CELL CYCLE AND DIFFERENTIATION IN GIARDIA

Department of Microbiology, Tumor- and Cell Biology Karolinska Institutet, Stockholm, Sweden

CELL CYCLE AND DIFFERENTIATION IN GIARDIA LAMBLIA

David S. Reiner

Stockholm 2008

Published by Karolinska Institutet. Printed by Larserics Digital Print AB, Sundbyberg, Stockholm, Sweden. © David S. Reiner, 2008 ISBN: 978-91-7409-026-0

Järn giver skärpa åt järn; så skärper den ena människan den andra. Ordspråksboken 27:17

ABSTRACT Giardia lamblia is the major cause of waterborne diarrhea worldwide. Giardiasis is initiated by ingestion of cysts, which after passing through the stomach are triggered to excyst. Excystation, or awakening from dormancy, is central to successful colonization of the host. An investigation of the role of calcium signaling throughout excystation was initiated to study the cellular regulation of this special differentiation. Calcium signaling was most crucial during late excystation where the excyzoite emerges. A calcium pump inhibitor, thapsigargin, inhibited excystation, calcium signaling and localized to a calcium storage compartment in cysts. Inhibitors of the calcium signaling protein calmodulin blocked excystation and calmodulin localized to Giardia’s basal bodies suggesting that the basal bodies are Giardia’s cellular control center. Basal bodies were isolated and 310 proteins identified using proteomics. Functional orthologs of these proteins were identified bioinformatically and used to build a network model. Differentiation-specific nodes were identified in the network using transcriptional data from the Giardia lifecycle. The model correctly predicts that calmodulin is involved in cytoskeletal remodeling and this was verified by affinity purifying 10 calmodulinspecific binding proteins. For cysts to survive in nature and the pass through the stomach successfully they need a protective wall. A study was undertaken to look for new cyst wall proteins. One cyst wall protein identified was identified by SAGE and localized to the cyst body. This new cyst wall protein was found to be an invariant cysteine-rich Type 1 membrane protein and a member of a larger cysteine-rich family. This new family of novel cysteine-rich Giardia proteins was shown bioinformatically to have homologs in two other cyst-forming protozoans. The initiation of differentiation is associated with cell cycle arrest in many cells. Giardia differentiates and forms cysts by arresting from the cell cycle and encysting. We looked at the role of the cell cycle in Giardia during encystation. We developed for the first time a method of synchronizing Giardia for use to determine where the encystation restriction point is in the cell cycle. We found using encystation-specific organelle biogenesis as a marker, that it was late in G2. In addition we used quantitative real-time PCR to determine the periodic cell cycle regulation of histones. Cyclin B is normally up-regulated in the late G2 stage of the cell cycle and promotes G2/M transition. We phylogenomically identified a Giardia cyclin B and found that expression gradually increased reaching a maximum at 3 h corresponding to G2, and decreased again with entry into mitosis after 4 h. We also identified bioinformatically 217 cell cycle orthologs and studies are in progress to verify these using synchronized populations and Giardia microarrays.

LIST OF PUBLICATIONS

I.

David S. Reiner, Michael L. Hetsko, J. Gary Meszaros, Chin-Hung Sun, Hilary G. Morrison, Laurence L. Brunton, and Frances D. Gillin: “Calcium signaling in excystation of the early diverging eukaryote, Giardia lamblia” J. Biol. Chem. (2003) 278: 2533–2540.

II.

Barbara J. Davids*, David S. Reiner*, Shanda R. Birkeland, Sarah P. Preheim, Michael J. Cipriano, Andrew G. McArthur, and Frances D. Gillin: “A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein” PLoS ONE. (2006) 1: e44

III.

David S. Reiner, Johan Ankarklev, Karin Troell, Daniel Palm, Rolf Bernander, Frances D. Gillin, Jan O. Andersson, and Staffan G. Svärd: “Synchronisation of Giardia lamblia: Identification of cell cycle stagespecific genes and a differentiation restriction point” Int. J. Parasitol. (2008) In press.

IV.

Tineke Lauwaet, Michael Baitaluk*, David S. Reiner*, Edwin P. Romijn, Catherine C. L. Wong, Hanna Skarin, Barbara J. Davids, Shanda R. Birkeland, Michael J. Cipriano, Daniel Palm, Sarah P. Preheim, Amarnath Gupta, Staffan G. Svärd, Andrew G. McArthur, John R. Yates 3rd, Animesh Ray, and Frances D. Gillin: “Unraveling the role of Giardia basal bodies in differentiation through proteome, transcriptome and interactome analyses” Submitted.

• These two authors contributed equally to the paper All published papers were reproduced with permission from the publisher.

