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

I Roxström-Lindquist, K., Ringqvist, E., Palm, D., Svärd, S. (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infection and Immunity, 73(12):8204-8

II Ringqvist, E., Palm, D., Skarin, H., Hehl, A., Weiland, M., Da- vids, B., Reiner, D., Griffiths, W., Eckmann, L., Gillin, F., Svard, S. (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Molecular and Biochemical Parasitology, 159(2):85-91

III Skarin, H., Ringqvist, E., Hellman, U., Svärd, SG. (2009) Giar- dia intestinalis Elongation Factor 1-alpha is released during host cell interaction. Manuscript

IV Ringqvist, E., Troell, K., Svärd, S. (2009) Transcriptional changes in Giardia during host-parasite interactions. Manu- script

Reprints were made with permission from the respective publishers.

Review articles, not included in the thesis: I Roxström-Lindquist, K., Palm, D., Reiner, D., Ringqvist, E., Svärd, S. (2006) Giardia immunity – an update. Trends in Pa- rasitology 22(1):26-31

II Ankarklev, J., Jerlström-Hultqvist, J., Ringqvist, E., Troell, K., Svärd, S. (2009) Behind the smile: cell biology and disease me- chanisms of Giardia. Nature Reviews Microbiology. Manu- script under revision.

Contents

Sammanfattning på svenska (Summary in Swedish) ...... 7 Introduction to Giardia ...... 11 The Giardia Cell ...... 12 Excystation: cyst to trophozoite ...... 12 The trophozoite – the living cell ...... 14 Encystation: trophozoite to cyst ...... 16 The cell biology of Giardia ...... 17 Parasitic metabolism – dependency on the host ...... 21 Introduction to Giardia infections and the human host ...... 24 Giardiasis ...... 24 Infection site ...... 27 Immune system of the upper intestinal tract...... 30 Innate immunity ...... 30 Adaptive Immunity ...... 31 Establishment of infection ...... 33 Host range and Zoonosis ...... 34 Assemblages or species/ subspecies ...... 34 Molecular mechanisms of Host – Parasite interactions ...... 36 Relevance of the thesis ...... 36 Model systems of infection ...... 36 The pathology from Giardia ...... 39 The human response to a Giardia infection (Paper I) ...... 42 Parasite mechanisms for host survival ...... 44 Characterization of released Giardia proteins (Paper II and III) ...... 46 The Giardia transcriptome of early infection (Paper IV) ...... 51 Conclusions and thoughts for the future ...... 54 Acknowledgements ...... 56 References ...... 58

Abbreviations

ADI Arginine deiminase CCL (-2, -20) Chemokine with double cysteine (CC) N-terminal motif CXCL (-1, -2, -3) Chemokine where the CC-motif is separated by 1 amino acid DC Dendritic cell EF-1 Elongation Factor 1 alpha ER Endoplasmatic reticulum HCMp High cysteine membrane protein IEC Intestinal epithelial cell IgA Immunoglobulin A IL (-10) Interleukin (-10), a cytokine MIP-3 Macrophage inflammatory protein 3 alpha MS Mass Spectrometry NO Nitric oxide NOS Nitric oxide synthase OCT Ornithine carbamoyl transferase ORF Open reading frame PLAUR/ uPAR Plasminogen activating urokinase receptor PV Peripheral vesicles qRT-PCR quantitative real time polymerase chain reaction ROS Reactive oxygen species 2D SDS-PAGE 2-dimensional SDS-polyacrylamide gel electrophoresis sIgA soluble IgA UTR Un-translated region VSP Variable surface protein ZO-1 Zonula Occludens protein 1

Sammanfattning på svenska (Summary in Swedish)

Giardia lamblia är en encellig parasit med urtida påbrå. Datoranalyser av parasitens arvsmassa visar att Giardia liknar den djurcell eller ”urdjuret” som en gång gav upphov till dagens alla växter och djur. Den här avhand- lingen behandlar dock inte den evolutionära aspekten av Giardia cellen, utan är helt inriktad på vad som gör att den kan infektera och överleva i männi- skor, och hur den orsakar sjukdomen giardiasis.

Att Giardia klassas som en parasit innebär den att utnyttjar näringen från sin värd (människor och djur) utan att ge någon positiv effekt tillbaka. För Giardia är inte parasitism ett val: utan sin värds tunntarm kan inte cellen överleva. För att kunna förflyttas från en värd till en annan kan parasiten skydda sig med ett tjockt skal uppbyggt av socker och protein och kallas då för cysta. Dessa cystor lämnar sin värd med avföringen, och sprids sedan via avloppsvatten och i naturen. I svenska vattenreningsverk förekommer mäng- der med Giardia cystor. Med hjälp av reningsmetoder och filtrering blir mängden cystor i slutliga dricksvattnet färre än vad som krävs för att skapa en infektion. Vid enstaka tillfällen händer det att rent vatten blir uppblandat med smutsigt vatten eller avloppsvatten. Då finns det stor risk att människor dricker tillräckligt många cystor för att utveckla sjukdom. I Sverige diagnos- tiseras ungefär 1500 Giardia infektioner varje år. De allra flesta av infektio- nerna har personer ådragit sig i samband med resor till länder med sämre vattenrening och vattenkontroll än Sverige.

Symptomen för en Giardia infektion, och sjukdomen giardiasis, är kraf- tig, illaluktande och fettrik diarré med tillhörande magkrämpor, aptitförlust och illamående. Magkrämpor och irriterad eller inflammerad tarm kan följa på en infektion, även om Giardia parasiten inte längre lever kvar. Hur starka symptom en infekterad människa får är väldigt olika från fall till fall, och orsakerna är inte helt fastställda även om mycket tyder på att både männi- skans ålder, hälsa och immunförsvar samt skillnader mellan olika Giardia stammar kan påverka. En stor del av de som blir infekterade av Giardia uppvisar inga symptom alls.

De stora skillnaderna i symptom och i hur tarmen ser ut inuti (på cellnivå i tunntarmen) under en Giardia infektion ledde till de grundläggande fråge-

7 ställningarna inför mina doktorandstudier. Vad är det som händer i tarmens celler när de blir infekterade av Giardia? Vad är det parasiten gör för att inte bli bortstötta från tarmen eller dödade av kroppens immunförsvar? Vad finns det för koppling mellan parasiten och sjukdomen den skapar? Avhandlingen bygger på fyra vetenskapliga studier som alla syftar till att besvara nämnda frågor. Två av artiklarna från studierna har publicerats i vetenskapliga tid- skrifter, två artiklar är nyligen fullbordade och har därför ännu inte skickats in för vetenskaplig granskning. Studierna i avhandlingen är gjorda tillsam- mans med kollegor i Sverige och utomlands, samt tillsammans med handle- daren för mina doktorandstudier; Professor Staffan Svärd.

I den första studien (appendix Paper I) granskade jag och mina kollegor hur människans tunntarm påverkas av en Giardia-infektion. För att kunna studera tarmens celler på molekylär nivå behövde vi tillgång till många tarmceller som med säkerhet aldrig hade infekterats av Giardia förut. Tarm- cellerna skulle dessutom vara fria från den stora mängd molekyler, matres- ter och bakterier som vi naturligt har i vår tarm. Vi valde att skapa ett infek- tionsförlopp med speciella tarmceller som klarar av att växa och odlas på plastytor i laboratoriet (kallat cellodling). Giardia parasiten kan odlas utan värddjur i laboratoriet genom att tillsätta en näringsrik vätska (näring som normalt tillförs av värdens celler i tunntarmen) tillsammans med parasiten i små plaströr med skruvkork. För att studera hur tarmcellerna reagerade på en Giardia infektion blandade vi de båda celltyperna i små plastskålar och lät dem reagera upp till 18 timmar. Därefter tog vi hand om tarmcellerna, plockade ur dem deras molekyl-kartor av översättningen från arvsmassan till cellaktivitet (egentligen mRNA; strukturerna som bestämmer vilka protein som kan skapas i en cell). Vi såg att tarmcellerna hade skapat en stor arsenal av immunförsvar-aktiverande mRNA efter det att de hade utsatts för Giar- dia. Detta var det första beviset på att mänskliga tarmceller påverkas stort av en Giardia infektion, samt att tarmen aktivt försöker avdöda parasiten ge- nom att tillkalla immunförsvarets celler.

Det var mycket spännande att se att Giardia aktiverade människans im- munförsvar. Det innebar att det måste finnas någon substans hos parasiten som känns igen av tarmcellerna samt sannolikt aktiva mekansimer host para- siten för att undkomma att bli dödad av kroppens immunförsvar. Det som kom att bli Paper II, III och IV i avhandlingen var studier som fokuserade på hur parasiten beter sig när den infekterar människans tarmceller och hur den förändrar kroppens immunförsvar.

I både Paper II och Paper III studerade vi vilka aktiva protein-molekyler som parasiten skickar ut i samband med en infektion. Proteiner är uppbygg- da av runt 20-talet byggstenar till olika långa kedjor, så att varje protein kan ha en egen funktion. Nästan alla aktiviteter som pågår i vår kropp och i våra

8 enskilda celler är kopplade till olika proteiner. Likaså fungerar Giardia cel- len in i minsta detalj tack vare en uppsjö av proteiner. När vi sorterade och studerade vad som förekom emellan och runtom Giardia och tarmceller i laboratoriets cellkultur-skålar kunde vi identifierade fyra stycken olika prote- in. Det är en väldigt liten mängd protein, då många funktioner inom celler kräver samspel mellan flera tiotals proteinsorter. De fyra proteinen vi fann, började vi sedan studera i detalj. Vad kunde de ha för effekt vid en infek- tion? Kunde de bidra till sjukdomen? Var det dessa protein som gjorde att tarmcellerna började tillkalla och aktivera immunförsvarets celler?

Ett utav proteinen, kallat Arginine Deiminase men förkortat till ADI, vi- sade sig vara högst intressant. Parasitens ADI kunde påverka tarmen så att en viktig del av immunförsvaret förblev avstängt trots att kroppen skulle dragit stor nytta av att ha det aktivt. ADI kunde även delvis kopplas till diarré- symptomen vid infektion. De andra tre proteinen som vi identifierade har alla möjlighet att underlätta för Giardia vid infektion och kan vara viktiga vaccinkandidater för framtiden. Tyvärr fann vi inte någon aktivering av im- munförsvaret hos tarmceller med något av dessa fyra protein, vilket ledde oss till att göra en större studie som presenteras i Paper IV.

Alla protein som skapas i celler byggs upp efter speciella kartor, så kalla- de mRNA. Dessa mRNA är helt beroende av arvsamassan i cellerna, och proteinen som skapas är i sin tur helt beroende av mRNAt. På så vis går en stark länk mellan arvsmassa, mRNA och proteiner. Istället för att fortsätta att söka efter protein från Giardia som på något sätt kunde vara viktiga för hur en infektion kan gå till, hur en infektion kan undkomma immunförsvaret och hur sjukdomen giardiasis uppkommer, gjorde vi en mRNA-studie. Arvsmas- san i en cell är konstant, men mRNA och proteiner förändras beroende på vad som händer i och runtomkring en cell. När Giardia celler flyttas till cell- odlingsskålar med tarmceller från människa skapas direkt nya mRNA för att parasiten optimalt ska kunna bibehålla en infektion. Precis som i Paper I, när vi studerade mRNA hos tarmcellerna, tog vi den här gången fram alla mRNA-kartor ur Giardia parasiterna. Vi såg en betydligt mindre aktivitet hos parasiten än vad vi hade sett hos människans tarmceller. Då parasiten enbart är skapt för att leva tillsammans med värdceller från tarm är det inte så märkligt att Giardia inte reagerar lika kraftigt som tarmcellerna, vilka blev utsatta för en onormal situation i och med infektionen. Trots den låga förändringen hos Giardia vid påträffandet av tarmceller kunde vi identifiera några unika enstaka och några grupper av sinsemellan liknande mRNA, som alla var direkt kopplade till infektionen. Exakt hur dessa mRNAs motsvaran- de proteiner fungerar på molekylär nivå vid infektion har vi ännu inte hunnit studera, men borgar för många intressanta forskningsår framöver.

9 För att sammanfatta; mina studier, i samarbete med utmärkta kollegor, har lett till en ökad förståelse för hur människans tarm påverkas av en Giardia infektion och vad parasiten gör för att dels kunna åstadkomma en infektion, dels för att skapa sjukdom. Mycket arbete återstår för att vi fullkomligt ska förstå vad som sker i männsikan efter upptag av sjukdomsframkallande Gi- ardia cystor, men utan den här avhandlingen hade forskningen haft en betyd- ligt längre väg att gå.

10 Introduction to Giardia

The first microbiological description of Giardia lamblia, also known as Giardia duodenalis and Giardia intestinalis appeared on the 4th of Novem- ber 1681 (Dobell, 1920). On this date, Antony van Leeuwenhoek wrote a letter to the secretary of the Royal Society in London describing how he took parts of his own diarrheal stool to look for potential ill-doers. Van Leeuwen- hoek was a skilled microscopist and described from his stool the presence of an animacule with paws. This was the first morphological description of the vegetative stage of the parasite Giardia (Dobell, 1920). The name Giardia was given two hundred years later by the researcher Kunstler in 1882-1883. The species name intestinalis was kept from the species description by Lambl in 1859 of Giardia as Cercomonas intestinalis while the species name lamblia was given as an honor to Lambl in 1888 by Blanchard (Adam, 2001). Additional names, such as duodenalis and the various sub-species names for Giardia infecting animals other than humans, have been given throughout the 20th century (Adam, 2001).

