARIAS-NEGRETE, S. & AVILA, E. E. 2008. Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity. Parasitol Int, 57, 417-23. GASCON, J. 2006. Epidemiology, etiology and pathophysiology of traveler's diarrhea. Digestion, 73 Suppl 1, 102-8. GERBABA, T. K., GUPTA, P., RIOUX, K., HANSEN, D. & BURET, A. G. 2015. Giardia duodenalis-induced alterations of commensal bacteria kill Caenorhabditis elegans: a new model to study microbial-microbial interactions in the gut. Am J Physiol Gastrointest Liver Physiol, 308, G550- 61.

57 GIBSON, C., SCHANEN, B., CHAKRABARTI, D. & CHAKRABARTI, R. 2006. Functional characterisation of the regulatory subunit of cyclic AMP- dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation. Int J Parasitol, 36, 791-9. GILLIN, F. D., HAGBLOM, P., HARWOOD, J., ALEY, S. B., REINER, D. S., MCCAFFERY, M., SO, M. & GUINEY, D. G. 1990. Isolation and expression of the gene for a major surface protein of Giardia lamblia. Proc Natl Acad Sci U S A, 87, 4463-7. GILLIN, F. D., REINER, D. S. & MCCAFFERY, J. M. 1996. Cell biology of the primitive eukaryote Giardia lamblia. Annu Rev Microbiol, 50, 679-705. GINGER, M. L., PORTMAN, N. & MCKEAN, P. G. 2008. Swimming with protists: perception, motility and flagellum assembly. Nat Rev Microbiol, 6, 838-50. GOYAL, N., TIWARI, R. P. & SHUKLA, G. 2011. Lactobacillus rhamnosus GG as an Effective Probiotic for Murine Giardiasis. Interdiscip Perspect Infect Dis, 2011, 795219. GRIT, G. H., DEVRIENDT, B., VAN COPPERNOLLE, S., GEURDEN, T., HOPE, J., VERCRUYSSE, J., COX, E., GELDHOF, P. & CLAEREBOUT, E. 2014. Giardia duodenalis stimulates partial maturation of bovine dendritic cells associated with altered cytokine secretion and induction of T-cell proliferation. Parasite Immunol, 36, 157-69. HALLIDAY, C. E., CLARK, C. & FARTHING, M. J. 1988. Giardia-bile salt interactions in vitro and in vivo. Trans R Soc Trop Med Hyg, 82, 428-32. HALLIEZ, M. C., MOTTA, J. P., FEENER, T. D., GUERIN, G., LEGOFF, L., FRANCOIS, A., COLASSE, E., FAVENNEC, L., GARGALA, G., LAPOINTE, T. K., ALTIER, C. & BURET, A. G. 2016. Giardia duodenalis induces paracellular bacterial translocation and causes postinfectious visceral hypersensitivity. Am J Physiol Gastrointest Liver Physiol, 310, G574-85. HANEVIK, K., WENSAAS, K. A., RORTVEIT, G., EIDE, G. E., MORCH, K. & LANGELAND, N. 2014. Irritable bowel syndrome and chronic fatigue 6 years after giardia infection: a controlled prospective cohort study. Clin Infect Dis, 59, 1394-400. HARDIN, W. R., LI, R., XU, J., SHELTON, A. M., ALAS, G. C. M., MININ, V. N. & PAREDEZ, A. R. 2017. Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking. Proc Natl Acad Sci U S A, 114, E5854-E5863. HERNANDEZ-SANCHEZ, J., LINAN, R. F., SALINAS-TOBON MDEL, R. & ORTEGA-PIERRES, G. 2008. Giardia duodenalis: adhesion-deficient clones have reduced ability to establish infection in Mongolian gerbils. Exp Parasitol, 119, 364-72. HERNANDEZ, H. M., MARCET, R. & SARRACENT, J. 2014. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis. Parasite, 21, 54.

58 HERRMANN, A., DAVIES, J. R., LINDELL, G., MARTENSSON, S., PACKER, N. H., SWALLOW, D. M. & CARLSTEDT, I. 1999. Studies on the "insoluble" glycoprotein complex from human colon. Identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage. J Biol Chem, 274, 15828-36. HOFSTETROVA, K., UZLIKOVA, M., TUMOVA, P., TROELL, K., SVARD, S. G. & NOHYNKOVA, E. 2010. Giardia intestinalis: aphidicolin influence on the trophozoite cell cycle. Exp Parasitol, 124, 159-66. HOUSE, S. A., RICHTER, D. J., PHAM, J. K. & DAWSON, S. C. 2011. Giardia flagellar motility is not directly required to maintain attachment to surfaces. PLoS Pathog, 7, e1002167. HUMEN, M. A., DE ANTONI, G. L., BENYACOUB, J., COSTAS, M. E., CARDOZO, M. I., KOZUBSKY, L., SAUDAN, K. Y., BOENZLI- BRUAND, A., BLUM, S., SCHIFFRIN, E. J. & PEREZ, P. F. 2005. Lactobacillus johnsonii La1 antagonizes Giardia intestinalis in vivo. Infect Immun, 73, 1265-9. HUMEN, M. A., PEREZ, P. F. & LIEVIN-LE MOAL, V. 2011. Lipid raft- dependent adhesion of Giardia intestinalis trophozoites to a cultured human enterocyte-like Caco-2/TC7 cell monolayer leads to cytoskeleton- dependent functional injuries. Cell Microbiol, 13, 1683-702. INGE, P. M., EDSON, C. M. & FARTHING, M. J. 1988. Attachment of Giardia lamblia to rat intestinal epithelial cells. Gut, 29, 795-801. JEDELSKY, P. L., DOLEZAL, P., RADA, P., PYRIH, J., SMID, O., HRDY, I., SEDINOVA, M., MARCINCIKOVA, M., VOLEMAN, L., PERRY, A. J., BELTRAN, N. C., LITHGOW, T. & TACHEZY, J. 2011. The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis. PLoS One, 6, e17285. JENIKOVA, G., HRUZ, P., ANDERSSON, M. K., TEJMAN-YARDEN, N., FERREIRA, P. C., ANDERSEN, Y. S., DAVIDS, B. J., GILLIN, F. D., SVARD, S. G., CURTISS, R., 3RD & ECKMANN, L. 2011. Alpha1- giardin based live heterologous vaccine protects against Giardia lamblia infection in a murine model. Vaccine, 29, 9529-37. JENKINS, M. C., O'BRIEN, C. N., MURPHY, C., SCHWARZ, R., MISKA, K., ROSENTHAL, B. & TROUT, J. M. 2009. Antibodies to the ventral disc protein delta-giardin prevent in vitro binding of Giardia lamblia trophozoites. J Parasitol, 95, 895-9. JIMENEZ, J. C., FONTAINE, J., GRZYCH, J. M., CAPRON, M. & DEI-CAS, E. 2009. Antibody and cytokine responses in BALB/c mice immunized with the excreted/secreted proteins of Giardia intestinalis: the role of cysteine proteases. Ann Trop Med Parasitol, 103, 693-703. JIMENEZ, J. C., FONTAINE, J., GRZYCH, J. M., DEI-CAS, E. & CAPRON, M. 2004. Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis. Clin Diagn Lab Immunol, 11, 152-60. JIMENEZ, J. C., MORELLE, W., MICHALSKY, J. C. & DEI-CAS, E. 2007. Excreted/secreted glycoproteins of G. intestinalis play an essential role in the antibody response. Parasitol Res, 100, 715-20.