TABLE OF CONTENTS 1 INTRODUCTION

1

2 THE DISEASE

2

3 THE PARASITE

3

3.1 Plasma membrane proteins

3

3.2 Cytoskeleton 3.2.1 Flagella 3.2.2 Basal bodies 3.2.3 Ventral disk 3.2.4 Median body

4 5 5 6 6

4 DIFFERENTIATION

7

4.1 Encystation 4.1.1 Encystation stimuli 4.1.2 Cyst wall biogenesis

7 8 8

4.2 Cyst 4.3 Excystation

9 9

5 CELL CYCLE

10

5.1 Growth

10

5.2 Ploidy

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6 COMPUTATIONAL BIOLOGY

12

6.1 Genome

12

6.2 Transcriptome

13

6.3 Proteomics

14

6.4 Interolog network

15

7 AIMS OF THE PRESENT STUDY

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7.1 Paper I

17

7.2 Paper II

18

7.3 Paper III

21

7.4 Paper IV

23

8 CONCLUSIONS

26

9 ACKNOWLEDGEMENTS

28

10 REFERENCES

31

LIST OF ABBREVIATIONS [Ca2+]i

intracellular calcium

2-DE

two-Dimensional gel Electrophoresis

BLAST

Basic Local Alignment Search Tool

CaM

CalModulin

CWP

Cyst Wall Protein

ERK1

Extracellular signal-Regulated Kinase 1

ESV

Encystation-Specific Vesicle(s)

GO

Gene Ontology

HCMp

High Cysteine Membrane protein

HCNCp

High Cysteine Non-variant Cyst protein

MALDI

Matrix-Assisted Laser Desorption/Ionization

MTOC

MicroTubule Organizing Center

MudPIT

Multidimensional Protein Identification Technology

NCBI

National Center for Biotechnology Information

NIH

National Institutes of Health, Bethesda, Maryland USA

PFR

ParaFlagellar dense Rod

PP2A

Protein Phosphatase 2A

QPCR

real-time Quantitative PCR

SAGE

Serial Analysis of Gene Expression

SALP1

Striated fiber Assemblin-Like Protein 1

SERCA

Sarco Endoplasmic Reticulum Ca2+ATPase

TG

Thapsigargin

TMHMM

TransMembrane Hidden Markov Model software

VSP

Variant Surface Protein(s)

1

INTRODUCTION

My first exposure to parasites was in Seoul, South Korea. Or at least that is what I told my interviewers when applying for a technical position working with Giardia lamblia and Entamoeba histolytica in the Laboratory of Parasite Growth and Differentiation at the NIH. After teaching a year of conversational English I had just returned to the United States with a sensitive and testy gut, months after an undiagnosed intestinal infection. This phenomenom, I now know is called irritable bowel syndrome, and is seen after viral, bacterial and parasitic outbreaks [1, 2]. Although I knew better, I had accepted (for social reasons), a gift of food from a student, which I promptly consumed in front of the class. Now 33 years later, I still vividly remember, how sick and ugly my very own “intestinal outbreak” from that innocent gift was. Looking back now, it is quite clear that one incident determined my research future. Looking back, I wouldn’t want it any other way. A majority of the diseases of the developing world, specifically, viruses, bacteria and parasites are diseases transmitted in the air, food and water, not the diseases of overconsumption and inactivity seen in the “developed world”. Besides poverty, these diseases exist for the most part, because of our inability to effectively counteract the countless “tricks and treats” of infectious organisms. By the time I gladly accepted a once-in-a-lifetime opportunity, to study in the Department of Tumor and Infection Biology, I was already familiar with some infectious diseases, but did not understand at all the connection to tumor biology. I have since (hopefully) learned, that the ability to differentiate, is what makes tumors and microbes, dangerous in the first place. They use the cell cycle (growth) to increase population density, and stack the odds in their favor, for continuing transmission and re-infection. Like a lazy relative, that visits but never leaves (without persuasion), they are masters of their own microuniverses, by fulfilling their needs, with minimal cellular effort. Intestinal parasites like Entamoeba and Giardia, are environmental survival specialists, equally adept in a cool mountain stream or the warm-dark-moist-anaerobic shelter of an unsuspecting host. The only barrier between the outside world and the interior of the human body, is a single epithelial cell layer [1]. By differentiating, Giardia thrives in this forbidding milieu, because of its unique morphology and mastery at exiting and re-entering its own cell cycle. This thesis examines specific aspects of Giardia’s transduction, survival and responses of known host stimuli, and proposes a network model of the cellular controller of those responses.

Dr. Louis Diamond, Section Chief of the Laboratory of Parasitic Diseases, from 1959-1994. Developed TYI-S-33 medium used growing Giardia, Entamoeba, Trichomonas and Spironucleus. Was the first of my two interviewers.