In recent times, the nomenclature of Giardia and its sub-species has been under debate. Monis, Caccio and Thompson proposed a revision of the Giardia nomenclature and urged the research community to use the name G. intestinalis when referring to one sub-species that infects humans (assem- blage A), and G. enterica for the other sub-species (Assemblage B) (Monis, et al., 2009). Since this thesis is focused on the host-parasite interplay of human infections in total, mainly working with assemblage A but not ex- cluding assemblage B, I have chosen to use Giardia as a combined species name for these two assemblages. Wherever a fact is true for only one of these species/ sub-species, the individual name is used. The Giardia clade also includes several sub-species with a non-human host range. These sub- species will only be mentioned briefly in the section on “Host range and zoonosis” and are in general excluded when I write about Giardia.

Giardia is an extracellular parasite of the upper intestinal tract of mam- mals. It has a peculiar morphology including two nuclei, a ventral suction cup, eight flagella, and very unusually for a eukaryote it lacks organelles like mitochondria and golgi cistarnae (Adam, 2001, Friend, 1966, Samuelson, et al., 2005). On the phylogenetic tree, the branch of excavata (including dip- lomonads such as Giardia) originates close to the point of divergence of

11 bacteria and eukaryotes (Dacks, et al., 2008). The apparent ancient origin of this lineage of dinuclear eukaryotes has been debated for several years (Lloyd and Harris, 2002). The discovery of the mitosome, a reduced mito- chondrion-like organelle, discredited the hypothesis that Giardia belonged to an ancestral lineage of eukaryotes that had never acquired the organelles typical of higher eukaryotes. This discovery of the mitosome has led to the alternative hypothesis that Giardia has lost its fully developed mitochondria during the course of evolution as a consequence of its parasitic life-style (Knight, 2004). Whether Giardia should be considered to be an example of an ancient and simple eukaryote or an example of a complex eukaryote that has subsequently undergone reductive evolution is still under debate. How- ever, the simplification and reduction of standard eukaryotic systems in Giardia enhances its relevance for studies as a model system for eukaryotic macromolecular machines.

The Giardia Cell The Giardia cell has a two-stage life cycle (figure 1) with additional short- lived intermediate forms present only during entry or exit of the host body. Inside a host’s upper small intestine lives the feeding and active cell form, the trophozoite. Outside the nutritional environment of the host, Giardia survives only as a resting, low activity cyst. The in-between stages, the ex- cyzoite and the encyzoite, are transient cell forms presenting features of both the cyst form and the trophozoite.

Excystation: cyst to trophozoite The Giardia cell occurs as a dormant cyst in water and on moist surfaces in the soil. The cyst form is protected by a rigid sugar rich cyst wall of two thirds -1,3-N-acetyl-D-Galactoseamine polymers and one third cysteine rich proteins (Gerwig, et al., 2002). The inner cyst cell is kept within a double membrane and consists of a dissembled viable parasite cell with four tetraploid nuclei (Bernander, et al., 2001). Once ingested, the cyst travels through the digestive system of the human or animal host and opens up by a process called excystation. This process was accomplished in vitro in 1979, by mimicking the passage through the stomach and entrance into the upper intestinal tract of a human (or more frequently in the early days of excysta- tion in the lab; the rat) (Bingham and Meyer, 1979, Boucher and Gillin, 1990, Rice and Schaefer, 1981, Schaefer, et al., 1984).

12 Figure 1. The life cycle of Giardia. The parasite is present within the host upper intestinal tract as trophozoites attaching to the epithelium lining of the lumen. During encystation and excystation transient intermediate cell forms are trans- forming into or exiting the dormant but infective cyst stage.

Upon passing through the acidic stomach of its host, the cell within the cyst wall can sense the low pH and initiate the assembly of the metabolic active form of the parasite, the trophozoite (Hautus, et al., 1988, Hetsko, et al., 1998). Upon exiting the stomach the rigidity of the cyst wall is lost due to the swift rise in pH and the influx of bile from the gall bladder, in concert with Giardia dephosphorylation of the cyst wall proteins (Reiner, et al., 2003, Slavin, et al., 2002). The content of peripheral vesicles (PVs), includ- ing a cathepsin B-like cysteine protease and an acid phosphatase, is released from the excyzoite into the space between the cell membrane and the protec- tive cyst wall. The released proteins are suggested to cause the de- phosphorylation and breakage of the cyst wall rigidity (Feely, et al., 1991, Slavin, et al., 2002, Ward, et al., 1997).

Entering the infectious state The trophozoite emerges from its degraded protective cyst wall shield with rapid movements of its body and the eight flagella (Hegner, 1927). In vitro, the whole procedure of excystation can be performed in as little as 15 mi- nutes (Svard, et al., 2003). Immediately after the first trophozoite has

13 emerged from its cyst shape, it begins to divide and simultaneously begins assembly of an adhesive disk in a spiral-like manner on the flattened ventral side of the cell body (Coggins and Schaefer, 1986, Palm, et al., 2005). The two newly formed trophozoite cells have duplicated genome content com- pared to regular trophozoites and can thus continue to grow and undergo a second round of division without the need to replicate their genomes (Ber- nander, et al., 2001). After completion of the second division, the one cyst- emerging trophozoite has given rise to four parasitic trophozoites living within the host’s upper small intestinal tract. By coordinating cell division with assembly of the attachment organelle (the adhesive disc) the daughter trophozoites are immediately able to attach to host surfaces after division.

The trophozoite – the living cell The Giardia trophozoite has a half-pear shaped body, 10 to 15 μm in length and about 5 μm in width, with a bilateral symmetry to the intracellular or- ganization (Adam, 2001). In scanning electron micrographs of the ventral and dorsal sides of a trophozoite, the body shape, the flagella and the adhe- sive disk are seen (figure 2) (Feely, et al., 1982). Prominent features of the trophozoite cell are schematically depicted in Figure 2.

Characteristic features of the trophozoite cell The adhesive disk is a structure specific for the Giardia species (Elmendorf, et al., 2003). It has a peculiar spiral cytoskeleton organization forming a circular flexible device used face down during attachment of the parasite (Feely, et al., 1982). In transverse sections, the spiral formation of the disk is noticeably stretched inwards the cell by parallel microtubule sheets or rib- bons (Holberton and Ward, 1981).The center of the spiral is called the bare zone, since it is empty of cytoskeletal proteins (Holberton and Ward, 1981). Occasionally a protrusion is seen in the bare zone, containing both glycogen particles and cytoskeletal proteins, but neither function nor frequency of occurrence of this protrusion is known (Lanfredi-Rangel, et al., 1999).

The two nuclei of trophozoites are ovoid and not identical in their forma- tion (Benchimol, 2005, de Souza, et al., 2004). The double nuclear mem- brane is, as usual for eukaryotes, perforated with nuclear pore complexes for cross-membrane transport (Benchimol, 2004). The cytoskeletal actin fila- ments seem important for the spatial rigidity of the nuclei positions and pos- sibly also for their segregation upon cell division (Benchimol, 2005). Within the nuclei the genomic DNA is folded around typical eukaryotic histones, albeit possibly lacking one out of the five subunits since a homologue to the Histone-1 linker is not found in the Giardia genome (Yee, et al., 2007).

14 Figure 2. The trophozoite. On the left, scanning electron micrographs of a Giar- dia lamblia trophozoite from the dorsal view (top picture) and the ventral side (bottom picture) (Feely, et al., 1982). The Giardia trophozoite contain two nuclei, an adhesive disk, basal bodies connecting to the flagella axonemes, a median body and several membrane-proximate peripheral vesicles. The flagella are organized as four pairs; Af, anterior, Plf, posterolateral, Vf, ventral, and Cf, caudal.

A prominent feature of Giardia, when seen through a microscope, is the presence of four pairs of flagella. The flagella are promoting movement of the free-swimming trophozoites. The flagella are constructed like the canonical motile flagella of eukaryotes with nine circular and two inner microtubule bundles with a covering of plasma membrane (Elmendorf, et al., 2003). The four flagella pairs are organized as anterior, ventral, posterior-lateral (postero- lateral) and caudal, with long intracellular parts called axonemes or dense rods. Transverse, and connecting to the caudal and posterior-lateral axonemes, stretches an uncharacterized microtubule sheet structure; the funis (Benchimol, et al., 2004). The ventral flagella have been implicated in attachment of the parasite to host surfaces, and are thought to be involved in acquisition of nutri- tion when the parasite is attached. Many proteins have been localized within or in between the four pairs of flagella. The cytoskeleton protein 19-giardin, for instance, is exclusively located at the extruding parts of the ventral flagella pair, suggesting that specific molecular functions may be associated with the different flagella of the Giardia cell (Saric, et al., 2009).

Eight basal bodies, the originating structures of the flagellar axonemes, are all located proximate to the two nuclei. The basal bodies have been impli- cated as the cellular signaling centers during trophozoite axenic growth, cell division and differentiation regulation (Abel, et al., 2001, Gibson, et al., 2006, Lauwaet, et al., 2007). The median body is located below the basal bodies. The median body is a cytoskeleton organelle exclusive to Giardia (de Souza,

15 et al., 2004) that seems to be cell-cycle regulated (Dawson, et al., 2007) (per- sonal communication K. Troell) but is as yet of unknown function. It consists of several sheets and stacks of microtubuli and has been proposed to serve as a microtubule storage reserve of the cell (Piva and Benchimol, 2004).

Canonical eukaryotic golgi are not found in Giardia. Instead, Giardia trophozoites have an intricate endoplasmatic reticulum (ER) and peripheral vesicles that function in the protein transport throughout the cell. The peri- pheral vesicles also transport proteins in the endocytotic direction, internaliz- ing extracellular and membrane bound proteins and macromolecules. The ER is most dense below the nuclei, with extensions of rough ribosome- bound ER protruding throughout the cell.

Encystation: trophozoite to cyst The trophozoites within the human intestinal tract are constantly shed as cysts with the feces of the host. Only very watery diarrhea will also hold trophozoite cells. Usually the passage through the lower parts of the small intestine will trigger and give the time necessary for trophozoites to enter the cyst stage. Replication occurs twice during encystation of a trophozoite in the G1 cell cycle stage. Simultaneously, the transcription and translation of cyst-wall proteins and polysaccharide pathway proteins are activated to ena- ble the assembly of the cyst wall. During the encystation processes the cell disassembles and compartmentalizes structural organelles and reforms to the shape of an oval cyst (Bernander, et al., 2001, Lauwaet, et al., 2007, Palm, et al., 2005, Sener, et al., 2004). The most optimal time-point for encystation induction in vitro is just prior to initiation of mitosis, in the late G2-phase of the cell cycle, when the cell material and genomic content is already dupli- cated in preparation for cell division (Reiner, et al., 2008).

Large changes in protein expression and protein modification occur upon encystation, altering cytoskeleton proteins as well as inducing stress- response proteins and metabolic enzymes expression (Kim, et al., 2009). When the cyst wall has formed, transcription and the metabolic activity within the cyst decreases greatly compared to the levels in the active tropho- zoite, whereas many protein levels remain relatively constant compared to the trophozoite (Palm, et al., 2005). The cyst can survive for months outside the host, as long as it is in water or on a moist surface (Hetsko, et al., 1998).

16 The cell biology of Giardia

The Giardia trophozoite contains two diploid nuclei, while the cyst contains four quadraploid nuclei (Bernander, et al., 2001). Each haploid genome of the most studied Giardia strain (WB-C6) is divided between five chromosomes with a total genome content of 11,7 Megabases (Morrison, et al., 2007). The ends of the linear chromosomes have telomeric repeats of (TAGGG)n and eight out of ten chromosome ends have characterized sub-telomeric repeats of pseudogenes with transcription in both directions (Prabhu, et al., 2007). Ge- nomes from both human-infecting Giardia assemblages, A and B, show ex- tremely reduced components of several eukaryotic machineries (Franzen, et al., 2009, Morrison, et al., 2007). Many genes are also more homologous, or only homologous, to archaeal or prokaryotic genes and systems. There is no doubt that Giardia is a true eukaryote albeit with the presence of non-typical eukaryotic features. The reduced cellular machineries have been proposed as model systems for understanding various cell functions of other, more com- plex, eukaryotic systems (Morrison, et al., 2007).

Open reading frames and transcription Most genes are interspersed with very short intergenic regions, with an aver- age of 130-370 bp between two adjacent open reading frames (ORFs) (Fran- zen, et al., 2009, Morrison, et al., 2007). Each gene is flanked by a very short (or no) upstream untranslated 5’-region (UTR) and a short 3’ UTR with a polyadenylation signal site usually positioned less than 15 bases from the stop codon. Unlike other eukaryotic translation machineries, translation in Giardia is most efficient with 5’ UTRs of less than 10 nucleotides in length (Li and Wang, 2004).

The genome is packed with predicted genes on both the sense and anti- sense strands. All three RNA polymerases found in eukaryotes have homo- logous enzymes in the Giardia genome, albeit not all the eukaryotic or arc- haeal subunits are present (Best, et al., 2004). The number of recognized transcription factor genes in the Giardia genome is lower than expected, based on archaeal and eukaryotic evolutionary studies (Best, et al., 2004). Giardia has a low stringency for transcription initiation and this tends to lead to bi-directional transcription not only for genes arranged head-to-head, but also producing vast amounts of sterile (predicted non-translated) transcripts from ORF initiation sites (Teodorovic, et al., 2007). Early characterizations

17 of the polyadenylated transcriptome of Giardia revealed an unusual abun- dance of putative sterile transcripts, up to 20% of total transcripts, highlight- ing the possibility of an atypically eukaryotic reduced or relaxed transcrip- tion control machinery (Elmendorf, et al., 2001).

Promoter sequences The promoter regions of Giardia genes are unusually small for eukaryotes, and the regulation of transcription initiation is unusually relaxed. An initia- tor-like AT-rich element located within 20 to 100 bases upstream of the start codon seems to be the only transcription activator needed, even if this dege- nerate stretch including 3 or more consecutive A or Ts is often accompanied by other regulatory elements (Elmendorf, et al., 2001, Sun and Tai, 1999). The promoter sequences of cytoskeleton protein genes were used to identify three sets of Giardia transcription related motifs; the AT-rich initiation re- gion, CAAG-like elements (later denoted gCAB) and a -30 box of 18 nuc- leotides, possibly unique to cytoskeleton genes (Holberton and Marshall, 1995, Kirk-Mason, et al., 1989).