59 JIMENEZ, J. C., UZCANGA, G., ZAMBRANO, A., DI PRISCO, M. C. & LYNCH, N. R. 2000. Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis. J Parasitol, 86, 859-62. JOHANSSON, M. E. & HANSSON, G. C. 2016. Immunological aspects of intestinal mucus and mucins. Nat Rev Immunol, 16, 639-49. JOHANSSON, M. E., PHILLIPSON, M., PETERSSON, J., VELCICH, A., HOLM, L. & HANSSON, G. C. 2008. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A, 105, 15064-9. JONES, D. E. & BEVINS, C. L. 1992. Paneth cells of the human small intestine express an antimicrobial peptide gene. J Biol Chem, 267, 23216-25. KABNICK, K. S. & PEATTIE, D. A. 1990. In situ analyses reveal that the two nuclei of Giardia lamblia are equivalent. J Cell Sci, 95 ( Pt 3), 353-60. KAMDA, J. D., NASH, T. E. & SINGER, S. M. 2012. Giardia duodenalis: dendritic cell defects in IL-6 deficient mice contribute to susceptibility to intestinal infection. Exp Parasitol, 130, 288-91. KAMDA, J. D. & SINGER, S. M. 2009. Phosphoinositide 3-kinase-dependent inhibition of dendritic cell interleukin-12 production by Giardia lamblia. Infect Immun, 77, 685-93. KANG, J. M., JU, H. L., LEE, K. H., KIM, T. S., PAK, J. H., SOHN, W. M. & NA, B. K. 2014. Identification and characterization of the second cysteine protease inhibitor of Clonorchis sinensis (CsStefin-2). Parasitol Res, 113, 47-58. KANG, J. M., LEE, K. H., SOHN, W. M. & NA, B. K. 2011. Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis. Mol Biochem Parasitol, 177, 126-34. KARANIS, P. & EY, P. L. 1998. Characterization of axenic isolates of Giardia intestinalis established from humans and animals in Germany. Parasitol Res, 84, 442-9. KARR, C. D. & JARROLL, E. L. 2004. Cyst wall synthase: N- acetylgalactosaminyltransferase activity is induced to form the novel N- acetylgalactosamine polysaccharide in the Giardia cyst wall. Microbiology, 150, 1237-43. KATELARIS, P. H., NAEEM, A. & FARTHING, M. J. 1995. Attachment of Giardia lamblia trophozoites to a cultured human intestinal cell line. Gut, 37, 512-8. KAUR, H., GHOSH, S., SAMRA, H., VINAYAK, V. K. & GANGULY, N. K. 2001. Identification and characterization of an excretory-secretory product from Giardia lamblia. Parasitology, 123, 347-56. KAUR, H., SAMRA, H., GHOSH, S., VINAYAK, V. K. & GANGULY, N. K. 1999. Immune effector responses to an excretory-secretory product of Giardia lamblia. FEMS Immunol Med Microbiol, 23, 93-105. KEENAN, K. P., SHARPNACK, D. D., COLLINS, H., FORMAL, S. B. & O'BRIEN, A. D. 1986. Morphologic evaluation of the effects of Shiga toxin and E coli Shiga-like toxin on the rabbit intestine. Am J Pathol, 125, 69-80.