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2

THE DISEASE

The parasitic protozoan Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is a leading cause of waterborne diarrhea worldwide [3]. Foodborne infections are less common. Fecal contamination in nursing homes and day care centers also leads to infection. In young children, acute giardiasis is responsible for rapid electrolyte loss, while chronic diarrhea causes malabsorption, often resulting in failure to thrive. Acute disease typically begins 1-2 weeks after infection and approximately half of the people infected with Giardia remain asymptomatic. Giardia‘s pathophysiology is poorly understood, as it does not invade or cause much inflammation. Typically infection with enteric pathogens induces the expression of chemokines and cytokines in the intestinal epithelium of the host. But a gene expression study analyzing several cytokines in human intestinal cells after a 5 hours infection with Giardia lamblia [1], did not reveal high levels of (IL)-8 cytokine release, which usually reflects inflammation during bacterial infections [2]. The low levels of (IL)-8 seen could help explain the known lack of inflammation, or alternatively, Giardia actively downregulates the inflammatory response [3]. Although many diseases mechanisms have been proposed, no conventional toxin or virulence factor has been identified. Therefore understanding the mechanisms of attachment, growth, colonization and differentiation is vitally important.

2

3

THE PARASITE

The parasite Giardia lamblia has two distinct and specialized forms, the trophozoite and the cyst. The trophozoite has a characteristic half pear-shaped body, 12–15 mm long and 5–9 mm wide with four pairs of flagella which exit the parasite at specific positions [4]. After staining, it presents as a face, with the nuclei being the eyes, the anterior flagellar rods the eyebrows and the median body appearing as a “frown. The prominent feature of the ventral surface is the attachment disk, used for anchoring to substrates, both natural and artificial. The infectious form of Giardia is the environmentally hardy cyst, with only 10 cysts required to transmit the disease, as first demonstrated in a Texas prison “volunteer” population in 1954 [5]. After ingestion and exposure to stomach acid, cysts are triggered to differentiate or excyst after passage into the small intestine. The emergent parasite or “excyzoite” quickly attaches to the host epithelium, using the specialized ventral disk in order to resist peristaltic elimination. Subsets of trophozoite populations exposed to specific upper intestinal factors are induced to encyst. These “encyzoites” develop novel organelles, the encystation-specific vesicles (ESV), which carry the components for forming the wall of the infectious cyst thus completing the lifecycle. Giardia lamblia has been suggested to comprise a species complex, with seven morphologically identical but genetically distinct assemblages. The role of zoonotic transmission is still uncertain, as is the connection between the severity of infection and different assemblages [6]. Giardia lamblia is the only species recovered from humans, and although apparently identical in morphology, there is a large genetic divergence, leading to the proposition that Giardia may be a species complex. The genotypes isolated include the original A and B assemblages, detected in humans (and hence potentially zoonotic), the other assemblages are: C and D (dogs), E (hoofed livestock), F (cats), G (rats and mice) [7]. 3.1

PLASMA MEMBRANE PROTEINS

The plasma membrane is a semipermeable lipid bilayer interface important for cell signaling and as an attachment point for the intracellular cytoskeleton. Giardia’s specialized plasma membrane interacts with the host environment and is crucial as it is the site of feeding (endocytosis), waste removal (exocytosis) and immune defense. A single layer of surface antigens coats the trophozoite and the ability to vary these coats is necessary for survival. This cellular defense mechanism is called “antigenic variation” and it involves gene activation and silencing to produce switching among the members of a multigene surface protein family [8], the variant surface proteins (VSP), which switch spontaneously and can be selected by immune pressure or physiologic conditions. Giardia is able to flourish in the proteolytic alkaline and bile-rich upper intestine because of the VSP that form a dense single molecule layer over the entire trophozoite surface, including the flagella. A striking feature of many protist variant surface antigens, including those of Giardia [9], Tetrahymena [10], and the parasitic fungus Pneumocystis carinii [11], is the conserved periodicity of cysteine residues. Giardia VSP molecular weights vary between 20 to 200 kDa, with an estimated repertoire of between 235 to 275 genes [12]. All are extremely cysteine-rich (about 12%) Type 1 integral membrane proteins with multiple CXXC motifs, and a C-terminal membrane-spanning region followed by the absolutely conserved CRGKA motif. All have a characteristic extended polyadenylation signal, and an N-terminal signal peptide (14-17 amino acids) for ER 3

transport [13]. The majority of cysteine residues are in intrachain bridges, and this internal crosslinking accounts for the resistance of Giardia to harsh host digestive conditions [14]. Only one VSP is expressed on the trophozoite surface at any one time except during VSP switching, which occurs every 6.5–13 generations [15]. The initially expressed VSP is gradually lost and replaced after 12–36 h [16]. Switching also occurs during differentiation (encystation–excystation) [17], offering an additional role in immune protection. Epigenetic mechanisms are now known to be responsible for VSP gene transcription activation, instead of special DNA rearrangements [18, 19]. Very few other membrane proteins have been identified in Giardia, but at least 278 proteins are predicted to have a signal sequence using SignalP [20] and at least one membrane spanning region using TMHMM (www.cbs.dtu.dk/services/TMHMM) (Reiner, Svard, Gillin unpublished). The heavily disulfide-bonded membrane proteins of Giardia provide a dual protective role against both environmental and immune challenges, while anchoring the parasites’ cytoskeleton. 3.2