The first putative Giardia promoter sequences were identified 56 nucleo- tides upstream of the encystation-induced cyst wall disaccharide-induced glucoseamine-6-phosphate isomerase (G6PI) gene (Knodler, et al., 1999). The specific sequence or promoter boxes were not identified at that time. Later, the promoter element of C(T/A)ACAG, directing the Giardia Myb2- protein containing two transcription activating myb-domains, was described for the G6PI and was also confirmed upstream in other encystation-related genes (Sun, et al., 2002).

The 3’ of transcripts The polyadenylation site of Giardia transcripts is slightly heterogenic and is spread over five nucleotides located down-stream of the AGUPuAA polya- denylation signal (Peattie, et al., 1989, Que, et al., 1996). Differentiation- induced alternative polyadenylation use was reported in Giardia but the function or implications for the different transcripts formed have not been studied (Que, et al., 1996). For transcripts of the variable surface protein family an extended polyadenylation signal of up to 15 bases is well con- served, and is not found among other Giardia transcripts, suggesting a link between VSP gene family regulation and polyadenylation (Svard, et al., 1998). The polyadenylating protein complex of Giardia is small compared to other eukaryotes, and its components have been cloned and purified as re- combinant proteins, but their functions in transcript regulation remain to be evaluated (personal unpublished data).

18 Translation Giardia genes show a transcription related codon usage bias, where highly expressed genes have C or G residues in the third position of the codon (La- fay and Sharp, 1999). Generally, coding regions are more GC-rich than the intergenic regions, but the overall GC content is about 46%. The extremely short 5’-UTRs of Giardia leave little to no sequence for ribosome or transla- tion initiation factors to bind. The 7-methylguanosine (m7G) cap of mRNA is bound by the initiation factor 4E (eukaryotic IF4E) that subsequently inte- racts with other initiation factors and ribosomal subunits to initiate transla- tion (Li and Wang, 2005). The Giardia ribosome does not scan the bound mRNA and always starts initiation of translation at the first AUG (Li and Wang, 2004).Translation can also be initiated without cap-binding, as with the Giardia virus transcripts that instead use complementary base pair bind- ing of a downstream box (located 65 nucleotides into the open reading frame) to the 16S ribosome RNA for initiation (Yu, et al., 1998).

Antisense regulation of gene transcripts RNA interference (RNAi) is a eukaryotic mechanism of post-transcriptional control of genes by mRNA binding of short antisense RNA oligonucleotides originating from double stranded precursors (small interfering RNA; siRNA and micro RNA; miRNA) (Fire, et al., 1998, Ullu, et al., 2004). Macromole- cular machineries within the RNAi pathway blocks or degrades the anti- sense-targeted mRNA, and the importance of RNAi has been described for eukaryote cell differentiation, cell control and stress response regulation (Ullu, et al., 2004). A homologue of Dicer, the precursor cleaving enzyme of RNAi, has been structurally and functionally described in Giardia (Macrae, et al., 2006). In vivo, knock-downs of gDicer and a homologue of RNA- dependent RNA polymerase (gRdRP) transcripts, by introduced constitutive expression of the antisense strand from either gene, did not render any ap- parent reduction in Giardia trophozoite function of growth, nor had any effect on in vitro differentiation into cysts (Prucca, et al., 2008). The endo- nuclease homologue gArgonaute, the cleaving enzyme of the RNAi pathway that destructs the target mRNA in other eukaryotes, is on the other hand essential for parasite growth and could be knocked down (Prucca, et al., 2008). The only function coupled to the RNAi machinery in Giardia so far identified is the selection of one transcript for translation at a time from ~ 200 genes encoding the variable surface proteins (VSPs), required for the immune-evasive surface coat switching of the parasite (Prucca, et al., 2008).

Protein modification and transport Giardia lacks the morphological golgi apparatus that other eukaryotic cells use for post-ER protein modification and cellular localization determination (Elias, et al., 2008). The lack of typical golgi cisternae however does not

19 exclude protein modifications and protein transport from the cells repertoire of functional mechanisms. Several proteins involved in protein/cargo vesicle transportation in other eukaryotes have homologues in the Giardia genome, for example clathrin, adaptor protein (AP) subunits, SNAREs (soluble N- ethylmalemide-sensitive factor attachment protein receptors), coat proteins (COP) I and II and Rab GTPases (Elias, et al., 2008, Marti, et al., 2003) and the corresponding proteins are localized in the nuclear membrane, in differ- ent portions of the ER membrane and in membrane-bound vesicles, but there is no evidence of any golgi-like apparatus (Elias, et al., 2008, Langford, et al., 2002, Marti, et al., 2003).

In non-encysting trophozoites, protein trafficking within and to the out- side of the cell is conveyed by peripheral vesicles (PVs), extruding from the ER (Abodeely, et al., 2009, Hehl and Marti, 2004). The PVs are also used in endocytosis. PV-mediated transport of proteins to the cell membrane or for extracellular release involves discrimination by the protein C-terminus in a tyrosine-motif based manner by an identified adaptor protein (AP1) of the PV membrane (Touz, et al., 2004). The PVs are generally only seen close to the plasma membrane indicating a long intracellular ER-transport route be- fore release of formed PVs (Abodeely, et al., 2009).

20 Parasitic metabolism – dependency on the host

Giardia is unable to produce many essential parts needed for fundamental cell biological processes. The Giardia cell is dependent on its host, i.e. is parasitoid, for general provision of nutrients and cell components. Uptake of essential molecules is conveyed by diffusion, passive and active membrane pump transportation and endocytosis into peripheral vesicles (PVs). The Giardia cell lack mitochondria and the mitosomes do not convey energy production. ADP conversion to produce ATP is strictly confined to the cy- toplasm of Giardia.

The parasite lacks the machinery for de novo synthesis of nucleotides, and both ribonucleotides and deoxynucleotides are salvaged through uptake of nucleosides or nucleobases from the host environment or through internal re- use (Jimenez and O'Sullivan, 1994). The parasite is also dependent on extra- cellular cholesterol for the biogenesis of cell membranes and are not able to synthesize cholesterol themselves (Jarroll, et al., 1981, Lujan, et al., 1998). Giardia has only limited ability to synthesize lipids in general de novo and relies on active uptake and modification of lipids from the intestinal lumen (Sonda, et al., 2008). The uptake of sterols, phospholipids and long-chain fatty acids is readily enhanced by the lipid-aggregating bile salts present in the upper small intestinal tract.

Mitosomes The mitosomes of Giardia, allegedly a reduced form of the eukaryotic mito- chondria, were firstly described in 2003 (Tovar, et al., 2003). The mitochon- drial cysteine desulphurase IscS, heat shock protein 70 (Hsp70) and chape- ronin 60 (Cpn60) had been cloned from the Giardia genome (Roger, et al., 1998, Tachezy, et al., 2001) and was characterized together with the interact- ing iron-binding protein IscU (Tovar, et al., 2003). The extreme mitochon- drion-reduction of the mitosomes does not allow respiration and is confined to the energy conversion by iron-sulfur (Fe-S) cluster proteins, proteins syn- thesized by the IscS and IscU (Tovar, et al., 2003). Similar Fe-S mitosomes have only been identified in the clades of Entamoeba, Cryptosporidium and Trachipleistophora to date. In other protists another double membrane orga- nelle with reduced mitochondria-like features has been identified; the hydro- genosome, with the name implicating the production of hydrogen as well as energy (Gray, 2005). The mitosomes of Giardia contain no DNA and it re-

21 lies on the genome for all required products. Within Giardia about 20 to 50 mitosomes are spread throughout the cytosol with some clustered in a band pattern between the two nuclei (Dagley, et al., 2009).

Energy convergence; arginine over glucose Giardia possesses the typically prokaryotic pathway of energy retrieval from arginine metabolism (the arginine dehydrolase pathway). In addition to this main energy pathway, the parasite also utilizes pyruvate rendered from gly- colysis to acquire energy. Depending on the oxygen pressure, the pyruvate will be metabolized to alanine (anaerobic), ethanol (microaerobic) or acetate and carbon dioxide (aerobic).In the intestinal tract, the aerobic glycolytic pathway with enzymes pyruvate:ferredoxin oxidoreductase (PFOR) and acetyl-CoA synthase in combination with the anaerobic pathway through enzymatic activity of glutamate dehydrogenase and alanine aminotransferase will be used for pyruvate metabolism (Nino and Wasserman, 2003). In the cyst form, the anaerobic pathway is dominating the pyruvate metabolism part of the extensively decreased metabolism of the Giardia cell. Glucose is about 4-fold less efficient in ATP yield than the arginine dehydrolase path- way (Schofield, et al., 1992). Noteworthy is that for in vitro culture of para- sites, glucose can be omitted as an energy source without causing growth arrest (Schofield, et al., 1990).

L-arginine is externally retrieved through an arginine-ornithine antiporter (Knodler, et al., 1995). Each arginine molecule metabolized renders one ATP through the arginine dehydrolase pathway with the conversion of argi- nine to citrulline to ornithine and carbamoyl phosphate to ammonia and car- bon dioxide by the enzymes Arginine Deiminase (ADI), Orntihine Carba- moyl Transferase (OCT) and Carbamate Kinase (CK) (Schofield, et al., 1992). The OCT enzyme generating carbamoyl phosphate is the rate limiting activity of the arginine dehydrolase pathway, and in addition Giardia is not able to acquire citrulline from the external milieu for processing (Schofield, et al., 1992). The CK is one of the most potent enzymes of Giardia, and conveys the substrate-level phosphorylation of ADP to ATP (Schofield, et al., 1992). ADI possesses the ability to also convert peptide bound arginine to citrulline, a function not linked to energy production (Touz, et al., 2008). In Paper II of this thesis, ADI and OCT were identified as released proteins and potential virulence factors and are further discussed in the chapter of “Parasite mechanisms for host survival”.

Oxygen stress Giardia shows a poor tolerance to oxygen and is categorized as a micro- aerophilic parasite, with low oxygen to anaerobic metabolism. The metabolic pathways of Giardia are oxygen-load sensitive and the parasite is functional- ly dependent on a low oxygen environment or internal oxygen reduction

22 systems. Under aerobic conditions, cellular DT-diaphorase (an oxidoreduc- tase) produces harmful hydrogen peroxide, a reactive oxygen species (ROS) (Li and Wang, 2006). While other organisms contain pathways for detoxify- ing internal ROS, Giardia lacks the conventional ROS scavenging systems of superoxide dismutase, catalase and peroxidases (Brown, et al., 1995). The major ROS scavenging mechanism of Giardia is conveyed by a cytoplasmic thioredoxin-like proteins and membrane bound NAD(P)H oxidase, both reducing oxygen through the thiol-group (sulfur-hydrogen) of imported cysteine (Brown, et al., 1998, Brown, et al., 1995). Giardia growth is obliga- tory dependent on external cysteine even if other reducing agents, such as ascorbic acid, also facilitates growth in vitro (Brown, et al., 1998) . In mi- croaerobic conditions pyruvate metabolism has some oxygen reducing capa- bilities by NADH oxidase, partly protecting the parasite from the accumula- tion of reactive oxygen species (ROS) Under oxygen rich conditions (studied in vitro) the Giardia cell membrane loses its electrochemical potential within a few hours and the parasite undergoes subsequent cell death due to the ac- cumulation of ROS and the related release of toxic hydroxyl radicals (Brown, et al., 1998, Lloyd, et al., 2000).

In parallel to oxidative stress from ROS, the intracellular presence of ni- tric oxide causes nitrosative stress by diminishing the available hydrogen and reductive donors leading to enhanced sensitivity of Giardia to oxygen (Lloyd, et al., 2003). Nitric oxide is a potent Giardia-cidal molecule readily activated in host epithelium during infections (Eckmann, et al., 2000) and is further described and discussed in the section of “Virulence by Arginine Deiminase” on page 48.

23 Introduction to Giardia infections and the human host

Giardia infections are established by the excystation of ingested cysts. Emerging trophozoites immediately attach to the host epithelium in the up- per one third of the small intestine (Campbell and Faubert, 1994). Over the following days colonization spreads further down in the small intestine and into the colon, with simultaneous initiation of encystation and shedding of Giardia cysts (Campbell and Faubert, 1994).

The luminal flow and the continuous replacement of endothelial cells re- quire Giardia cells to detach, swim and re-attach in order to efficiently co- lonize the intestine (Campanati, et al., 2002). The parasite utilizes both the flagella and the caudal part of the cell body for movement and they are able to swim against the current of the intestinal flow to keep within the preferred growth niche in the upper parts of the small intestine (Carvalho and Montei- ro-Leal, 2004, Ghosh, et al., 2001).

Giardiasis Giardia was included in the World Health Organization (WHO) initiative for neglected disease in 2004, due to its link to poverty by the endemic state of undeveloped countries and the lack of knowledge of molecular mechanisms for the disease (Savioli, et al., 2006). The disease of Giardia, when giving rise to symptoms, is called giardiasis. Seemingly independent of the amount of trophozoites in the intestine, a Giardia infection can give severe to no symptoms. The infectious dose for Giardia was reported to be as low as 10 cysts in experimental humans by Rendtorff and Holt in 1954 (Rose, et al., 1991). Every cyst will upon excystation in the upper intestinal tract divide into four trophozoites. The trophozoite load will then increase steadily be- fore onset of any disease symptoms (Muller and von Allmen, 2005).