60 KESELMAN, A., LI, E., MALONEY, J. & SINGER, S. M. 2016. The Microbiota Contributes to CD8+ T Cell Activation and Nutrient Malabsorption following Intestinal Infection with Giardia duodenalis. Infect Immun, 84, 2853-60. KOFOID, C. A., CHRISTENSEN, E. B. 1915. On binary and multiple fission in Giardia muris (Grassi). Univ Calif Publ Zool, 16, 30-54. KOH, W. H., GEURDEN, T., PAGET, T., O'HANDLEY, R., STEUART, R. F., THOMPSON, R. C. & BURET, A. G. 2013. Giardia duodenalis assemblage-specific induction of apoptosis and tight junction disruption in human intestinal epithelial cells: effects of mixed infections. J Parasitol, 99, 353-8. LAMBL, W. 1859. Mikroskopische Untersuchungen der Darmexcrete. Vierteljahrsschr. Prakst. Heikunde (Prague), 61:1-58. LANE, S. & LLOYD, D. 2002. Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol, 28, 123-47. LANGFORD, T. D., HOUSLEY, M. P., BOES, M., CHEN, J., KAGNOFF, M. F., GILLIN, F. D. & ECKMANN, L. 2002. Central importance of immunoglobulin A in host defense against Giardia spp. Infect Immun, 70, 11-8. LASSERRE, C., COLNOT, C., BRECHOT, C. & POIRIER, F. 1999. HIP/PAP gene, encoding a C-type lectin overexpressed in primary liver cancer, is expressed in nervous system as well as in intestine and pancreas of the postimplantation mouse embryo. Am J Pathol, 154, 1601-10. LAUWAET, T., DAVIDS, B. J., TORRES-ESCOBAR, A., BIRKELAND, S. R., CIPRIANO, M. J., PREHEIM, S. P., PALM, D., SVARD, S. G., MCARTHUR, A. G. & GILLIN, F. D. 2007. Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol, 152, 80-9. LEE, H. Y., KIM, J., NOH, H. J., KIM, H. P. & PARK, S. J. 2014a. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs. Parasite Immunol, 36, 627-46. LEE, J., KIM, J. H., SOHN, H. J., YANG, H. J., NA, B. K., CHWAE, Y. J., PARK, S., KIM, K. & SHIN, H. J. 2014b. Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties. Parasitol Res, 113, 2765-76. LI, E., ZHOU, P. & SINGER, S. M. 2006. Neuronal nitric oxide synthase is necessary for elimination of Giardia lamblia infections in mice. J Immunol, 176, 516-21. LIDELL, M. E., MONCADA, D. M., CHADEE, K. & HANSSON, G. C. 2006. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C- terminal domain and dissolve the protective colonic mucus gel. Proc Natl Acad Sci U S A, 103, 9298-303. LU, S. Q., BARUCH, A. C. & ADAM, R. D. 1998. Molecular comparison of Giardia lamblia isolates. Int J Parasitol, 28, 1341-5.

61 LUJAN, H. D., MOWATT, M. R., BYRD, L. G. & NASH, T. E. 1996. Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci U S A, 93, 7628-33. MA'AYEH, S. Y. & BROOK-CARTER, P. T. 2012. Representational difference analysis identifies specific genes in the interaction of Giardia duodenalis with the murine intestinal epithelial cell line, IEC-6. Int J Parasitol, 42, 501-9. MA'AYEH, S. Y., KNORR, L., SKOLD, K., GARNHAM, A., ANSELL, B. R. E., JEX, A. R. & SVARD, S. G. 2018. Corrigendum: Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate. Front Cell Infect Microbiol, 8, 297. MAASER, C. & KAGNOFF, M. F. 2002. Role of the intestinal epithelium in orchestrating innate and adaptive mucosal immunity. Z Gastroenterol, 40, 525-9. MAGNE, D., FAVENNEC, L., CHOCHILLON, C., GORENFLOT, A., MEILLET, D., KAPEL, N., RAICHVARG, D., SAVEL, J. & GOBERT, J. G. 1991. Role of cytoskeleton and surface lectins in Giardia duodenalis attachment to Caco2 cells. Parasitol Res, 77, 659-62. MAIA-BRIGAGAO, C., MORGADO-DIAZ, J. A. & DE SOUZA, W. 2012. Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells. Parasitol Int, 61, 280-7. MANKO, A., MOTTA, J. P., COTTON, J. A., FEENER, T., OYEYEMI, A., VALLANCE, B. A., WALLACE, J. L. & BURET, A. G. 2017. Giardia co- infection promotes the secretion of antimicrobial peptides beta-defensin 2 and trefoil factor 3 and attenuates attaching and effacing bacteria-induced intestinal disease. PLoS One, 12, e0178647. MARTI, M., LI, Y., SCHRANER, E. M., WILD, P., KOHLER, P. & HEHL, A. B. 2003. The secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient Golgi-like compartments. Mol Biol Cell, 14, 1433-47. MATOWICKA-KARNA, J., KRALISZ, M. & KEMONA, H. 2011. Assessment of the levels of nitric oxide (NO) and cytokines (IL-5, IL-6, IL-13, TNF, IFN- gamma) in giardiosis. Folia Histochem Cytobiol, 49, 280-4. MCCAFFERY, J. M. & GILLIN, F. D. 1994. Giardia lamblia: ultrastructural basis of protein transport during growth and encystation. Exp Parasitol, 79, 220- 35. MCCOLE, D. F., ECKMANN, L., LAURENT, F. & KAGNOFF, M. F. 2000. Intestinal epithelial cell apoptosis following Cryptosporidium parvum infection. Infect Immun, 68, 1710-3. MISTRY, D. & STOCKLEY, R. A. 2006. IgA1 protease. Int J Biochem Cell Biol, 38, 1244-8. MOKRZYCKA, M., KOLASA, A., KOSIERKIEWICZ, A. & WISZNIEWSKA, B. 2010. Inducible nitric oxide synthase in duodenum of children with Giardia lamblia infection. Folia Histochem Cytobiol, 48, 191-6. MOLAU, U., NORDENHALL, U. & ERIKSEN, B. 2005. Onset of flowering and climate variability in an alpine landscape: a 10-year study from Swedish Lapland. Am J Bot, 92, 422-31.