CYTOSKELETON

Giardia’s cytoskeleton is a dynamic cellular "scaffolding" that is required for multiple processes, including nuclear division, cytokinesis, maintaining cell shape, motility, flagellar movement, and intracellular transport [21]. During mitosis, a big challenge is to correctly segregate the two nuclei and multiple cytoskeletal components. The parasite cytoskeleton resists membrane leakage and structural collapse [22], and is composed of both classic cytoskeletal (microtubules and microfilaments) and Giardia-specific organelles and proteins [23]. There are five types of microtubular structures: the mitotic spindle, flagella/axonemes, ventral disc, median body and funis. The first two are universal cytoskeletal organelles, but the ventral attachment disk, median body and funis (described below) are Giardiaspecific. The Giardia cytoskeletal proteins giardins [24, 25], belong to at least three separate gene families: the a-giardins or annexins [26, 27], b-giardin, a striated fibre assemblin (SFA) homolog [28] and g-giardin, a protein without notable homologs [29]. The presence of 21 annexin homologs in such a simple eukaryote is surprising and emphasizes their functional importance to the parasite. Annexins in other organisms are calcium (Ca2+) dependent phospholipid-binding proteins, which interact with structural cell-membrane components, occasionally acting as atypical Ca2+ channels [30]. In Giardia, they have been localized to flagella during growth and presumably excystation, and are close to the cyst wall during encystation, and are highly expressed proteins [31]. b-giardin assembles into 2.5 nm filaments that further assemble into the superstructures of the ventral ribbons of the ventral disk [32]. Extensive searches of the Giardia genome failed to identify actin-associated proteins, microfilament-specific motor protein family or myosin [12]. The main microtubule organizing center (MTOC) of cells, and nucleating center for flagella is the basal body which would be expected to play a central role in the organization of Giardia’s complex cytoskeleton and eight flagella. Giardia’s complex cytoskeleton undergoes dramatic re-arrangements during both differentiations, and it is important to understand, which signaling proteins localize to the cytoskeleton. Once these proteins are identified, their specific function can be examined.

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3.2.1

Flagella

Eukaryotic flagella are motile organelles built on a framework of nine microtubule fibers surrounding two central microtubule fibers. The “9 + 2” axoneme, selectively incorporates specific receptors and ion channels, and is powered by dynein ATPase motors [33]. Beyond simple propulsion, flagella have sensory functions as mechanotransducers and chemoreceptors. These biological “antenna” are capable of a stimulus-response coupling of environmental stimuli into changes of flagellar beat frequency mediated by a rise in intracellular Ca2+ levels ([Ca2+]i ) [34, 35]. Giardia’s eight flagella are covered by the plasma membrane and arrayed in four pairs: the anterior, posterolateral, caudal and ventral, which originate from basal bodies located between the two nuclei [22]. Each flagellum has a characteristic motion, and exits the cell body at a specific location. The intracellular portion of each flagellum is accompanied by a unique paraflagellar dense rod (PFR) while the extracellular portion is membrane-bounded [22]. The kinetoplastid flagellum also contains a paraflagellar rod structure which is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating [36], and has homologs in the Giardia genome (Reiner, Svard, Gillin unpublished). The anterior, posterolateral, and ventral flagella beat with a similar frequency, but with different amplitudes [37]. The caudal flagella have a single set of dorsal and ventral short arrays of microtubules, which run parallel to the caudal flagella from the disc to the tip of the tail called the funis [22, 38, 39]. Giardia’s flagella are involved in all aspects of the parasites lifecyle. During growth they function in attachment [40], detachment, swimming, and feeding [37], They undergo a complex migration and maturation over the course of three cell divisions (18-24 h) [41]. During encystation, flagella become internalized and quiescent [42]. Resumption of flagellar motion during excystation helps enlarge the opening at one pole of the cyst, where excyzoites emerge, posterior end first [43]. Typically flagella are thought of as propulsion organelles, but some could play concurrent roles in environmental monitoring and signal transduction. 3.2.2