The clearance of infection is usually mentioned as spontaneous in the cases where medication is not used. Genetic differences between hosts are thought to be important factors for whether infections are established and whether the established infections are cleared, but only a few marker genes have been de-

24 Figure 3. Overview of a Giardia infection. Giardia cysts infect a host and trans- form through the excyzoite to the active trophozoite in the upper intestinal tract. The initial colonization phase includes overcoming the host’s natural barriers and innate immune response. After established infection the parasite is under addition- al combat with the host adaptive immune response. The trophozoite load is rapidly increasing early in infection and is naturally cleared after some weeks (as depicted in the figure) or persists for longer or chronic infections due to host and parasite factors. Figure from (Roxstrom-Lindquist, et al., 2006)

scribed, and these studies have not been repeated. A study from 1980 indicated a slight difference in representations of HLA-classes among Giardia infected individuals contra the overall representation within the population (Roberts- Thomson, et al., 1980), and some association between Giardia prevalence and blood group A was presented in 1977 (Barnes and Kay, 1977). The host’s clinical status in concert with nutritional state and age are most certainly fac- tors implicated in disease outcome (Faubert, 2000), and the intestinal com- mensal microflora or co-infections can prevent attachment and survivability of trophozoites (McFarland, 2000, Singer and Nash, 2000).

Symptoms Giardiasis symptoms vary in severity on an individual basis as does the dura- tion of the Giardia infection. The wide range of disease outcome from an established infection include asymptomatic carry to severe (persistent) and chronic diarrhea. Symptoms caused by Giardia, aside from diarrhea, are abdominal pain, nausea, flatulence, vomiting, anorexia, weight loss, dehy- dration and general exhaustion (Farthing, 1996, Gascon, 2006, Panaro, et al., 2007). Persistent diarrhea is defined as the “continuous passage of loose stool for more than two weeks”. If the diarrheal state is continuous for more than four weeks the term chronic diarrhea is used (Pawlowski, et al., 2009).

25 In addition to direct infection-duration effects, secondary long term symp- toms have been correlated to Giardia infections (Morch, et al., 2009). Fol- low-up studies 2 years after an outbreak in the Norwegian town of Bergen, noted increased abdominal problems along with diagnosed functional ga- strointestinal disorders (FGID) and irritable bowel syndrome (IBS) in the treated, but once infected, individuals (Hanevik, et al., 2009, Morch, et al., 2009). Studies on infected children of endemic countries have emphasized the problems from early child-hood diarrhea. Generally, children with recur- ring infections, or anti-Giardia antibody titers indicating previous infections, show a poorer weight-for-height or -age ratio than other children. A reduced performance in developmental evaluations, or documented problems with learning in the first school years, are also correlated to reoccurring childhood diarrhea (Berkman, et al., 2002, Goto, et al., 2008, Guerrant, et al., 2002).

Prevalence The infectious rate of Giardia, based on diagnosis of symptomatic or sus- pected infected individuals is about 0.015% in the western industrialized world (Public Health Agency of Canada 2007, Yoder, 2007). Approximately 1500 Swedish citizens are diagnosed annually (Smittskyddsinstitutet, 2009). Assuming the same infection rate worldwide the amount of Giardia infec- tions in people would be almost 700,000 annually. This is probably an un- der-estimate since infection rates in countries with endemic giardiasis are far greater than in western countries with high quality clean water, good hygiene and temperate climate. In 1996, the world health organization (WHO) esti- mated that there were 250 million people infected with Giardia worldwide (Palm, et al., 2003, Upcroft and Upcroft, 1998). Humans do not readily ac- quire immunity to Giardia infections which is suggested by the high re- infection rates observed in treated children in the suburbs of Lima, Peru where Giardia is endemic (Gilman, et al., 1988).

Diagnostics Generally Giardia infections are diagnosed by finding cysts within stool sample(s) from individuals with suspected infection. Diagnosis in Sweden is mainly made by visual cyst detection using light microscopy to analyze sin- gle or repeated fecal sample(s) fixated in sodium acetate, acetic acid and formalin (SAF) (2006). When required, immunolocalization of cysts in SAF- samples is also used in Swedish diagnosis. Cyst wall protein 1 is the usual target protein for diagnostic monoclonal antibodies to Giardia that are used both in immunolocalization of cysts and in commercial antigen detection ELISA tests (June 2005, Ratner, et al., 2008). Polymerase Chain Reaction (PCR) analysis or quantitative real time PCR (qRT-PCR) of Giardia specific genes are more sensitive, specific and fast methods for parasite detection than microscopy detection, but are mainly used in research laboratories and not in routine clinical diagnostics (Bruijnesteijn van Coppenraet, et al., 2009,

26 Pawlowski, et al., 2009). Sub-speciation is not a standard routine of diagnos- tics of today, since no differences in disease outcome or treatability have been reported for the different Giardia assemblages or sub-assemblages.

Treatment Giardia infections, both symptomatic and asymptomatic, are treated with antibiotics, usually nitroimidazoles (metronidazole or tinidazole) or benzi- midazoles (albendazole) (Ali and Nozaki, 2007, Savioli, et al., 2006). In addition, thiazolides (nitazoxanide) are prescribed in the USA to patients with prolonged Giardia associated diarrhea or metronidazole-resistant infec- tions. The benzimidazoles inhibit the formation of tubulin bundles while the nitroimidazoles and thiazoles are activated by compound reduction within the parasite (Rossignol, 2009).

Nitroimidazoles are activated by obtaining an electron from proteins of the anaerobic pyruvate:ferredoxin oxidoreductase (PFOR) pathway and pos- sibly other nitroreductases of the cytoplasm of Giardia (Ali and Nozaki, 2007, Dan, et al., 2000). The activated form of nitroimidazoles binds tran- siently to DNA and induces strand breaks, leading to death of the parasite (Rossignol, 2009). While nitroimidazoles are taken up by passive diffusion in the intestine as inactive compounds, the thiazolide nitazoxanide is rapidly deacetylated and later metabolized to tizoaxinide glucoronide (TIG) in the liver. The TIG-form of thiazolide is excreted via bile to the upper small in- testine and is activated within Giardia as an electron acceptor (Muller, et al., 2007). PFOR seems to be the activator of metronidazole, while the activator for TIG is likely to be another nitro-reducing enzyme since metronidazole- resistant clones can be killed by nitazoxanide (Ali and Nozaki, 2007).

Infection site The human small intestine is composed of three parts, the short duodenum connecting to the stomach, the jejunum , and the ileum connecting to the large intestine with the ileocecal sphincter (Marieb and Hoehn, 2007). The small intestine is extensively folded, and the inside layer is bulged in circu- lar protrusions of 1 mm long villi lined with epithelial cells, where each sur- face epithelial cell is covered in microvilli spikes. The folds, protrusions and spikes together provide an extensive surface area enabling the adsorptive requisite for water and nutrition uptake. The duodenum, the twelve inch long upper part of the small intestinal tract is the location for release of bile salts and pancreatic digestive enzymes from the proximate gall bladder and the pancreas (Marieb and Hoehn, 2007).

27 The main function of the small intestine is to mechanically and enzymati- cally break down food and to absorb nutrients and water (Marieb and Hoehn, 2007). The Giardia cell is not just able to survive in the degrading environ- ment, but thrives in the presence of host accumulated nutrients and intestine- specific molecules of the human duodenum to ileum. A mucus layer forms a thick coat of proteins, lipids and membrane-bound and soluble glycoproteins (mucins) that together protect the epithelial cells, as well as attaching tro- phozoites from enzymatic damage (Faubert, 2000, N'Dow, et al., 2004). The mucus also protects the intestine from invasive pathogens by hiding surface glycolipid and glycoprotein receptors on the epithelial cells (Navaneethan and Giannella, 2008). Mucus is secreted from goblet cells dispersed among the epithelial cells in the inner lining of the small intestine (N'Dow, et al., 2004). The number of goblet cells increases with distance from the stomach, being more abundant in the ileum compared to the duodenum (Karam, 1999).

Epithelial cell to cell connections The epithelial lining of the small intestine provides a flexible semi-permeable barrier between the intestinal lumen and the rest of the body. Proliferating epithelial cells of the crypts migrate upwards the villi surface while differen- tiating, and are shed from the villus tips at a constant rate resulting in a total replacement of the outer surface cells every three to six days (Marieb and Hoehn, 2007). The cells are strictly polarized at the tip of the villi, with a baso- lateral side facing the inside of the villus and the apical side covered with mi- crovilli facing the lumen. The differentiated villus cells are linked together and structurally ordered in relation to each other by tight, adherens and desmo- some junctions consisting of intra- end intercellular protein interactions.

The tight junctions are located between the apical upper half of adjacent epithelial cells and contains four identified classes of intracellular connecting proteins with at least one membrane spanning region; occludin, claudins, junctional adhesion molecules (JAMs) and the viral receptor CAR (Hossain and Hirata, 2008). These membrane proteins bind through zonula occludens (ZO) membrane adaptors to the actin cytoskeleton in the cytoplasm of the cells. The ZO proteins are, together with catenin and afadin of the adherens junctions, both signal transducers and structural proteins (Hossain and Hira- ta, 2008). Adherens junctions are less tightly formed and are shaped by nec- tin and e-cadherin extracellular connections with nectin to afadin and e- cadherin to catenin intercellular connections (Hossain and Hirata, 2008). Desmosomes are similar to and locate basally to the adherens junctions but connect outer proteins to the cytoskeleton intermediate filaments instead of the actin cytoskeleton (Koch and Nusrat, 2009).

28 Complex cellular signaling networks regulate the dynamic contraction and relaxation of the tight and adherens junctions during the uptake of nu- trients, immune cell activation and also cell death. Gap junctions are located on the more basal side of the polarized epithelium and convey cell to cell cross talk of ions and small molecules through protein channels or pores (called connexons) (Koch and Nusrat, 2009). When under proapoptotic sti- muli, outside-in stimuli cause intracellular cysteine protease cleavage of desmosomal proteins and stimulate apoptotic pathways (Koch and Nusrat, 2009). The different junction connections are essential for intestine function and individual cell survival, and are maintained also during the extrusion of cells during cell shedding from the villus tips (Watson, et al., 2009).

The environment of intestine lumen Every time the stomach empties into the duodenum, the pH is lowered by the extreme acidity of the influx and alkaline bile and pancreatic fluid is stimu- lated to be released. The partly digested acidic food and the bile and pan- creatic fluid are mixed and a weak alkaline pH is reached. The mixing is accounted for by intestinal motility, achieved by neural regulation of smooth muscle layers surrounding a thick layer of connective tissues, blood and lymphatic vessels and nerves adjacent to the inner epithelial lining (Marieb and Hoehn, 2007). A dozen constrictions per minute in the duodenum, to less frequent segmented movements further down in the small intestine, constantly twirl and push everything within the lumen of the intestine up and down, with a net flow downwards. Additionally, every release from the sto- mach stimulates peristaltic waves flowing through the intestine (Marieb and Hoehn, 2007). Occasionally entrapped food-particles also provoke local movement of the intestine for complete homogeneity and breakdown of the luminal content.

Host nutrient retrieval The Giardia cells attach to the epithelial lining of the small intestine. These cells, and the tight junctions holding them together, are responsible for the final step of degradation of food, and for the passage from the intestine lu- men to the afferent blood and lymphatic vessels for further nutritional cycl- ing through the liver. Starch and disaccharides are cleaved by pancreatic amylase and epithelial membrane-bound disaccharidases. The monosaccha- rides glucose and galactose are actively transported into and through the epithelial cytoplasm, while fructose diffuses through the tight junctions. Pancreatic enzymes and membrane-bound peptidases break polypeptides that are actively internalized into the epithelial cells. Both sugars and amino ac- ids are passed through the epithelial cells to the blood vessels. Bile salts disrupt large lipid aggregates into small lipid and salt formations, which in proximity to the intestinal lining gradually release the lipids to diffuse through the epithelial cell membrane. By cycling through the endoplasmatic

29 reticulum and golgi apparatus of the epithelial cells, the lipids are conjugated with proteins that are exocytosed on the basolateral side of the cells, where the conjugates are taken up for redistribution by lymphatic vessels. Water is absorbed by osmosis throughout the small intestine, while ions are actively transported.

Immune system of the upper intestinal tract Innate immunity The innate immunity is readily described as all parts of the host’s body that is always present and actively working against intruders (Wood, 2006). The epithelial lining of the intestinal tract possess a true barrier for many patho- gens by the impermeability of the tight junctions between the individual cells. Obviously, many pathogens are able to invade the epithelial cells, break the tight junctions or slide through the tight junctions by well adapted virulence mechanisms (Mims, et al., 2002). Other pathogens, like Giardia, do not need the entry into or through the epithelial cells for active parasitism and have found their niche in the lumen of the small intestine.

Several molecules and small proteins are constantly or immediately pro- duced upon infection in the upper small intestinal tract. In pockets between the villi, the crypts, Paneth cells differentiate from progenitor stem cells to constitutively secrete digestive enzymes and small antimicrobial peptides (defensins/cryptidins) (Karam, 1999). Digestive enzymes and molecules are also constantly provided to the intestinal lumen from the gall bladder and pancreas. With every constriction of the gall bladder, high amounts of the protein lactoferrin are released. Much lower concentrations of lactoferrin are cytotoxic to Giardia in vitro, implying protection of the parasite by means only present in vivo (Turchany, et al., 1995). Nitric oxide along with reactive oxygen species (NO and ROS) are produced by epithelial cells and are known to be harmful substances for in vitro growth of trophozoites. Many defensins of human, mice or rabbit origin have been proven to be cytotoxic for both trophozoites and cysts in vitro (Aley, et al., 1994), but the release of defensins in vivo have been shown to be entirely coupled to co-infections or presence of commensal bacteria, and not due to the infecting trophozoites themselves (El-Shewy and Eid, 2005).