62 MONCADA, D., YU, Y., KELLER, K. & CHADEE, K. 2000. Entamoeba histolytica cysteine proteinases degrade human colonic mucin and alter its function. Arch Med Res, 31, S224-5. MONIS, P. T., ANDREWS, R. H., MAYRHOFER, G. & EY, P. L. 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol, 16, 1135-44. MONIS, P. T., ANDREWS, R. H., MAYRHOFER, G., MACKRILL, J., KULDA, J., ISAAC-RENTON, J. L. & EY, P. L. 1998. Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology, 116 ( Pt 1), 7-19. MONIS, P. T., CACCIO, S. M. & THOMPSON, R. C. 2009. Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol, 25, 93-100. MORALES, F. C., FURTADO, D. R. & RUMJANEK, F. D. 2004. The N-terminus moiety of the cystatin SmCys from Schistosoma mansoni regulates its inhibitory activity in vitro and in vivo. Mol Biochem Parasitol, 134, 65-73. MORRIS, S. M., JR. 2010. Arginine: master and commander in innate immune responses. Sci Signal, 3, pe27. MORRISON, H. G., MCARTHUR, A. G., GILLIN, F. D., ALEY, S. B., ADAM, R. D., OLSEN, G. J., BEST, A. A., CANDE, W. Z., CHEN, F., CIPRIANO, M. J., DAVIDS, B. J., DAWSON, S. C., ELMENDORF, H. G., HEHL, A. B., HOLDER, M. E., HUSE, S. M., KIM, U. U., LASEK-NESSELQUIST, E., MANNING, G., NIGAM, A., NIXON, J. E., PALM, D., PASSAMANECK, N. E., PRABHU, A., REICH, C. I., REINER, D. S., SAMUELSON, J., SVARD, S. G. & SOGIN, M. L. 2007. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science, 317, 1921-6. MOTTRAM, J. C., SOUZA, A. E., HUTCHISON, J. E., CARTER, R., FRAME, M. J. & COOMBS, G. H. 1996. Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors. Proc Natl Acad Sci U S A, 93, 6008-13. MOWATT, M. R., AGGARWAL, A. & NASH, T. E. 1991. Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia. Mol Biochem Parasitol, 49, 215-27. MUNDODI, V., KUCKNOOR, A. S. & GEDAMU, L. 2005. Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition. BMC Mol Biol, 6, 3. MUNIZ, L. R., KNOSP, C. & YERETSSIAN, G. 2012. Intestinal antimicrobial peptides during homeostasis, infection, and disease. Front Immunol, 3, 310. MURTAGH, J. J., JR., MOWATT, M. R., LEE, C. M., LEE, F. J., MISHIMA, K., NASH, T. E., MOSS, J. & VAUGHAN, M. 1992. Guanine nucleotide- binding proteins in the intestinal parasite Giardia lamblia. Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor. J Biol Chem, 267, 9654-62.

63 MUSIL, D., ZUCIC, D., TURK, D., ENGH, R. A., MAYR, I., HUBER, R., POPOVIC, T., TURK, V., TOWATARI, T., KATUNUMA, N. & ET AL. 1991. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J, 10, 2321-30. NAKAO, J. H., COLLIER, S. A. & GARGANO, J. W. 2017. Giardiasis and Subsequent Irritable Bowel Syndrome: A Longitudinal Cohort Study Using Health Insurance Data. J Infect Dis, 215, 798-805. NANDAN, D. & REINER, N. E. 2005. Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1. Clin Immunol, 114, 266-77. NASH, T. E. 2002. Surface antigenic variation in Giardia lamblia. Mol Microbiol, 45, 585-90. NASH, T. E., AGGARWAL, A., ADAM, R. D., CONRAD, J. T. & MERRITT, J. W., JR. 1988. Antigenic variation in Giardia lamblia. J Immunol, 141, 636- 41. NASH, T. E., HERRINGTON, D. A., LEVINE, M. M., CONRAD, J. T. & MERRITT, J. W., JR. 1990. Antigenic variation of Giardia lamblia in experimental human infections. J Immunol, 144, 4362-9. NASH, T. E., HERRINGTON, D. A., LOSONSKY, G. A. & LEVINE, M. M. 1987. Experimental human infections with Giardia lamblia. J Infect Dis, 156, 974-84. NASH, T. E. & KEISTER, D. B. 1985. Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. J Infect Dis, 152, 1166- 71. NASH, T. E., MCCUTCHAN, T., KEISTER, D., DAME, J. B., CONRAD, J. D. & GILLIN, F. D. 1985. Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J Infect Dis, 152, 64- 73. NASH, T. E., MERRITT, J. W., JR. & CONRAD, J. T. 1991. Isolate and epitope variability in susceptibility of Giardia lamblia to intestinal proteases. Infect Immun, 59, 1334-40. NASH, T. E. & MOWATT, M. R. 1992. Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size. Mol Biochem Parasitol, 51, 219-27. NDAO, M., BEAULIEU, C., BLACK, W. C., ISABEL, E., VASQUEZ- CAMARGO, F., NATH-CHOWDHURY, M., MASSE, F., MELLON, C., METHOT, N. & NICOLL-GRIFFITH, D. A. 2014. Reversible cysteine protease inhibitors show promise for a Chagas disease cure. Antimicrob Agents Chemother, 58, 1167-78. NISHIMURA, T. & TAKEICHI, M. 2009. Remodeling of the adherens junctions during morphogenesis. Curr Top Dev Biol, 89, 33-54. NOHRIA, A., ALONSO, R. A. & PEATTIE, D. A. 1992. Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol Biochem Parasitol, 56, 27-37.