Basal bodies

Basal bodies are highly conserved, self-replicating, cylindrical organelles that provide the template for flagellar assembly. One basal body directly nucleates each cilium (flagellum), and is thought to be involved, in cell-cycle progression, morphogenesis, and motility [44]. Giardia’s eight flagella are each nucleated by a basal body [45], each located between and slightly anterior to the two nuclei [46, 47]. Currently three signaling proteins that are required for and also upregulated in excystation have been shown to localize to the basal bodies: calmodulin (CaM) [48], protein kinase A (PKA) [49, 50] and protein phosphatase 2A (PP2A) [51], with aurora kinase localizing to the basal bodies during mitosis [52]. Recent basal body proteomic analyses have identified many proteins in other organisms [53-56], but until now only six other Giardia basal body proteins were known: a, b-, and g-tubulins, centrins 1 and 2, and ERK1 [52, 57-60]. Beyond the traditional role as nucleating centers for flagellar biogenesis, Giardia’s basal bodies may act as signal transduction and control centers during both growth (cell cycle) and differentiation (life cycle).

5

3.2.3

Ventral disk

A unique anatomical feature is Giardia’s prominent ventral attachment disk. There is a strong link between the ventral disk and disease since attachment to host epithelial cells is essential for Giardia’s ability to avoid peristaltic elimination [22, 61]. The ventrolateral flange of the disk interdigitates between microvilli of the small intestine, but is also capable of attaching to inert surfaces [62]. During growth in vitro Giardia alternates between attached and free swimming phases, depending on the competence of the parent or newly assembled disks [63]. Division starts in attached cells by detachment of the disk microtubules from basal bodies, the b-giardin microribbons are lost, and the microtubular layer unfolds, resulting in detachment. Then two daughter discs assemble on the dorsal side of the attached cell during a freeswimming phase. Finally, the daughter cells with fully developed disks, but still connected tail to tail by a cytoplasmic bridge, attach to a substrate and terminate the division by a process resembling adhesion-dependent cytokinesis [63]. a- and btubulin and b-giardin, SALP-1, delta-giardin and aurora kinase all localize to the ventral disk [22, 52]. b-giardin and SALP-1 (striated fiber assemblin-like protein 1) are both immunoreactive in serum from patients with giardiasis. They are also are homologous to the striated fiber assemblins, which are microtubule-associated fibers that emerge from Chlamydomonas reinhardtii basal bodies [64]. During encystation both proteins localize to the four disassembled ventral disk fragments in the cyst, which is rapidly reassembled during excystation, and used by the excyzoite to attach [65]. A central theme for Giardia’s survival during growth and differentiation is defeating peristaltic elimination. 3.2.4

Median body

The median body is a Giardia-specific cytoskeletal organelle, which has been widely used for species classification. Proposed cellular functions of this puzzling organelle include: 1) a microtubule reservoir, 2) ventral disk progenesis, 3) immobilization of microtubules between cell divisions, 4) a microtubule nucleation site and 5) vertical “tail” flexing [66]. By light microscopy, the median body in the trophozoite is shaped like a claw hammer [67], in electron micrographs it is seen as roughly aligned “fascicles” (bundle or cluster) [22] and like clusters of unorganized microtubules in the cyst [68]. Because the presence of median bodies varies between 50-86% in interphasic populations, a role in Giardia’s cell cycle has also been proposed [66, 69]. The Ca2+-binding basal body protein centrin and the ventral disk protein b-giardin, aurora kinase and an uncharacterized coiled-coil 101 kDa median body-specific protein [70], have been localized to this non-membrane bounded organelle [52, 58, 59, 71]. Post-translational modifications of median body-specific tubulin include acetylation, mono and polyglycylation, and tyrosinylation [72-74] have been observed. Whatever its eventual function proves to be, it is quite curious that the median body shares proteins with both the basal body and ventral disk.