Innate immune cells Several immune cells are present just beneath the epithelial lining in the lamina propria of the intestine and within pockets of the basolateral side of specialized epithelial cells, lacking microvilli, called M-cells (Takahashi, et al., 2009). Particles are constantly being transported through the M-cells to

30 resident phagocytic dendritic cells of the lymphocyte enriched pockets (the Peyer’s patches). The antigen presenting dendritic cells will in turn activate other parts of the immune system upon recognition of foreign non-nutritional particles or entire invading cells. Dendritic cells can also sample the intestin- al lumen by penetrating the adherens and tight junctions, that forms the semi-permeable links between the epithelial cells, without disrupting the protective barrier (Fasano and Shea-Donohue, 2005). Cytotoxic T-cells and Natural Killer Cells are also residing within the Peyer’s patches and lamina propria, although activation of these cells with release of their cytotoxic granular enzymes have not been seen in human or animal Giardia infections to date. Experimental mouse infections have indicated an accumulation of and importance of activation of granular mast cells during Giardia eradica- tion. Mast cells can be instantly activated by a wide variety of pathogen moieties and released granular enzymes display antimicrobial functions as well as inflammation and immune stimulatory effects (Mims, et al., 2002). The innate chain reaction peptide pathway of complement system, induced by the cleavage of parasite-bound C1 peptide can kill Giardia parasites in vitro, and the pathway has recently been re-confirmed by the lab of S. Singer (manuscript under revision) as a Giardia-eliminating host defense, but was initially described as mechanism of Giardia killing during the 1980’s (De- guchi, et al., 1987).

Adaptive Immunity Adaptive immunity is at large the activating-dependent cell mediated im- mune system controlling later stages of infection. Compared to the ever- present innate immunity, adaptive immunity has an arsenal of stand-by im- mune cells with highly specific targets. Within two weeks of infection the host’s adaptive immune system will be fully activated.

The main two types of lymphocytes (white blood cells) are thymus- derived T-cells and bone marrow derived B-cells. T-cells are sub-classed into several different classes in dependence of their surface molecules and immune function, while B-cells are mainly divided due to their activity sta- tus. The foremost receptor or surface molecules of T-cells are the antigen- recognizing TcRs, the T-cell receptors. In combination with the surface TcR, each T-cell presents an array of additional receptors validating the antigen binding upon contact with potential foreign molecules. The CD4 positive and CD8 positive T-cells respectively is one of the most prominent divisions of the T-cell population. Whereas CD8+ T-cells will recognize antigens pre- sented from viral or intracellular infections and become cytotoxic, the CD4+ T-cells detect antigens presented from engulfed, but natural extracellular pathogens, and are activated as helper (further immune modulating) cells.

31 All types of T-cells need the antigens to be presented by antigen present- ing cells (APCs). Within the small intestinal tract and the location of Giardia colonization, the epithelial cells are able to present soluble or internalized antigens to resident T-cells or pass antigens through the specialized M-cell epithelial cells, overlaying the mucosal associated lymph tract with resident dendritic cells (DCs). DCs are, like several other leukocytes are professional antigen presenting cells in the sense that they have the ability to engulf or phagocytos entire pathogens. For instance, the polymorpho- and mono- nuclear lymphocytes present in the milk of nursing mothers are able to ingest trophozoites in vitro (Franca-Botelho, et al., 2006). When being activated within the tissue site, the professional antigen presenting cell will migrate to germinal centers for further activation of immune cells. The many different subtypes of T-cells all activate subsequent local responses and immune reac- tions in different manners and with different outcome. In general terms, the responses can be said to lead to either an inflammatory or non-inflammatory response.

+ The CD4 T-cells, also called the helper T-cells (Th-cells), are potentially important during Giardia infection since they activate and stimulate B-cells to produce antibodies (immunoglobulins). B-cells are derived from the bone marrow, and are constitutively presenting a surface-attached basic immunog- lobulin (Ig) with a specific, although extremely cell to cell inter-variable antigen recognizing domain. The B-cells are controlled for activity during maturation and are ideally only present as competently non-self recognizing cells in the follicles of the lymph system. The lymphatic system runs sepa- rately to the blood system of the entire human body, with specified lymph nodes (recognizing centers) with the resident B-cells and high content of specialized antigen presenting cells and active T-cells. Activated B-cells leave the lymph node and are recruited to infection sites where they special- ize; in the intestine to secrete soluble IgA (sIgA), that are transported to the intestinal lumen for binding, blocking and potential killing of the infecting pathogen. Other antibody isoforms are also produced but are not transported across the epithelium and remain in the blood.

Although humans are not protected against recurrent infections, some pro- teins of Giardia (cytoskeleton, metabolic and surface proteins) are frequently recognized by patient sera immunoglobulins (Palm, et al., 2003, Taylor and Wenman, 1987). Hypogammaglobulinemic individuals suffer extensively more during infection than patients with functional B-cells and soluble IgA antibody delivery into the infected intestine, with extreme prolongation of disease symptoms and infection (Taylor and Wenman, 1987). Infants up to 6 months of age are significantly protected by Giardia detecting antibodies in the mother’s milk of previously infected mothers though (Tellez, et al., 2003). The antigen recognition patterns and levels of serum IgG does not strictly

32 Figure 4. Giardia lamblia attach to host epithelium of the small intestine and leave a suction mark on the brush border after detachment. Scanning electron mi- croscopy show a trophozoite attached to a human jejunal biopsy (a) and a recently detached trophozoite in the upper left corner with the remaining imprint on the in vitro polarized human Caco-2 epithelial cell (b). Pictures from (Magne, et al., 1991) and (Erlandsen and Chase, 1974).

correlate to the sIgA present in mother’s milk or to the sIgA present in duo- denal aspirates (Tellez, et al., 2005, Zinneman and Kaplan, 1972).

Establishment of infection Trophozoites penetrate the mucus layer of the intestinal lumen and attach to the endothelial cell lining of the villi. Attachment is primarily achieved by the structural ventral disk and the adhesive membrane of the shapeable ven- trolateral flange (Céu Sousa, et al., 2001, Chavez, et al., 1995, Chavez and Martinez-Palomo, 1995, Erlandsen, et al., 2004, Katelaris, et al., 1995). Dur- ing approximation to surfaces, the trophozoite’s ventral flagella pair beats in a spiral fashion aiding, at least theoretically, in creating a under pressure of the disk upon attachment (Elmendorf, et al., 2003, Ghosh, et al., 2001). The disk has an outer rim of actin filaments possibly contracting during attach- ment to shape

The pronounced concave form and a suction cup effect of the adhesive disk during attachment (Elmendorf, et al., 2003, Feely, et al., 1982, Narcisi, et al., 1994). The ventrolateral flange, located around and above the disk, has a surface adhesive function in itself that can be seen as filamentous extru- sions of the plasma membrane attaching to in vitro surfaces (Erlandsen, et al., 2004). The attachment of excyzoites with incomplete adhesive discs and occasional trophozoite dorsal attachment is, at least in part, achieved by sur- face adhesive lectins (Inge, et al., 1988, Katelaris, et al., 1995, Pegado and de Souza, 1994, Scott, et al., 2000, Weiland, et al., 2003). Attachment can be impaired by binding of the entire parasite surface with different anti-surface

33 protein antibodies or by pre-coating parasites and epithelial cells with sever- al kinds of monosaccharides (Gillin, et al., 1990, Hernández-Sánchez, et al., 2008, Magne, et al., 1991).

Experimental centrifugation forced detachment showed that the parasite attachment is insensitive to the net charge or hydrophobicity of the attach- ment surface in vitro (Hansen, et al., 2006). The under pressure created be- tween the disk and the surface is not essential for trophozoite attachment, but the parasite load in experimental in vitro experiments is severely reduced when the suction cup-like disk is sterically impaired by attachment experi- ments on spiky surfaces (Erlandsen, et al., 2004).

Host range and Zoonosis Assemblages or species/ subspecies Giardia parasites infect various animals and are classified into so-called assemblages based on genetic differences and on their host preference (Ta- ble 1). The general morphology of cysts from different Giardia species ap- pear similar on the surface by microscopy (Erlandsen, et al., 1989), but the internal structures can look drastically different. Giardia microti cysts for instance contain two completely assembled trophozoites, with intact adhe- sive discs, to be compared with the fully disassembled oval cell structure of Giardia lamblia cysts (Januschka, et al., 1988, Palm, et al., 2005). Typically the genes for the small subunit ribosomal RNA, variable surface proteins (VSPs), glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 alpha and beta-giardin are used for assemblage distinction (Wielinga and Thompson, 2007).

With completion of the sequencing of two Giardia genomes, large scale comparative studies between the two human infecting assemblages (A and B) could be performed (Franzen, et al., 2009). The overall similarity between assemblage A strain WB-C6 and assemblage B strain GS/M is 78% on the amino acid level, and slightly less on the nucleotide level, within the coding regions (Franzen, et al., 2009). Since the specific protein coding capacity, apart from the VSPs and other multi-gene families with high in-family similar- ity, are exceedingly small between the assemblages A and B the transcription- al or translational differences might be what distinguish the two sub-species in efficiency during establishment of infection. The immunodominant protein content of WB, GS/M and P-1 were similar when compared by human serum on Western blots of 2D-gel protein electrophoresis (Palm, et al., 2003). The sera with Giardia reactivity used for the 2D mapping were collected from one distinct outbreak and might have restricted the detection of differences pre-

34 sented between isolates. Future studies might be able to connect assemblage specific genes, gene regulation or amino acid changes within proteins to host specificity or different outcomes of infection. Currently at least three different sequencing projects are on-going; one for clinical isolates of assemblage A, one of an assemblage E isolate from a pig-infection, and one more extensive assembly of GS/M (Svärd SG and Adam RD, unpublished data).

Table 1. The species of Giardia including host specificity and origin of the most common isolates for cell biology and infection studies. Information summarized from references (Nash, et al., 1985), (Monis, et al., 2009) and www.atcc.org.

Species Sub-species Host Axenized isolates Giardia lamblia Assemblage A A variety of mammals, Human isolates; WB including humans (Afghanistan), Isr (MD, USA), P-1 (OR, USA) Assemblage B A variety of mammals, Human isolate GS/M including humans (Alaska, USA) Assemblage C and D Canids (dogs) Assemblage E Hoofed livestock S-2 (lamb) Assemblage F Cat Assemblage G Rats Giardia muris Rodents Giardia microti Rodents Giardia ardeae Birds Giardia psittaci Birds Giardia agilis Amphibians

35 Molecular mechanisms of Host – Parasite interactions

Relevance of the thesis The molecular grounds for giardiasis and asymptomatic manifestations of Giardia infections were not well understood at the initiation of the work of this thesis back in 2004. Several studies had focused on, and established the outcome and pathophysiology of Giardia colonization but the exact cause was far from elucidated. Recent (at that time) publications revealed the pres- ence of unexpected Giardia proteins with an immunological response, de- tected as antibody recognition to Giardia extracts. During the five years of compiling this doctoral thesis several interesting and important conclusions have emerged from work by others and from the work included in this thesis. The joint efforts have taken us, and the society at large, a great step towards revealing the molecular world of Giardia – host interactions.

Model systems of infection The study of Giardia infections is not straight forward. Naturally, controlled experimental human infections in the laboratory are rare. The pathophysiol- ogy of human infection have been studied through small intestine biopsies from patients with natural infections (Hanevik, et al., 2007, Troeger, et al., 2007). Model animals can only be transiently infected and most animal sys- tems never develop giardiasis. The available epithelial cell lines are general- ly of carcinoma origin and are mostly not recovered from the upper intestinal tract, even if small intestine key characteristics can be induced. To the host issue, the question of parasite similarities or dissimilarities is added. Differ- ent strains show different infectiveness in human and animal trials, and some pathologic changes described in vitro are clearly Giardia strain dependent.

Host-Parasite relationship in vitro There is no optimal system for studying Giardia infections. The available animal models are inadequate due to differences to the human body and the in vitro interaction systems lack in environmental and nutritional complexity of a body. Whereas human epithelial cell lines require oxygen (provided by

36 carbon dioxide monitored airflow incubators, with cells grown in a few mil- limeters of medium in open-cap cell culture flasks) the Giardia cells are micro-aerophilic or aero-tolerant and are axenically cultivated with limited amounts of environmental air, in completely filled screw-cap tubes. The growth media of the two cell types are also drastically different with Giardia growing in luminal content mimicking medium with supplemental arginine and cysteine, and epithelial cells grown according to nutrients and growth factors provided by the blood and the intestinal lumen (amino acids, vita- mins and minerals).

The “ideal system”, where both cell types live in their preferred niche has not been established or published yet. Instead, we and others have sought for the best feasible system for each occasion of study. For Paper I in this thesis, our main interest was the human epithelial cell response, and accordingly we optimized the cell growth medium and the airflow for the human cells (these conditions were just tolerable for the Giardia cells). When on the other hand we were most interested in the Giardia response to the interaction (as in Paper IV), we prioritized the parasite’s well-being by decreasing the oxygen pressure (less air in the culture flasks). In our third experimental set-up (Pa- pers II and III) we aimed to identify released or secreted virulence factors and accordingly we used a serum-free growth medium optimal for both the parasite and the host (Paper II and III).

Several cell lines have been successfully used as models of the small in- testine and infections thereof with pathogens other than Giardia. The best described cell line, Caco-2, is an adenocarcinomal colonic epithelial cell line that is constricted in propagation by cell to cell contact and can be induced to express small intestinal features (such as apical to basolateral polarization, formation of tight junctions and appearance of microvilli) by growth post- confluence for around 20 days (Sambuy, et al., 2005). Sub-cloning of the ATCC Caco-2 cell line can render populations with homogenous villi fea- tures as with the cell line dominantly used in Papers I to IV (Chantret, et al., 1994). Other cell lines used are primarily the Ht-29 and T-84 cell line where T-84 epithelial cells show a crypt like feature compared to the Caco-2 villi- like epithelial cells (Bernet-Camard, et al., 1996). Small intestinal human epithelial cells can be recovered and used as primary cell lines, but have not yet been used in Giardia interaction studies. One cell line, SCBN, was long presented as a “small intestine epithelial cell line of human origin”, but this had to be revised when chromosome and feature analysis instead identified the cell line as of canine origin. Thus, when viewing work with SCBN (pub- lications from the lab of A. Buret) one should take this into consideration in the interpretation of data. However, the SCBN does show different response to different Giardia cell lines, and is capable of both tight junction alteration and apoptotic induction and resistance (Chin, et al., 2002). One study have

37 been published with the use of the HCT-8 cell line in responses to Giardia (Panaro, et al., 2007). The apoptosis rate during in vitro infections with HCT-8 was much higher than what has been reported in the human host (Troeger, et al., 2007), which potentially partly undermine the value of achieved results.