64 NOURRISSON, C., WAWRZYNIAK, I., CIAN, A., LIVRELLI, V., VISCOGLIOSI, E., DELBAC, F. & POIRIER, P. 2016. On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers. Parasitology, 143, 1713-1722. NYINDODO-OGARI, L., SCHWARTZBACH, S. D. & ESTRANO, C. E. 2014. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals. Infect Disord Drug Targets, 14, 23-9. O'BRIEN, T. C., MACKEY, Z. B., FETTER, R. D., CHOE, Y., O'DONOGHUE, A. J., ZHOU, M., CRAIK, C. S., CAFFREY, C. R. & MCKERROW, J. H. 2008. A parasite cysteine protease is key to host protein degradation and iron acquisition. J Biol Chem, 283, 28934-43. O'HANDLEY, R. M., CERI, H., ANETTE, C. & OLSON, M. E. 2003. Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections. Vet Parasitol, 113, 89-98. O'NEIL, D. A., PORTER, E. M., ELEWAUT, D., ANDERSON, G. M., ECKMANN, L., GANZ, T. & KAGNOFF, M. F. 1999. Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol, 163, 6718-24. OLMEZ, S., ASLAN, M., YAVUZ, A., BULUT, G. & DULGER, A. C. 2014. Diffuse nodular lymphoid hyperplasia of the small bowel associated with common variable immunodeficiency and giardiasis: a rare case report. Wien Klin Wochenschr, 126, 294-7. ORTEGA-PIERRES, G., ARGUELLO-GARCIA, R., LAREDO-CISNEROS, M. S., FONSECA-LINAN, R., GOMEZ-MONDRAGON, M., INZUNZA- ARROYO, R., FLORES-BENITEZ, D., RAYA-SANDINO, A., CHAVEZ- MUNGUIA, B., VENTURA-GALLEGOS, J. L., ZENTELLA-DEHESA, A., BERMUDEZ-CRUZ, R. M. & GONZALEZ-MARISCAL, L. 2018. Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers. Int J Parasitol, 48, 621-639. OUELLETTE, A. J., DARMOUL, D., TRAN, D., HUTTNER, K. M., YUAN, J. & SELSTED, M. E. 1999. Peptide localization and gene structure of cryptdin 4, a differentially expressed mouse paneth cell alpha-defensin. Infect Immun, 67, 6643-51. PAGET, T. A. & JAMES, S. L. 1994. The mucolytic activity of polyamines and mucosal invasion. Biochem Soc Trans, 22, 394S. PALM, D., WEILAND, M., MCARTHUR, A. G., WINIECKA-KRUSNELL, J., CIPRIANO, M. J., BIRKELAND, S. R., PACOCHA, S. E., DAVIDS, B., GILLIN, F., LINDER, E. & SVARD, S. 2005. Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol, 141, 199-207. PALM, J. E., WEILAND, M. E., GRIFFITHS, W. J., LJUNGSTROM, I. & SVARD, S. G. 2003. Identification of immunoreactive proteins during acute human giardiasis. J Infect Dis, 187, 1849-59.

65 PANARO, M. A., CIANCIULLI, A., MITOLO, V., MITOLO, C. I., ACQUAFREDDA, A., BRANDONISIO, O. & CAVALLO, P. 2007. Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis. FEMS Immunol Med Microbiol, 51, 302-9. PARENTI, D. M. 1989. Characterization of a thiol proteinase in Giardia lamblia. J Infect Dis, 160, 1076-80. PAWLOWSKI, S. W., WARREN, C. A. & GUERRANT, R. 2009. Diagnosis and treatment of acute or persistent diarrhea. Gastroenterology, 136, 1874-86. PEREZ, P. F., MINNAARD, J., ROUVET, M., KNABENHANS, C., BRASSART, D., DE ANTONI, G. L. & SCHIFFRIN, E. J. 2001. Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study. Appl Environ Microbiol, 67, 5037-42. PIMENTA, P. F., DA SILVA, P. P. & NASH, T. 1991. Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and label fracture immunocytochemistry survey. Infect Immun, 59, 3989-96. POKUTTA, S. & WEIS, W. I. 2007. Structure and mechanism of cadherins and catenins in cell-cell contacts. Annu Rev Cell Dev Biol, 23, 237-61. PORTER, E. M., LIU, L., OREN, A., ANTON, P. A. & GANZ, T. 1997a. Localization of human intestinal defensin 5 in Paneth cell granules. Infect Immun, 65, 2389-95. PORTER, E. M., VAN DAM, E., VALORE, E. V. & GANZ, T. 1997b. Broad- spectrum antimicrobial activity of human intestinal defensin 5. Infect Immun, 65, 2396-401. POTOKA, D. A., UPPERMAN, J. S., ZHANG, X. R., KAPLAN, J. R., COREY, S. J., GRISHIN, A., ZAMORA, R. & FORD, H. R. 2003. Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro. Am J Physiol Gastrointest Liver Physiol, 285, G861-9. POXLEITNER, M. K., CARPENTER, M. L., MANCUSO, J. J., WANG, C. J., DAWSON, S. C. & CANDE, W. Z. 2008. Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science, 319, 1530-3. PRUCCA, C. G. & LUJAN, H. D. 2009. Antigenic variation in Giardia lamblia. Cell Microbiol, 11, 1706-15. PUENTE-RIVERA, J., RAMON-LUING LDE, L., FIGUEROA-ANGULO, E. E., ORTEGA-LOPEZ, J. & ARROYO, R. 2014. Trichocystatin-2 (TC-2): an endogenous inhibitor of cysteine proteinases in Trichomonas vaginalis is associated with TvCP39. Int J Biochem Cell Biol, 54, 255-65. QUE, X., ENGEL, J. C., FERGUSON, D., WUNDERLICH, A., TOMAVO, S. & REED, S. L. 2007. Cathepsin Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii. J Biol Chem, 282, 4994-5003. REGOES, R. R., CROTTY, S., ANTIA, R. & TANAKA, M. M. 2005. Optimal replication of poliovirus within cells. Am Nat, 165, 364-73. RENDTORFF, R. C. 1954. The experimental transmission of human intestinal protozoan parasites. II. Giardia lamblia cysts given in capsules. Am J Hyg, 59, 209-20.