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4 DIFFERENTIATION Differentiation in Giardia involves two very different developmental transitions, from the motile, replicative trophozoite to the dormant cyst (encystation), and from the ingested cyst to the emergent excyzoite (excystation), which is accomplished by exiting from and re-entering the parasite’s cell cycle [75-77]. During differentiation there is a regulated switching of gene expression programs in response to host and environmental stimuli. Giardia’s differentiations are among the simplest eukaryotic developmental processes known and are experimentally tractable [78-81]. Equally important, Giardia’s completed in vitro lifecycle is and excellent system for modeling other uncompleted parasite lifecycles and for studies of protozoan differentiation in general. 4.1 ENCYSTATION The cyst is essential for survival of the parasite outside the host [82] and the persistence of endemic infection is due to the transmission of the cyst from host to host through fecal contamination. Encystation is the gradual transformation of the motile trophozoite into the immotile, dormant and refractile cyst in response to host-specific factors [42]. As the trophozoites’ flagella are internalized, the parasite loses the ability to attach [83], the ventral disk fragments [65] and the differentiating encyzoite gradually rounds up and enters into a hypometabolic dormancy. The cyst wall is the environmentally resistant, but actively transducing, biological boundary responsible for Giardia’s survival during dormancy. The highly insoluble nature of the Giardia cyst wall (CW) is due to the strong carbohydrate interchain interactions and cyst wall sugar associations with cyst wall proteins. The wall is synthesized de novo from endogenous glucose via a pathway of inducible enzymes, which are transcriptionally as well as allosterically regulated [82]. The synthesis of cyst wall proteins (CWP) that begins early in encystation leads to the formation of novel large encystation secretory vesicles (ESV) [84-87], which are the CWP export organelles. Late in encystation, after the cyst wall has been laid down, nuclear division occurs producing the four cyst nuclei [84, 88]. Giardial encystation is reminiscent of the sexual process of meiosis [75]. Until recently, Giardia has been considered strictly asexual, but the presence of meiotic genes [12, 89], low levels of allelic heterozygosity [90], and new evidence of recombination from population genetics [91] argues otherwise. Logsdon recently reported that to “unlock sex” in Giardia, we need investigations of genetic exchange at the cellular level [92, 93] and to “catch them in the act” [94]. Recent evidence for this suggests that encystation-specific karyogamy (fusion of nuclei), with subsequent somatic homologous recombination occurs in cysts. This ancestral parasexual process, is coined “diplomixis”, and is presently unique to Giardia, but predicted to occur in other diplomonads [95]. Both pathogenic microbes and parasites are beginning to disclose some shared principles. By limiting genetic exchange, but retaining the sexual or parasexual reproduction machinery, pathogens are able to generate highly clonal, but at the same time, recombining populations. This is a constantly changing balance between clonality versus diversity, and is in direct response to host, environmental, or antimicrobial challenges [96]. Encystation therefore is a key virulence and survival mechanism with a fine-tuned genetic response to host stimuli.

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4.1.1 Encystation stimuli It was long observed that there is a relationship between bile secretion and the number of colonizing trophozoites [97], and when a low concentration of bile is added to parasite culture medium, trophozoite growth increases dramatically [98, 99]. We hypothesized that mimicing the human upper intestinal environment would trigger encystation, and we were the first to observe encystation in vitro after adding high concentrations of bile at the physiologic pH 7.8 [78, 80]. Cyst formation has alternatively, been induced in vitro by starving trophozoites of cholesterol. The physical state of the bile salt molecules in solution (monomers or micelles) was suggested as the stimulus of encystation [100]. When physiological levels of bile are reduced in mice with giardiasis, using either surgical cholestasis or feeding a diet of bile-binding resins (cholestyramine), significant reductions (103-104) in fecal excretion of Giardia cysts are observed [101]. Whether encystation is induced by high amounts of bile directly or by cholesterol deprivation, or whether “It is likely that the former induces the latter” [102], there is still no identified molecular mechanism for induction of Giardia encystation. 4.1.2 Cyst wall biogenesis Giardia’s cyst wall is the single structure that protects and preserves the parasite during dormancy. It is a model immunoprotective extracellular matrix excluding small molecules like water while efficiently transmitting host stimuli. The fibrillar extracellular matrix is lined by a double inner membrane and composed of an outer 300 nm thick filamentous wall [103], which is 43% carbohydrates [104], at least 86% of which is a b(1–3)-N-acetyl-D-galactosamine homopolymer [82]. The cyst wall filaments measure 7–20 nm in diameter and are arranged in a tightly packed meshwork [105]. The wall is assembled during encystation by the encyzoite using an elaborate CWP secretory system [85, 86, 106, 107]. There are currently four known CWP’s, three of which are leucine-rich repeat-containing proteins with positionally conserved cysteine residues, while the fourth resembles trophozoite variant surface proteins (VSP) [108-111]. All form extensive intermolecular disulfide bonded complexes, are sorted, concentrated and exported to the nascent CW by encystation-specific vesicle (ESV), but their supramolecular architecture is incompletely understood. CWP are either synthesized in a dilated ER cisternae region known as the cleft [87, 88, 106], which gradually widens to form the novel (ESV), or ER vesicles containing CWP use each other to form the ESV [85, 112]. The importance of the ESV cargo is supported by our finding that chemically reducing these complexes in situ with DTT reversibly disrupts the ESV [113], transforming them to flattened ER-like cisternae [114]. The ESV contents must attain their insoluble architecture after secretion [115]. Several enzymes, mainly localized to the ESV are needed for post-translational modifications of the CWPs that have been implicated in CW formation. These include three protein disulfide isomerases [116], a lysosomal cysteine proteinase [117] and a Ca2+-binding granule-specific protein (gGSP). Knockdown of gGSP inhibits ESV release, suggesting a Ca2+-dependent process [118]. Thus, a number of independent studies show that the ESV are central to CW biogenesis, since any manipulation that interferes with the ESV pathway, blocks all downstream events. Despite cellular differences between the human intestinal parasites Entameba histolytica and Giardia lamblia, and the opportunistic free-living soil amoeba Acanthamoeba castellani, recent morphological studies support the idea that 8