Giardia cells can be encysted, excysted and axenically grown in vitro in buffered growth medium mimicking the upper intestinal tract (Boucher and Gillin, 1990, Gillin, et al., 1987). The medium for axenic growth is rich on yeast extracts and glucose, and is supplemented with both heat-inactivated bovine serum, bile, cysteine, salt, buffers and an iron-source (Keister, 1983). Most Giardia strains can be grown in the same medium, with a batch- dependency on the choice of serum and peptone (hydrolyzed yeast extract). Some newly axenized human isolates are sensitive to the bovine serum and require the use of human serum instead (Ankarklev, personal communica- tion). Several human isolates have been axenized and are used in Giardia studies in vitro, with strains WB, GS/M and P-1 being the most common. Apart from the common human isolates other isolates have also been used, some with human origin (NF) and others with animal origin (S-2) or even separate species of Giardia (G. muris, a restrictively murine pathogen).

Host-Parasite relationship in vivo In recent times, only one study of experimental human infections has been published. The voluntary subjection of Giardia cysts led to 100% infection rate of assemblage B strain GS/M, compared to no established infections with the assemblage A strain Isr (Nash, et al., 1987), highlighting the differ- ence in virulence between Giardia strains. Due to the ethical questionability of human experimental infections, the studies of human Giardia infections have since then been restricted to studying biopsy specimen from naturally infected individuals (as in Oberhuber 1997 and Troeger 2007). The absolute majority of host – Giardia studies has thus been conducted in different ani- mal models.

Identification of molecular mechanisms of giardiasis in animal models are largely restricted to infections of Mongolian gerbils (Meriones ungucilatus), the only model animal showing diarrheal disease in response to human iso- lated Giardia strains (Eckmann, 2003). Much in vivo and ex vivo work has been performed in murine animal models with human isolates of Giardia. Rats and mice spontaneously clear infections within 10 to 14 days and do not show disease symptoms from assemblage B. The assemblage A isolates are not able to colonize the small intestinal tract of mice or rats, except for in new-born, suckling mice where a transient infection can be established (Hill, et al., 1983). In many cases, the murine animal model is used with the mu- rine-restricted Giardia muris parasite, which is different from the human

38 infecting isolates, and causes no symptoms, nor infection duration of equali- ty to human infections. Differences between rat, mouse and human intestine in ion channel composition (Kiela and Ghishan, 2009), which is directly related to ion and water adsorption, and differences in cell-based immunity could be important factors in the absence of persistent infection and diarrheal symptoms in murine animal models. Furthermore, the murine model animals acquire immunity to Giardia, which is not the case for humans that can rea- dily be re-infected by identical strains or different Giardia strain upon treat- ment or spontaneous resolving of a prior infection (Eckmann, 2003, Lang- ford, et al., 2002, Nash, et al., 1987).

If deciding to use the murine animal model, the choice of mouse strain for experimental Giardia infections is important. The immune response for dif- ferent common genetic background mice, as Balb/c and C57BL/6, varies slightly with a bias towards a Th2 response in Balb/c compared to a Th1- response for C57BL/6 mice. For infections with Giardia muris the Th1- response mice clears infections faster than the Balb/c mice (Li, et al., 2004).

Notwithstanding the differences between animal model infections and human infections, murine animals are still the most commonly used in vivo model for infection studies for Giardia. Hopefully, murine studies will be- come complemented with indicative in vitro studies on human epithelial cells to a larger extent, and improved gerbil models will be developed. Fur- thermore, human ex vivo experiments with surgical or biopsy material from the upper intestinal tract could be more commonly used.

The pathology from Giardia An established Giardia infection generally causes little to no immediate in- flammation of the infectious site (Oberhuber, et al., 1997). The anti- inflammatory effects of Giardia also decrease inflammatory responses of co- infections, as seen by in vitro alterations of activity in dendritic cells pre- activated through various toll-like receptors (Kamda and Singer, 2009). The pathophysiology of infection shows a general shortening of villi and diffuse, local or general shortening to depletion of the microvilli brush border leading to an overall reduction in mucosal surfaces by up to 75% (Oberhuber, et al., 1997, Troeger, et al., 2007). Prolonged infections can cause long-term in- flammation of the intestine (Hanevik, et al., 2007). In experimental mouse infections the reduction in epithelial brush border is directly linked to CD8+ T- cells, but the effector mechanism(s) remains unresolved (Scott, et al., 2004).

39 Giardia causes a leaky intestine Chloride adsorption reduction and/or increased anion secretion has been reported to accompany Giardia infections in experimental mouse infections with human isolates, but has not yet been reported to occur in humans (Ce- vallos, et al., 1995). An increased leakiness of the small intestine as a conse- quence of infection has been documented though (Dagci, et al., 2002, Troeg- er, et al., 2007), specifying the diarrhea of Giardia as not only being a result of improper adsorption, by reduced epithelial surfaces and increased chloride secretion, but also as dysfunction of the cell to cell junctions. Tight junction integrity has generally been described as the absence of reorganization of the ZO-1 protein (Graham, et al., 2009), however recent publications show the possibility of intact ZO-1 localization with the delocalization of other junc- tional proteins rendering the diarrheal fluxes (Graham, et al., 2009). Fur- thermore, as demonstrated by Troeger et al. in 2007, the claudin-1 protein (of adherens junctions) is significantly less expressed in Giardia infected human duodenum than in control duodenum, while ZO-1 was if anything more expressed than in controls (Troeger, et al., 2007). It should be noted though, that ZO-1 delocalization together with F-actin rearrangement has been demonstrated in vitro for infections with the human-retrieved NF Giar- dia isolate on kidney epithelial cells, whereas this change does not occur on the same epithelial cell type with WB or P-1 Giardia strains (Chin, et al., 2002). There is thus a plausibility of differences in pathophysiology from different infections also in vivo.

Apoptosis and pathology The level of apoptotic cells is increased from the naturally occurring 1% to 1.5% in the duodenum of chronic Giardia infected individuals (Troeger, et al., 2007). The increase of apoptotic cells is small but significant and might be more prominent in acute infections and be of impact in mediating disease. Molecular characterizations identified Giardia-mediated in vitro induced apoptosis to be activated through the caspase-3 pathway (Yu, et al., 2008). The activation of the apoptosis pathway in vitro is linked to the internal glu- cose level of the host epithelial cells, as higher uptake of glucose protects the cells whereas lower concentrations increase the apoptotic level (Yu, et al., 2008). A low glucose environment is plausible during Giardia infections. Luminal disaccharidase activities are lowered in a parasite protease- mediated fashion in mouse and gerbil infection models, much depending on the choice of Giardia strain (Belosevic, et al., 1989, Cevallos, et al., 1995, Scott, et al., 2004, Scott, et al., 2000). The lactase activity is lower in human infections, as seen as enhanced fecal lactose in infected individuals (Goto, et al., 2002). Although the active protease(s) has not been identified, the host inability to hydrolyze and break bonds within larger sugar molecules may lead to a decrease in accessible sugars and to a decreased import, thus to

40 more apoptosis-susceptible epithelial cells (Yu, et al., 2008). It remains to be investigated whether or not lowered disaccharidase activity occurs in human infections, and if so, if apoptosis of human giardiasis shares the mechanism of the low-glucose enhanced Giardia-induced apoptosis seen in vitro. In addition to the possibly decreased glucose levels during Giardia infection, the local arginine is consumed by the parasite. Lack of arginine has a known anti-proliferating effect and causes apoptosis in healthy human tissue (Philip, et al., 2003). Although not investigated yet, the arginine depletion by Giar- dia presents a likely inducer of the apoptosis during giardiasis.

Intestinal movement and parasite eradication Anorexia, or aversion to eating, and the nausea of giardiasis in humans has been coupled to hypersecretion of cholecystokinin (CCK) in the upper intes- tine. In healthy individuals, increased levels of CCK are produced in re- sponse to elevated nutrient influx to the upper intestinal tract as a signal to prohibit further food intake. The CCK levels of giardiasis patients are greatly elevated compared to the levels in healthy individuals (Leslie, et al., 2003). In a mouse model of Giardia infections, the increase in CCK-levels was coupled to neuronal nitric oxide synthase (nNOS) signaling in the smooth muscle cells of the intestine, and the consequent enhancement of intestinal movement was shown to be important for the naturally occurring clearance of infection in mice (Li, et al., 2006).

41 The human response to a Giardia infection (Paper I)

Early studies of the human response to a Giardia infection had mainly had two focuses: the general pathology and the antibody response. Little to no information had been presented on the molecular level of infection responses in the human host. A first step in understanding the host-parasite interplay upon interactions was thus to understand at least parts of what the host cells goes through during Giardia colonization of the small intestinal epithelium (Paper I). We used intestinal epithelial cells with small intestine characteris- tics for in vitro interactions with the Giardia WB cell line belonging to as- semblage A. The time course was set to include the first 18 hours of coloni- zation, and the total transcriptome of the host cells were analyzed on Af- fymetrix whole human genome arrays. From our experiments, we could conclude that less than 5% of the human transcriptome changed during early infection (Paper I), and that this change was highly specific for the Giardia infection. Gene families of immunological, cell sustainability and apoptosis related proteins were regulated in response to the colonization. With the acquired information of host molecular gene responses we could in Paper II – IV continue to evaluate and open up a much needed research field of Giar- dia cell biology and giardiasis.

The immune response to Giardia The human gene that shows the strongest response upon early Giardia infec- tions is chemokine CCL-20 encoding the Macrophage Inflammatory Protein 3-alpha (MIP-3) (Paper I). The MIP-3 receptor CCR6 is constitutively expressed on small intestinal resident dendritic cells and on circulating T- cells, with a higher presence on CD4+ T-cells than on CD8+ T-cells, correlat- ing well to the amount of recruited cells in experimental mouse infections (Iwasaki and Kelsall, 2000, Schutyser, et al., 2003). Duodenal biopsies of giardiasis patients reveal an increase of CD8- T-cells in infected epithelial lining of the intestine, while resident cytotoxic CD8+ T-cells do not become activated upon human infections (Oberhuber, et al., 1996). From the micro- array experiments of the human transcriptome during early infections, a few other chemokines were also identified in Paper I as induced by Giardia co- lonization; the CCL-2, CXCL-1, -2, -3 and the plasminogen activating uro- kinase receptor PLAUR. Taken together, these chemokines provide potential activation and recruitment of antigen presenting cells as dendritic cells

42 (DCs) and macrophages, and activation of T- and B-cell mediated immunity. The lack of pro-inflammatory cytokines, are in line with the pathology of Giardia showing little to no inflammation in infected host. In addition, in vivo experiments in mice showed the de-activation of inflammatory path- ways by Giardia with the induction of IL-10 within dendritic cells, when these cells were co-stimulated with other pathogens (Kamda and Singer, 2009), indicating how prominent the anti-inflammatory reactivity seem to be. Furthermore, an increase of IL-10 production leads to up-regulation of the CCR6 receptor, as an autocrine activating molecule of CCR6 expression in DCs, additionally promoting MIP-3 responsiveness of the infection (Schutyser, et al., 2003).

Activation without parasite attachment When the mapping of general immunodominant proteins of Giardia was performed in 2003 many of the identified Giardia antigens were structural proteins of trophozoites and soluble, cytoplasmic proteins, not known to be presented on the parasite surface nor secreted (Palm, et al., 2003). A possible explanation was major trophozoite lysis during infections, something which was not studied at the time. From the characterization of the human epitheli- al cell response to Giardia infections in vitro (Paper I) we could conclude that trophozoite lysis was not a major factor and not the reason for the acti- vated immunological response. In fact, we showed that not even attachment to or cell-to-cell contact between the human host and the live and intact tro- phozoite was needed for the immune activation. In continued but unpub- lished work we could conclude that the activation of the host immune system (measured as induction of the gene CCL-20 and subsequent secretion of corresponding chemokine MIP3 into culture medium), was activated by concentrated proteins from spent, filtered, Giardia and human IEC co- culture medium.

Comment on studies of Giardia infections in mice A substantial amount of work on elucidating the mechanism of Giardia in- fections have been performed with murine models, primarily infections in mice. As mentioned earlier, the use of the murine animal model is proble- matic. Infections in humans are often asymptomatic (in approximately 60% of the cases) (Rose, et al., 1991). However, in mouse and rat models infec- tions are always asymptomatic and these models can only be infected with a small subset of human infecting Giardia strains or the murine specific G. muris. Furthermore, the infections of mice are always cleared, unlike the infections in humans that can persist for years if not treated, with or without accompanying symptoms (Buret, 2005). It is thus possible that the differenc- es between the murine small intestine and its immune defense, compared to the human counterparts, are too great to actually draw conclusions about giardiasis and Giardia infections.

43 Parasite mechanisms for host survival

Since Giardia infections do occur, and not all Giardia infections are cleared naturally, the parasite must have ways of overcoming the intestinal defenses of humans. The upper intestinal tract produces many lethal mediators of host origin that could kill the parasite in the absence of proper protection. Me- chanical features, such as the movement of the parasite and trophozoite at- tachment, are essential ways of counter-acting the natural replenishment of the gut lining and the constant flow in the intestine. The attachment of tro- phozoites directly to the epithelial cells confers protection by the thick mu- cus layer from extensive activity of some harmful substances in the lumen. On the other hand, the close proximity to the host cells exposes the parasite to host cell activities.