66 RIEKENBERG, S., WITJES, B., SARIC, M., BRUCHHAUS, I. & SCHOLZE, H. 2005. Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica. FEBS Lett, 579, 1573-8. RINGQVIST, E., AVESSON, L., SODERBOM, F. & SVARD, S. G. 2011. Transcriptional changes in Giardia during host-parasite interactions. Int J Parasitol, 41, 277-85. RINGQVIST, E., PALM, J. E., SKARIN, H., HEHL, A. B., WEILAND, M., DAVIDS, B. J., REINER, D. S., GRIFFITHS, W. J., ECKMANN, L., GILLIN, F. D. & SVARD, S. G. 2008. Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol, 159, 85-91. RIVERO, F. D., SAURA, A., PRUCCA, C. G., CARRANZA, P. G., TORRI, A. & LUJAN, H. D. 2010a. Disruption of antigenic variation is crucial for effective parasite vaccine. Nat Med, 16, 551-7, 1p following 557. RIVERO, M. R., VRANYCH, C. V., BISBAL, M., MALETTO, B. A., ROPOLO, A. S. & TOUZ, M. C. 2010b. Adaptor protein 2 regulates receptor- mediated endocytosis and cyst formation in Giardia lamblia. Biochem J, 428, 33-45. ROBBINS, P. W. & SAMUELSON, J. 2005. Asparagine linked glycosylation in Giardia. Glycobiology, 15, 15G-16G. RODRIGUEZ-FUENTES, G. B., CEDILLO-RIVERA, R., FONSECA-LINAN, R., ARGUELLO-GARCIA, R., MUNOZ, O., ORTEGA-PIERRES, G. & YEPEZ-MULIA, L. 2006. Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro. Mem Inst Oswaldo Cruz, 101, 693-6. RODRIGUEZ, O. L., HAGEL, I., GONZALEZ, Y., ROQUE, M. E., VASQUEZ, N., LOPEZ, E. & DI PRISCO, M. C. 2004. Secretory IgA antibody responses in Venezuelan children infected with Giardia duodenalis. J Trop Pediatr, 50, 68-72. ROGAWSKI, E. T., BARTELT, L. A., PLATTS-MILLS, J. A., SEIDMAN, J. C., SAMIE, A., HAVT, A., BABJI, S., TRIGOSO, D. R., QURESHI, S., SHAKOOR, S., HAQUE, R., MDUMA, E., BAJRACHARYA, S., GAFFAR, S. M. A., LIMA, A. A. M., KANG, G., KOSEK, M. N., AHMED, T., SVENSEN, E., MASON, C., BHUTTA, Z. A., LANG, D. R., GOTTLIEB, M., GUERRANT, R. L., HOUPT, E. R. & BESSONG, P. O. 2017. Determinants and Impact of Giardia Infection in the First 2 Years of Life in the MAL-ED Birth Cohort. J Pediatric Infect Dis Soc, 6, 153-160. ROSKENS, H. & ERLANDSEN, S. L. 2002. Inhibition of in vitro attachment of Giardia trophozoites by mucin. J Parasitol, 88, 869-73. ROXSTROM-LINDQUIST, K., RINGQVIST, E., PALM, D. & SVARD, S. 2005. Giardia lamblia-induced changes in gene expression in differentiated Caco- 2 human intestinal epithelial cells. Infect Immun, 73, 8204-8. SAGHAUG, C. S., SORNES, S., PEIRASMAKI, D., SVARD, S., LANGELAND, N. & HANEVIK, K. 2016. Human Memory CD4+ T Cell Immune Responses against Giardia lamblia. Clin Vaccine Immunol, 23, 11-8. SAJID, M. & MCKERROW, J. H. 2002. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol, 120, 1-21.

67 SAMRA, H. K., GANGULY, N. K., GARG, U. C., GOYAL, J. & MAHAJAN, R. C. 1988. Effect of excretory-secretory products of Giardia lamblia on glucose and phenylalanine transport in the small intestine of Swiss albino mice. Biochem Int, 17, 801-12. SARAIYA, A. A. & WANG, C. C. 2008. snoRNA, a novel precursor of microRNA in Giardia lamblia. PLoS Pathog, 4, e1000224. SATO, D., NAKADA-TSUKUI, K., OKADA, M. & NOZAKI, T. 2006. Two cysteine protease inhibitors, EhICP1 and 2, localized in distinct compartments, negatively regulate secretion in Entamoeba histolytica. FEBS Lett, 580, 5306-12. SCHOFIELD, P. J., EDWARDS, M. R., MATTHEWS, J. & WILSON, J. R. 1992. The pathway of arginine catabolism in Giardia intestinalis. Mol Biochem Parasitol, 51, 29-36. SCHUMANN, M., SIEGMUND, B., SCHULZKE, J. D. & FROMM, M. 2017. Celiac Disease: Role of the Epithelial Barrier. Cell Mol Gastroenterol Hepatol, 3, 150-162. SCHUTTE, A., ERMUND, A., BECKER-PAULY, C., JOHANSSON, M. E., RODRIGUEZ-PINEIRO, A. M., BACKHED, F., MULLER, S., LOTTAZ, D., BOND, J. S. & HANSSON, G. C. 2014. Microbial-induced meprin beta cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus. Proc Natl Acad Sci U S A, 111, 12396-401. SCOTT, K. G., MEDDINGS, J. B., KIRK, D. R., LEES-MILLER, S. P. & BURET, A. G. 2002. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology, 123, 1179-90. SCOTT, K. G., YU, L. C. & BURET, A. G. 2004. Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis. Infect Immun, 72, 3536-42. SHANT, J., BHATTACHARYYA, S., GHOSH, S., GANGULY, N. K. & MAJUMDAR, S. 2002. A potentially important excretory-secretory product of Giardia lamblia. Exp Parasitol, 102, 178-86. SHANT, J., GHOSH, S., BHATTACHARYYA, S., GANGULY, N. K. & MAJUMDAR, S. 2004. The alteration in signal transduction parameters induced by the excretory-secretory product from Giardia lamblia. Parasitology, 129, 421-30. SHANT, J., GHOSH, S., BHATTACHARYYA, S., GANGULY, N. K. & MAJUMDAR, S. 2005. Mode of action of a potentially important excretory--secretory product from Giardia lamblia in mice enterocytes. Parasitology, 131, 57-69. SHUKLA, G., DEVI, P. & SEHGAL, R. 2008. Effect of Lactobacillus casei as a probiotic on modulation of giardiasis. Dig Dis Sci, 53, 2671-9. SHUKLA, G. & SIDHU, R. K. 2011. Lactobacillus casei as a probiotic in malnourished Giardia lamblia-infected mice: a biochemical and histopathological study. Can J Microbiol, 57, 127-35.