ESV’s may correspond to a common mechanism of synthesis, transport, and deposition of cyst wall components [119]. 4.2 CYST Giardia lamblia cysts are nonmotile and oval shaped with refractile walls. They measure 8-12 mm long by 7-10 mm wide and are secreted in the feces. The first ultrastructural observation of Giardia cysts revealed a thin layer of cytoplasm separating the wall from the cyst periphery, with four nuclei and basal bodies in between and ribbon-like microtubule structures from disassembled attachment disks and flagella [120]. Environmental communication is essential for cyst survival and even after being dormant for months in water, they are poised for rapid awakening upon ingestion. Proteins required for excystation are upregulated during encystation, and stored within the cyst body. The parasite emerges in or near the duodenal region after passage through the stomach in a regulated awakening from dormancy. The cysts’ re-entry into the cell cycle requires accurate transduction of host signals and timing of cellular re-assembly. This differentiation, called excystation is a crucial, but incompletely understood, aspect of the giardal lifecycle. 4.3 EXCYSTATION Excystation is a dramatic awakening from dormancy that is required to initiate infection. excystation in vitro involves at least a two-step process that is initiated by exposing cysts to “conditions closely approximating the organism's in vivo environment", an acidic environment, [121, 122] followed by the slightly alkaline and proteolytic conditions mimicking the duodenal-jejunal region [81, 123, 124]. Excystation is a rapid process [125], the cyst is first triggered to excyst by host stomach acids or activation (5-10 minutes), and then passes into the small intestine before rupturing (20-60 seconds). During the rapid emergence (70%) excystation, implicating Ca2+ homeostasis as an important factor for excystation. We hypothesized that the greatest effects of Ca2+ perturbations would be during cellular activation that occurs late in excystation during emergence, when the excyzoite becomes motile and goes through rapid cytokinesis. To test that hypothesis, we used specific inhibitors of the cellular machinery required for maintaining Ca2+ homeostasis. We first lowered [Ca2+]i levels using the Ca2+channel blocker verapamil , and found strong inhibition (>75%) of excystation, specifically in Stage II. We then raised [Ca2+]i levels by allowing Ca2+ influx from the culture medium, using two different Ca2+ carboxylic ionophores, ionomycin (1 mM) and A23187 (1 mM), we again found the greatest inhibition during Stage II. This confirmed the importance of Ca2+ signaling and homeostasis during differentiation, specifically when the excyzoite is emerging from the cyst during excystation. Ca2+ easily precipitates phosphate, and cells invest much energy to exert control over Ca2+, by either chelating, compartmentalizing, or extruding it, to maintain a 20,000-fold gradient between their intracellular (~100 nM free Ca2+) and extracellular (mM) concentrations. These gradients are maintained by Ca2+ pumps which are Ca2+ ATPases which transfer Ca2+ from the cytosol of the cell to the lumen of the endoplasmic reticulum at the expense of ATP hydrolysis [166]. Cellular control of cytosolic Ca2+ is regulated in other cells by the endoplasmic reticulum (ER) Ca2+ pump called SERCA (Sarco Endoplasmic Reticulum Ca2+ATPase) which plays a vital role in cell growth and differentiation [168]. We hypothesized that Giardia had a SERCA pump which played the same vital role during its growth and differentiation. SERCA pumps are specifically inhibited by the selective and irreversible sesquiterpene lactone thapsigargin (TG) [169]. The lipophilic TG crosses cell membranes and prevents sequestration of Ca2+ from the cytosol, causing a massive secondary influx of extracellular Ca2+ into the cell [170]. Therefore TG is a highly versatile tool for studies of [Ca2+]i levels including inhibition of [Ca2+]i dependent functions and localization of cellular Ca2+ stores. To look at the 17