From the studies of Paper I we could conclude that Giardia provokes a response in the host epithelial cells, that the response activates the immune system, and that the activating factor was a soluble Giardia factor. In papers II to IV, we sought to identify the immune-activating, and more importantly immune and general host-modifying Giardia factor(s).

Immunodominant proteins of Giardia A complete survey of Giardia proteins possessing epitopes for serum anti- bodies of acute infected humans identified the major immunodominant pro- teins by 2D SDS-PAGE and mass spectrometry (Palm, et al., 2003). Later, 2D protein maps of proteins reacting with IgA from mother’s milk of Giar- dia positive lactating women was compared to immunoreactivity maps from the serum samples of the same mothers, and to the immunodominant pro- teins previously identified (Tellez, et al., 2005). In both studies, some of the identified proteins were known cytoskeleton or surface proteins of Giardia while others were proteins only known to possess an intracellular localiza- tion or previously unidentified proteins. Similar immunoreactive protein maps as with human serum or mother’s milk were seen with antibodies from Giardia WB infected mice (Davids, et al., 2006).

Variable surface proteins does not explain the pathology A large family of highly antibody-reactive proteins was the Variable Surface Proteins (VSPs). These cysteine-rich proteins cover the entire cell surface of trophozoites and are thought to be a resistance mechanism protecting the

44 parasite from intestinal proteases (Nash, 2002). During the 1980’s, the sur- face proteins were found to vary from clone to clone of Giardia in the labor- atories, and also within a clone over time. The family of proteins was called Variable or Variant Surface Proteins (VSPs) and is represented by approx- imately 200 genes in the WB genome (Morrison, et al., 2007). At any time, one VSP is predominantly presented on the parasite surface, and over time the expressed VSP will be exchanged for another from the 200 VSP-coding genes (switching). Extensive work on the mechanism of switching (Prucca, et al., 2008), the induction and regulation of the switching mechanism, and of functions of VSPs has proven the importance of switching in antibody (sIgA) evasion. However, switching is not the only mechanism for parasite survival and the VSPs are not causing the pathophysiology of giardiasis.

Screening for Virulence Factors Several of the immunodominant proteins that had been described had no known function in the pathogenesis of Giardia and were at large uncharacte- rized. We speculated that the immune-activating soluble factor(s) of Giardia noted in Paper I could be proteins, and that these then probably were among the immunodominant proteins identified previously (Palm, et al., 2003). To analyze the interaction proteome (presented in Paper I and II), initial ques- tions needed to be raised and answered. Firstly, the time frame of immune activation was less than 1.5 hours for an immense increase in cytokine tran- scription (Paper I), thus we were interested in the early secretome of the parasite. Secondly, the interaction setup used for the study of the human interaction transcriptome in Paper I had to be modified. It proved impossible to separate secreted proteins from the medium serum proteins present when co-incubating the Giardia cells with polarized Caco-2 epithelial cells in se- rum containing Dulbeco’s Modified Eagles Medium (the medium recom- mended for growth of Caco-2 cells). In complete, serum containing culture medium the Giardia parasites live on happily for at least 24 hours together with the epithelial cells. When omitting the serum on the other hand, the parasite cells rapidly die off. To compensate for the rougher environment of a no-serum medium, the oxygen pressure was decreased by making co- cultures in medium-filled cell culture flasks, and the medium was supple- mented with cysteine and ascorbic acid, both having strong reducing activity thus aiding the parasite defense against harmful reactive oxygen species (see section on oxygen stress, page 22). In the modified interaction setup, the parasites remained unharmed for many hours, while the epithelial cells started to round up and die off at six hours. In aiming to characterize the early interaction response proteins of the parasite, we settled for protein de- tection of up to 2.5 hours in vitro.

By the use of an established method of protein concentration and separa- tion on 2 dimensional SDS-protein polyacrylamide electrophoresis gels (2D

45 SDS-PAGE) several proteins were detected from filtered, cell free, culture supernatants (for details see Paper II). The resolution from separate sets of gels varied, but the same protein pattern was seen from both interactions with Giardia WB and polarized Caco-2 epithelial cells or Ht-29 epithelial cells. All visible spots from Coomassie stained gels were excised by pipette tips, and dissolved, trypsin-digested and sent for mass spectrometry. Most protein spots revealed the contamination of serum proteins, even though both the human epithelial cells and the Giardia trophozoites were well washed in PBS prior to interactions. In total, only three proteins of Giardia origin were detected from the 2D SDS-PAGE/ mass spectrometry setup in Paper II.

All of the three had previously been described as immunodominant pro- teins (Palm, et al., 2003, Tellez, et al., 2005), and they were known to be important metabolic enzymes of the parasite. Arginine deiminase (ADI) and ornithine carbamoyl transferase (OCT) are enzymes of the primary metabol- ic pathway (arginine dehydrolase pathway) and Enolase is active in the gly- colytic pathway. In sealed illeal loop experiments of Balb/c mice and NOD mice (similar results, only Balb/c results included in Paper II) two of the proteins (ADI and Enolase) were recognized as secreted in vivo by the Giar- dia strain GS/M. This potentiates an activity of release common for both of the human-infecting assemblages (A; WB, and B; GS/M) and might prove important in future treatment or vaccination trials against Giardia.

By improving the protein detection by using a modified silver protein staining procedure, and excellent mass spectrometry, we could excise low abundance bands from one dimensional SDS-PAGE gels for protein identifi- cation (Paper III, mass spectrometry by Ulf Hellman). In addition to the previously detected proteins, Elongation Factor 1-alpha was identified in the supernatant of parasites grown alone in serum-free medium, and the EF-1a was also shown to be released during co-cultures with Caco-2 cells, and to be immunodominant for sera from acute Giardia infected individuals (Paper III). Along with many more serum proteins a 58 kDa version of ADI, that is a slightly shorter version than had previously been identified in Paper II, was also detected by the experiment setup in Paper II.

Characterization of released Giardia proteins (Paper II and III) Preliminary data showed a strong increase of MIP3- (the chemokine en- coded by CCL-20) through addition of recombinant EF-1 to Caco-2 cells, but the activation might have been due to contaminating prokaryotic prod- ucts (i.e. LPS; lipopolysaccharide) from the expression in and protein purifi- cation from Escherichia coli. Polymyxin-binding and neutralization of con- taminating LPS and tagged protein expression from transgenic Giardia could answer the question of whether EF-1 truly is involved in CCL-20 transcrip-

46 tion or not, but those experiments remains to be performed. ADI, OCT and Enolase, also purified from E. coli did however not induce CCL-20 or MIP- 3 levels separately.

The Giardia proteins identified in Paper II had already been cloned for protein expression (Palm, et al., 2003), while EF-1 was created for Paper III. Monoclonal antibodies from mouse against the four recombinant pro- teins were raised and used for localization of the proteins within axenically grown Giardia and in parasite and human epithelial cell interactions. All proteins showed a relocalization within the parasite upon interaction with human epithelial cells, most prominently seen for EF-1 where the interac- tion induced a change from strict endoplasmatic reticulum localization to a cytoplasmatic dispersion (Paper II and III).

Virulence by Arginine Deiminase Neither of the identified proteins had previously been reported or characte- rized in the extracellular environment of a Giardia infection. For ADI and OCT, the intracellular, metabolic arginine dehydrolase pathway degrading arginine to citrulline and carbamate, rendering ATP through consecutive carbamate kinase substrate and AMP conversion, was well studied ((Knodler, et al., 1998) and previous publications cited therein). An addi- tional intracellular function of Giardia ADI has recently been suggested, involving ADI in controlling the antigenic variation of variable surface pro- teins (VSPs) through citrullination of the arginine in the cytoplasmic CRGKA-tail of VSPs (Touz, et al., 2008), although this enzymatic activity could not be detected by others (Li, et al., 2009).

With the active scavenging of arginine, Giardia will consume high rates from the local environment, presumably causing arginine depletion for host cells. Several reports have highlighted the importance of arginine for euka- ryotic (human) cell growth, and recombinant non-Giardia ADI has been used for arginine depletion in clinical trials, delivering ADI as an anti- proliferating agent for cancer cell treatment (Shen, et al., 2006). Interesting- ly, arginine depletion for non-cancerous cells can induce apoptosis (Philip, et al., 2003). Whether the release of Giardia ADI and OCT could convey the apoptosis related to pathophysiology of a Giardia infection remains to be elucidated, but hold additional promising investigations of this metabolic enzyme.

Related to arginine depletion, a publication from the labs of Eckmann and Gillin had reported the peculiar non-presence of the innate immune molecule nitric oxide in Giardia infections (Eckmann, et al., 2000). Nitric oxide (NO) is cytostatic for Giardia trophozoite growth and inhibits differentiation of both encystation and excystation (Eckmann, et al., 2000). In addition, se-

47 creted NO stimulates natural killer cell activity (Sarti, et al., 2004), thus NO production would severely impair Giardia infections and spread of disease. NO is produced by epithelial cells by arginine conversion to nitrite and ni- trate by the enzyme family of NO synthases (NOS). For Giardia infections the absence of secreted NO from epithelial cells was unrelated to regulation of NOS transcription and was instead associated with depletion of arginine from the extracellular environment. Furthermore, consumption did not re- quire Giardia trophozoites to be attached to, or be in close proximity to the epithelial cells (Eckmann, et al., 2000). Eckmann postulated the immense uptake of arginine by the parasite as a sole cause for lack of NO production. With the identification from paper II that ADI and OCT also are present extracellulary, the arginine levels could be speculated to be further reduced than they would have been with only intracellular arginine consumption of Giardia.

Recombinant and enzymatically active ADI, purified from cloned and ex- pression-induced E. coli, was able to convey the Giardia-protective mechan- ism of local arginine depletion (Paper II). Epithelial cells were stimulated for NO production by cytokines IL-1, TNF- and INF- and exposed to enzy- matically active ADI. NO production was reduced by 50% in the presence of 100ng of ADI (Paper II). The amount and reactivity of ADI added was sig- nificantly less than what is released by the parasite during in vitro reactions (measured as a rough estimate from supernatants on protein gels, calcula- tions not presented in Paper II). The results of in vitro decrease of NO pro- duction in stimulated polarized epithelial cells, in cohort with the local ac- cumulation through binding of ADI to the epithelial cells (see figure 2 in Paper II), are indicative of that Giardia ADI also in vivo could be the key to inhibition of nitric oxide production. This would then present an elegant way for the parasite to circumvent the production and secretion of a toxic mole- cule and shape a favorable growth niche upon the epithelial cells.

Tempting roles for enolase In Paper II we do not present any model for secreted enolase activity during infection. Some experiments were however conducted but excluded from the manuscript since they were not repeated or completed. Enolase has been described as a surface protein of Group A Streptococci, conveying adherence and invasion of human pharyngeal epithelial cells (Mundodi, et al., 2008). The enolase of Streptococcus mutans has a mucin binding ability and is in addition to its surface localization also detected in the culture supernatant (Ge, et al., 2004). Preliminary experiments showed mucin binding of Giar- dia enolase (unpublished data). The biological relevance for this binding remains to be further studied. One activity of mucins is the capability of binding to the antimicrobial peptide lactoferrin and to sIgA antibodies (Soares, et al., 2003). Lactoferrin was proven to be Giardia-cidal at physio-

48 logical levels, but no mechanism for in vivo survival or escape of the parasite was presented (Turchany, et al., 1995). Initial tests with recombinant enolase and MUC2 mucin showed a protection of the parasite from lactoferrin killing (unpublished data). These preliminary data would be interesting to perform in a larger scale and with multiple cultures.

The Trichomonas vaginalis enolase has been characterized as a host- interaction induced plasminogen-binding protein that cause cleaving of plasminogen to active plasmin (Mundodi, et al., 2008). The importance of TvEnolase during establishment of infections is not clear, but infected patient sera acquire enolase recognizing antibodies (Mundodi, et al., 2008). Interes- tingly, the Streptococci enolase also binds to plasminogen, and the epithelial receptor for urokinase plasminogen (PLAUR or uPAR) is coupled to epi- thelial junction disruption by plasminogen activation (Pancholi, et al., 2003). If the plasminogen binding, the PLAUR up-regulation noted in Paper I of interacting Caco-2 cells, or the leakiness of an infected intestine is in any way linked to the released Giardia enolase, was not tested during the project resulting in Paper II. Additionally, enolase from Streptococcus sobrinus was shown to be an anti-inflammatory cytokine stimulating protein (Veiga- Malta, et al., 2004), opening up for yet another plausible function of Giardia enolase.

EF-1, an immunodominant protein of Giardia In Paper III we could identify a fourth protein of Giardia; Elongation Factor 1-alpha (EF-1), that was both immunodominant and released into interac- tion growth medium. EF-1 is a translation regulating and activating factor in all eukaryotic cells, and the homologue EF-Tu is active in prokaryotes. In addition to translation catalysis, eukaryotic EF-1 has been shown to be involved in diverse mechanisms such as binding of microtubular cytoskele- ton, regulating activities of the actin cytoskeleton, nuclear transcription regu- lation as well as involvement in the Rho signaling pathway (Nandan and Reiner, 2005). For several pathogens the distribution of EF-1 upon infec- tion, with implications in virulence to host cells, has been documented (Nan- dan, et al., 2002). The function of Giardia EF-1 in correlation to infection remains to be studied with the investigations in Paper III restricted to de- scribing the general characteristics of Giardia EF-1 in combination with identifying a specific detection by human serum from acute infected patients (Paper III).