68 SIJWALI, P. S., KATO, K., SEYDEL, K. B., GUT, J., LEHMAN, J., KLEMBA, M., GOLDBERG, D. E., MILLER, L. H. & ROSENTHAL, P. J. 2004. Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites. Proc Natl Acad Sci U S A, 101, 8721- 6. SIJWALI, P. S., KOO, J., SINGH, N. & ROSENTHAL, P. J. 2006. Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum. Mol Biochem Parasitol, 150, 96-106. SIJWALI, P. S. & ROSENTHAL, P. J. 2004. Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum. Proc Natl Acad Sci U S A, 101, 4384-9. SINGER, S. M., ELMENDORF, H. G., CONRAD, J. T. & NASH, T. E. 2001. Biological selection of variant-specific surface proteins in Giardia lamblia. J Infect Dis, 183, 119-24. SINGER, S. M. & NASH, T. E. 2000. The role of normal flora in Giardia lamblia infections in mice. J Infect Dis, 181, 1510-2. SKARIN, H., RINGQVIST, E., HELLMAN, U. & SVARD, S. G. 2011. Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites. Exp Parasitol, 127, 804-10. SMITH, P. D., GILLIN, F. D., BROWN, W. R. & NASH, T. E. 1981. IgG antibody to Giardia lamblia detected by enzyme-linked immunosorbent assay. Gastroenterology, 80, 1476-80. SMITH, P. D., GILLIN, F. D., KAUSHAL, N. A. & NASH, T. E. 1982. Antigenic analysis of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon. Infect Immun, 36, 714-9. SOLAYMANI-MOHAMMADI, S. & SINGER, S. M. 2013. Regulation of intestinal epithelial cell cytoskeletal remodeling by cellular immunity following gut infection. Mucosal Immunol, 6, 369-78. SOUSA, M. C., GONCALVES, C. A., BAIROS, V. A. & POIARES-DA-SILVA, J. 2001. Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin Diagn Lab Immunol, 8, 258-65. STADELMANN, B., MERINO, M. C., PERSSON, L. & SVARD, S. G. 2012. Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells. PLoS One, 7, e45325. STAGER, S., GOTTSTEIN, B., SAGER, H., JUNGI, T. W. & MULLER, N. 1998. Influence of antibodies in mother's milk on antigenic variation of Giardia lamblia in the murine mother-offspring model of infection. Infect Immun, 66, 1287-92. STARK, D., BARRATT, J. L., VAN HAL, S., MARRIOTT, D., HARKNESS, J. & ELLIS, J. T. 2009. Clinical significance of enteric protozoa in the immunosuppressed human population. Clin Microbiol Rev, 22, 634-50. STEVERDING, D., SEXTON, D. W., WANG, X., GEHRKE, S. S., WAGNER, G. K. & CAFFREY, C. R. 2012. Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target. Int J Parasitol, 42, 481-8. STILES, C. W. 1902. The type species of certain genera of parasitic flagellates, particularly Grassi's genera of 1879 and 1881. Zool Anz, 25:689.