In the Giardia genome, the EF-1 gene is present in two copies, with identical open reading frames and poly-adenylated 3’-UTRs, but with dis- tinct promoter sequences in the 5’ upstream region (Paper III). For Trypano- soma brucei the EF-1 protein is present in several isoforms, and is tran- scribed as a tandem-transcript together with another gene, and subsequently

49 regulated both at transcription and post-transcription level (Ridgley, et al., 1996). It should be mentioned that T. brucei possesses frequent multiple open reading frame-containing transcripts and extensive post-transcriptional modifications, completely remote from Giardia transcription and translation control. In Plasmodium species EF-1 is present as two identical gene copies with similar or identical expression over the erythrocytic life cycle (Vinke- noog, et al., 1998). The presence of transcriptional EF-1 regulation in other protists prompted an evaluation of the distinct promoters of Giardia EF-1 (Paper III). The two promoters were cloned to drive luciferase expression from epigenomic plasmids in Giardia. Under axenic growth conditions both of the promoters are equally active in transfected Giardia (selected by plas- mid-conveyed puromycin resistance) (Paper III). It is possible that the dupli- cate Giardia EF-1 confer merely a gene dosage effect as is the case in Plasmodium (Vinkenoog, et al., 1998).

EF-1 has been reported as a virulence factor for other infectious organ- isms, both as a surface adhesive molecule on Lactobacillus johnsonii, and as a secreted antigen by the nematode Ascaris suum. The protist Leishmania donovani down-regulate inflammatory signaling by EF-1 localization with- in the host. Interestingly, Giardia, Entamoeba histolytica, Cryptosporidium parvum and Trypanosomal and Leishmania EF-1 share a structural diver- gence in the lack of one 12 amino acid long stem-loop compared to human EF-1, potentially conferring virulence and difference enough that is seen as specific antibody detection of the parasite protein.

The 58kDa E/S-protein of Giardia; is it ADI? In the early 2000’s, the first report of excretory-secretory (ES) products pro- ducing morphological alterations in host cells was published (Kaur, et al., 2001). Fluid accumulation at the infection site in mice, and fluid accumula- tion in rabbit illeal loops was connected to one fraction of ES proteins of Giardia strain Portland-1, eluted by ion exchange chromatography (Shant, et al., 2002). A major glycosylated protein of 58kDa was noted on SDS-protein gels in the activated fraction (Jimenez, et al., 2007). Unfortunately the pro- tein has not been identified, and the partial amino acid sequence of ADFVPQVST is more similar to bovine serum albumin (BSA) than the tox- ins of other organisms (Shant, et al., 2002)(Paper II). A specific 58kDa pro- tein is neither detected on protein gels nor in mass spectrometry characteri- zations of excretory/secretory products presented in Paper II. However, with enhanced sensitivity on 1D protein gels, working in serum free medium with non-interacting Giardia trophozoites, a 58kDa fragment was confirmed to be arginine deiminase (Paper III). The serum protein BSA is virtually impossi- ble to wash off of Giardia trophozoites, and contaminate in vitro studies of the Giardia and human-interactive secretome (Paper III), and usually mi- grates at the same size as ADI in protein gels (Paper II). Robbins and Samu-

50 elson further argued that the description of excretory/secretory glycans, in- cluding the alleged 58kDa protein of Jimenez et al. were in reality characte- rizations of serum contaminants, as the Giardia genome does not convey logical explanations for how the parasite would process and compile identi- fied glycosylations (i.e. derivatives of mannose and galactose) (Robbins and Samuelson, 2005). Whether ADI possesses the toxin-like features of the 58 kDa protein described, or if the 58 kDa protein actually is a P-1 strain- specific protein not detectable by us (working with strain WB) remains to be investigated. Immunoreactive proteins from ES-fractions have been seen to differ some between Giardia strains (Nash and Keister, 1985).

The Giardia transcriptome of early infection (Paper IV) The limited amount of Giardia proteins detected in the interaction superna- tant of parasites co-cultured with intestinal epithelial cells in vitro prompted a different way to search for interaction related virulence mechanisms of Giardia. By using a third setup of in vitro interactions between polarized Caco-2 cells and the WB strain of Giardia, small but interesting changes of the parasite transcriptome during establishment of infection were detected by microarray analysis (Paper IV). In general, many genes were highly tran- scribed under the axenic growth of Giardia trophozoites, and showed an initial decrease in transcript levels when the cells were transferred to interac- tion conditions. Some genes were less decreased when interacting tropho- zoites with Caco-2 cells, compared to trophozoites alone in the interaction medium without host cells. However, genes only specifically altered by the presence of host epithelial cells were also up- and down-regulated.

Environmental induced changes The interaction setup with Giardia and Caco-2 cells co-cultured for up to 18 hours in 5% CO2 and with only semi-filled flasks is apparently a strong stress for the parasite. Already after a few hours the trophozoites loses via- bility without the host cells in interaction medium. With the presence of host epithelial cells on the other hand, the parasites survive for more than 18 hours. Compared to when studying the interaction transcriptome within the host (in Paper I), the medium amount were doubled to at least slightly com- pensate for the extensive oxygen pressure for the Giardia trophozoites. Dur- ing the first 1.5 hours of interaction, a vast amount of genes were decreased that seem to have a strong correlation to the change of environment (Paper IV). Many genes were shared in expression changes between axenic growth (low oxygen, Giardia adapted growth medium) and in vitro interaction me- dium, either containing or lacking host cells. The genes can be generally be described as coding for oxygen and osmotic stress proteins and include gly- cerol kinase (described in yeast; (Westfall, et al., 2008)) and several oxygen

51 scavenging related proteins (eg. NADPH-generating enzymes and thioredox- in-like proteins).

New virulence factors identified To compensate for the initial change in environment the microarray data was normalized to the transcript rates within trophozoites grown for 1.5 hours in interaction medium without host cells. The most up-regulated genes seen without compensation between the data were still dominating after the nor- malization (Paper IV, tables 1 and 3). Few genes were down-regulated more than two-fold, but the list including the top 30 with most decreased expres- sion (Paper IV, table 4) contain genes for released interaction-related pro- teins identified in Paper II and III. The decrease of these genes has a strong coupling to the decreased metabolic activity and the general lowered cell activity rate in the interaction setup compared to axenic growth. Noteworthy, the protein release is very rapid during interactions, and the transcript de- crease is seen mostly in later time-points (up to 18 hours). Thus the data does not have to be viewed as contradictory.

The most striking changes in expression, strictly connected to the pres- ence of host epithelial cells, were the up-regulation of several high cysteine membrane (HCM) proteins and increased detection of small open reading frames. After studying the genome location, and noting the absence of ho- mologues with other Giardia assemblages than A (assemblage B; GS/M (Franzen, et al., 2009) and assemblage E; P15, Jerlström-Hultquist, unpub- lished data) and with all other gene resemblance data available through BLAST at NCBI the increase in expression was considered a result of re- laxed transcription control in Giardia. The short open reading frames seem to be strictly located in proximity to strongly expressed promoters of de- scribed genes. Some allegedly specific probes on the microarrays (provided by the PFGRC, J. Craig Venter Institute, Rockville, MD, USA) for these short open reading frames revealed to be cross-reactive to larger, more plausibly translated open reading frame transcripts.

The HCMp protein family was recently recognized as different from the variant surface protein family (Davids, et al., 2006). Both protein families contain a high abundance of cysteine-rich repeats, and a membrane spanning region, but the HCMps lack the cytoplasmic CRGKA-tail, that has been implied as important for antigen switching of the parasite surface protein coat. The role of the high cysteine membrane proteins and the dominancy (7 out of 30 top up-regulated genes) of the transcription induction during in vitro host relation detected in Paper IV remains to be further studied. It holds an interesting promise that we are closer to understanding the molecular mechanism of Giardia infections and giardiasis.

52 The potential importance of Cysteine proteases The only protein family of previously suggested virulence factors in Giardia that are identified as transcriptionally up-regulated during the in vitro inte- ractions, are genes of cysteine proteases (Paper IV). Proteases, and cysteine proteases specifically, have been described as virulence factors for other unicellular eukaryotic pathogens as Entamoeba and has been proposed to be important for the pathogenicity of Giardia-related protist Trichomonas vagi- nalis (De Jesus, et al., 2009, Santi-Rocca, et al., 2009). Protease activities have been reported in excretory/secretory products of Giardia. Cysteine protease activities have been detected in the supernatants of in vitro cultures of different Giardia human isolates (de Carvalho, et al., 2008). For the WB strain, protease activity during interactions with rat intestinal epithelial cells (IEC-6) or canine kidney epithelial cells (MDCK) is elevated intracellulary, but the extracellular presence or activity was not measured in the studies (Rodriguez-Fuentes, et al., 2006). The presence of extracellular cysteine proteases during Giardia infections in concert with the increase of protease transcription during in vitro interactions as seen in Paper IV do point to a protease involvement in Giardia host colonization.

53 Conclusions and thoughts for the future

The research included in this thesis covers well the molecular aspect of the crucial establishment of infection by Giardia lamblia at host epithelial cells. From the start in the year of 2004 with little known of the molecular aspects of giardiasis, the picture is now much greater with the knowledge of immune system activation in the host and several described virulence factors of the parasite. With this said, there is still much more to learn about the infections of Giardia.

In Paper I, the activation of immune attracting chemokines was described. Mice, rat and gerbil infection studies in vivo and the use of peripheral lym- phocytes or even biopsies of infected humans have not led to any clear con- clusions about the attraction and activation of especially dendritic cells. The murine animal model widely used for Giardia-host studies inconsistent compared to Giardia infections of humans on an immunological level, not only obvious by the difference in infection outcome between murine animals (no disease, obligate clearance, protection against secondary infection) and humans. Human biopsies show the truth about infection. However, the dif- ferences in Giardia isolates as well as infection duration of sampled patients make stage specificity and molecular mechanisms difficult to study in hu- man infections. Hopefully, the future holds several studies on mucosa asso- ciated lymphocyte activation, dendritic cell recruitment studies and ex vivo experimental infections of human upper small intestine biopsies.

In Paper II and III, the proteome of early infection released proteins were under investigation. The immune activation of human intestinal epithelial cells, seen in Paper I, was concluded to be due to a released factor. We were not able to characterize this/these activating proteins, but instead docu- mented the presence of four potential virulence factors of Giardia. The viru- lence potential of the proteins that were identified in Paper II and III can be studied in more depth. The putative apoptosis relation of ADI and OCT re- mains mostly speculative, but highly plausible, and would be of interest for future studies. Furthermore, the implications of enolase and EF-1 in host alterations seen from studies of other pathogens also prompt for further in- vestigations.

54 A multidimensional proteome screening by liquid chromatography and tandem mass spectrometry, with an initial cleaning step reducing serum pro- tein contaminations through sequestering columns, might enable the identifi- cation of immune stimulating protein(s) of Giardia and also identification of other virulence factors that are lost in our gel-based protein separations of Paper II and III.

The transcriptional analysis of Giardia in response to human intestinal epithelial cells in Paper IV is a major stepping stone for future research. The close to undescribed protein family of high cysteine membrane proteins are highly regulated at transcription level during interactions. The function of the HCM proteins as membrane molecules could be important for sensing and signaling during infections, or compose an infection specific surface protein family. The guesses are at this point unfounded but intriguing. Fur- ther transcripts of interest from the interaction transcriptome of the parasite include proteases and metabolic regulation. Naturally, finding the mediators of pathophysiology from Giardia and possible giardiasis-treatments or anti- Giardia targets motivate why the interaction transcriptome should be further investigated on protein level and investigated as in vivo mechanisms.

In conclusion, the last five years have been intriguing times for all of us interested in Giardia – host mechanisms. With the insights from this thesis into the molecular mechanisms of the parasite and the human responses to infection the research community might soon be able to describe the major key players of giardiasis and thus come closer to a true elucidation of the infection and disease.

55 Acknowledgements

There are naturally many people to thank, out of whom a few have been extensively important for making me able to conduct the research and com- pile this thesis. Firstly, I would like to thank Uppsala University and Vetens- kapsrådet for doctoral research funding and Svensk förening för Tropikme- dicin, Resemedicin och Internationell Hälsa, Anna Maria Lundins stipen- dienämnd at Smålands nation in Uppsala, and Uppsala University C F Lilje- walch and Wallenberg stipends for travel funding. By saying that, I will also thank the Swedish tax payers for directly enabling me to spend five years on doing something that I love. Thank you!

A PhD-student is nothing without the supervisor; thank you Staffan for ask- ing me to join you lab, for listening to my ideas, for scientific discussions and for letting me work and grow together with you. Ups and downs come and go, but all in all it is like you wrote; “Staffan é bra, ibland i alla fall”.

I got a smooth start thanks to the previous members of the Svärd group and colleagues at MTC, KI and PMV, SMI. It was highly appreciated. Since the move to Uppsala, with the growing crowd of Giardia people in the lab the last year(s), the research environment and the enormous thirst for knowledge that spread in the group has felt exceptional! I have had so much fun with you both inside and outside the lab! Thank you so much, and keep up the good work.

The Giardia – Uppsala community has also included work and discussions together with Prof. Rolf Bernander and his group and Dr. Jan Andersson at EBC, Uppsala University. I will especially thank the Bernander group for all the help with microarray setup and analysis.

Working with Giardia, I have also been fortunate enough to collaborate and make a short exchange in the lab of Dr. Hugo Lujan, then located at IN- IMEC-CONICET, Córdoba, Argentina. I am very grateful to Hugo, Maria Touz and Andrea Ropolo and the students in your groups in late 2005. I had so much fun, and learnt a lot!

56 The ICM at Uppsala University, and especially the microbiology depart- ment, is a very stimulating and productive work place. Thank you to all the colleagues at the department! I have enjoyed to constantly learn more about prokaryotes and the RNA world through discussions and project presenta- tions and the journal club (although compiling the booklet always felt too time consuming…).

I am immensely thankful for the many and very good friends I have gotten to know during my time at ICM. You are all very dear to me, and frankly there have been times when you were the ones that kept me going. I would also like to direct a special thank you to my friends from outside ICM that have carried and supported me when needed. All of you have given me many happy memories and I hope for many more in the future!

Finally, I would like to thank my beloved boyfriend and my family for all the warmth, kindness and support. You have shaped me into this person, and I am grateful for what I can accomplish with you by my side.

57 References

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