69 SUN, C. H., PALM, D., MCARTHUR, A. G., SVARD, S. G. & GILLIN, F. D. 2002. A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia. Mol Microbiol, 46, 971-84. SVARD, S. G., MENG, T. C., HETSKO, M. L., MCCAFFERY, J. M. & GILLIN, F. D. 1998. Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. Mol Microbiol, 30, 979-89. TAKO, E. A., HASSIMI, M. F., LI, E. & SINGER, S. M. 2013. Transcriptomic analysis of the host response to Giardia duodenalis infection reveals redundant mechanisms for parasite control. MBio, 4, e00660-13. TELLEZ, A., PALM, D., WEILAND, M., ALEMAN, J., WINIECKA-KRUSNELL, J., LINDER, E. & SVARD, S. 2005. Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women. Parasite Immunol, 27, 163-9. TEOH, D. A., KAMIENIECKI, D., PANG, G. & BURET, A. G. 2000. Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol, 86, 800-6. THIRION, J., WATTIAUX, R. & JADOT, M. 2003. The acid phosphatase positive organelles of the Giardia lamblia trophozoite contain a membrane bound cathepsin C activity. Biol Cell, 95, 99-105. THORNBERRY, N. A. & LAZEBNIK, Y. 1998. Caspases: enemies within. Science, 281, 1312-6. THORPE, M., AKULA, S. & HELLMAN, L. 2016. Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells. Dev Comp Immunol, 63, 84-95. TORRES, M. F., UETANABARO, A. P., COSTA, A. F., ALVES, C. A., FARIAS, L. M., BAMBIRRA, E. A., PENNA, F. J., VIEIRA, E. C. & NICOLI, J. R. 2000. Influence of bacteria from the duodenal microbiota of patients with symptomatic giardiasis on the pathogenicity of Giardia duodenalis in gnotoxenic mice. J Med Microbiol, 49, 209-15. TOUZ, M. C., KULAKOVA, L. & NASH, T. E. 2004. Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia. Mol Biol Cell, 15, 3053-60. TOUZ, M. C., ROPOLO, A. S., RIVERO, M. R., VRANYCH, C. V., CONRAD, J. T., SVARD, S. G. & NASH, T. E. 2008. Arginine deiminase has multiple regulatory roles in the biology of Giardia lamblia. J Cell Sci, 121, 2930-8. TOVAR, J., LEON-AVILA, G., SANCHEZ, L. B., SUTAK, R., TACHEZY, J., VAN DER GIEZEN, M., HERNANDEZ, M., MULLER, M. & LUCOCQ, J. M. 2003. Mitochondrial remnant organelles of Giardia function in iron- sulphur protein maturation. Nature, 426, 172-6. TRAVERS, M. A., SOW, C., ZIRAH, S., DEREGNAUCOURT, C., CHAOUCH, S., QUEIROZ, R. M., CHARNEAU, S., ALLAIN, T., FLORENT, I. & GRELLIER, P. 2016. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth. Front Microbiol, 7, 1453.

70 TROEGER, H., EPPLE, H. J., SCHNEIDER, T., WAHNSCHAFFE, U., ULLRICH, R., BURCHARD, G. D., JELINEK, T., ZEITZ, M., FROMM, M. & SCHULZKE, J. D. 2007. Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut, 56, 328- 35. TSE, S. K. & CHADEE, K. 1992. Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica. Infect Immun, 60, 1603-12. TUMANOV, A. V., KOROLEVA, E. P., GUO, X., WANG, Y., KRUGLOV, A., NEDOSPASOV, S. & FU, Y. X. 2011. Lymphotoxin controls the IL-22 protection pathway in gut innate lymphoid cells during mucosal pathogen challenge. Cell Host Microbe, 10, 44-53. TUMOVA, P., HOFSTETROVA, K., NOHYNKOVA, E., HOVORKA, O. & KRAL, J. 2007. Cytogenetic evidence for diversity of two nuclei within a single diplomonad cell of Giardia. Chromosoma, 116, 65-78. TURK, B., TURK, D. & TURK, V. 2000. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta, 1477, 98-111. WALTERSPIEL, J. N., MORROW, A. L., GUERRERO, M. L., RUIZ-PALACIOS, G. M. & PICKERING, L. K. 1994. Secretory anti-Giardia lamblia antibodies in human milk: protective effect against diarrhea. Pediatrics, 93, 28-31. WAMPFLER, P. B., TOSEVSKI, V., NANNI, P., SPYCHER, C. & HEHL, A. B. 2014. Proteomics of secretory and endocytic organelles in Giardia lamblia. PLoS One, 9, e94089. WARD, W., ALVARADO, L., RAWLINGS, N. D., ENGEL, J. C., FRANKLIN, C. & MCKERROW, J. H. 1997. A primitive enzyme for a primitive cell: the protease required for excystation of Giardia. Cell, 89, 437-44. WEERATUNGA, S. K., OSMAN, A., HU, N. J., WANG, C. K., MASON, L., SVARD, S., HOPE, G., JONES, M. K. & HOFMANN, A. 2012. Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms. J Mol Biol, 423, 169-81. WEILAND, M. E., PALM, J. E., GRIFFITHS, W. J., MCCAFFERY, J. M. & SVARD, S. G. 2003. Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan- binding activity. Int J Parasitol, 33, 1341-51. WENSAAS, K. A., LANGELAND, N., HANEVIK, K., MORCH, K., EIDE, G. E. & RORTVEIT, G. 2012. Irritable bowel syndrome and chronic fatigue 3 years after acute giardiasis: historic cohort study. Gut, 61, 214-9. WOMMACK, A. J., ZIAREK, J. J., TOMARAS, J., CHILEVERU, H. R., ZHANG, Y., WAGNER, G. & NOLAN, E. M. 2014. Discovery and characterization of a disulfide-locked C(2)-symmetric defensin peptide. J Am Chem Soc, 136, 13494-7. YOSHIDA, M., CLAYPOOL, S. M., WAGNER, J. S., MIZOGUCHI, E., MIZOGUCHI, A., ROOPENIAN, D. C., LENCER, W. I. & BLUMBERG, R. S. 2004. Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells. Immunity, 20, 769-83.

71 YU, L. C., HUANG, C. Y., KUO, W. T., SAYER, H., TURNER, J. R. & BURET, A. G. 2008. SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis. Int J Parasitol, 38, 923-34. YU, L. Z., BIRKY, C. W., JR. & ADAM, R. D. 2002. The two nuclei of Giardia each have complete copies of the genome and are partitioned equationally at cytokinesis. Eukaryot Cell, 1, 191-9. ZHOU, P., LI, E., SHEA-DONOHUE, T. & SINGER, S. M. 2007. Tumour necrosis factor alpha contributes to protection against Giardia lamblia infection in mice. Parasite Immunol, 29, 367-74. ZHOU, P., LI, E., ZHU, N., ROBERTSON, J., NASH, T. & SINGER, S. M. 2003. Role of interleukin-6 in the control of acute and chronic Giardia lamblia infections in mice. Infect Immun, 71, 1566-8.

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