effects of by the SERCA pump we used Indo 1, a fluorescent Ca2+ dual-emission dye [171]. As Ca2+ progressively increases from 100 to 1000 nM, the peak emission of Indo 1 (a UV-excitable fluorescent Ca2+ sensor) decreases, and shifts to lower wavelengths. It is then possible to derive the [Ca2+]i by measuring the intensity of emission at two wavelengths (405 and 480 nm), and calculating the ratio of intensities at these wavelengths [172]. We added TG (5 mM) to trophozoites, cysts, or excysting cells that were pre-loaded with Indo-1. This immediately produced a sustained rise in fluorescence, corresponding to an increase in free [Ca2+]i . We found that Giardia maintains a low basal cytosolic Ca2+ concentration, ranging from 40 to 85 nM, and that the addition of TG caused significant increases in cytosolic Ca2+ levels. The magnitude of peak Ca2+ TGinduced release increased during excystation and TG (10mM) also inhibited excystation strongly at Stage II. Fluorescently labeled TG was used to identify a likely Ca2+ storage compartment in cysts. This confirms that Ca2+ is stored in the cyst and that [Ca2+]i levels in Giardia are maintained by SERCA pumps throughout growth and differentiation and again emphasizes the importance of Ca2+ signaling during emergence, late in excystation. The professional Ca2+ chelator in proteins is the EF hand domain (named after the E and F regions of parvalbumin). The affinities of EF hand domains for Ca2+ vary ~100,000fold [173]. Ca2+ binding regulates protein function by triggering shape changes in EF hand domains, and altering local electrostatic fields, efficiently amplifying a molecular signal to the size scale of proteins [174]. Calmodulin is the archetypal Ca2+ sensor and Ca2+ adaptor protein [166] and one of the most highly conserved proteins in nature. It was previously known that CaM and PKA were involved in excystation [49, 175], that endogenous CaM is present at a level sufficient to activate cyclic AMP phosphodiesterase [176]. We hypothesized that Giardia’s CaM would play an important role during excystation especially during emergence, because of our previous experiments. We found that three different specific calmodulin inhibitors were indeed effective at blocking excystation, especially during excyzoite emergence. The calculated IC50 values were: chlorpromazine (28 mM), trifluoperazine (15 mM) and calmidazolium (1 mM). We identified Giardia’s CaM homolog (gCaM; GL50803_5333, 2.4e-52) using the genome database and cloned it. When gCaM was expressed with either an N or C-terminal AU-1 epitope tag it specifically localized to the basal bodies in trophozoites and cysts. This is an important finding, because it suggests that the basal bodies are a Ca2+/CaM-regulated cellular control center in Giardia, with a special role in awakening from dormancy (excystation) to re-enter the cell cycle. 7.2 PAPER II Currently there are only three known giardial cyst wall proteins, one of which CWP3 (GL50803_2421) we previously identified using database mining [110]. We hypothesized that more than three CWP would be required to provide the necessary sophistication and accurate signal transduction required during Giardia’s differentiation. We used a bioinformatic approach to identify a novel cyst protein and found that it was part of a larger group of unique cysteine-rich proteins. Paper II describes the bioinformatic, molecular and cellular approaches that were taken to both validate and classify this potential differentiation-specific gene family.

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We first mined the SAGE transcriptome database for expression patterns suggestive of encystation or cyst wall specific genes, and identified one (GL50803_40376) for further functional validation. The encystation-specific SAGE expression profile was confirmed using Northern analysis, and we named the 169, 320 kDa (1609 aa) gene product, HCNCp. HCNCp strongly resembles the 235-275 genes encoding classical giardial VSP protein family: cysteine-rich (13.9%), Type 1 integral membrane protein, multiple CxxC and GGCY motifs, a typical N-terminal cleavage site, a C-terminal membrane spanning region and a short cytoplasmic tail. Distinct from VSP’s, however, HCNCp has a divergent C-terminal transmembrane (TM) region, and an unusual C-terminal amino acid tail, CRRSKAV versus the CRGKA motif found in every VSP. To test the hypothesis that HCNCp would be increasingly expressed on the trophozoite’s membrane during encystation, HCNCp was expressed under its own promoter with a C-terminal AU1 epitope tag for monitoring expression and localization during encystation. In immunoblots similar to the stable mRNA levels, HCNCp (170 kDa) was faintly detected in trophozoites, increasing at 21-42 hours with the highest levels in cysts. There were also apparently physiologically processed C-terminal fragments of 21 and 42 kDa with the same pattern of protein expression. In vegetative trophozoites, HCNCp localizes to the nuclear envelopes/ER. During encystation, HCNCP traffics through ESV’s, but unlike the exclusive cyst wall localization of CWP1-3, HCNCp localizes to the excyzoite cell body. This not only identifies a new cyst protein but also suggests that the cysteine-rich protein families in Giardia may be necessary for environmental survival both during growth and differentiation. If confirmed, this would also be the first excyzoite-specific protein and an excellent vaccine candidate due to its invariant nature. To determine whether HCNCP is a variant protein, we used HCNCp-specific internal PCR primers and amplified genes and mRNA transcripts from 7 different Giardia isolates from Assemblage A including WB C6 subclones, unrelated human isolates, one dog isolate and one isolate from Assemblage B, and detected HCNCp expression in all except the Assemblage B isolate (GS/M). Assemblage A isolates are highly homogenous, while assemblage B isolates are genetically highly variable [6]. This suggests that HCNCp is not a classical VSP, but a new invariant cyst protein. Using custom Perl scripts (McArthur, unpublished) all proteins ≥ 10% cysteine were identified and this high cysteine membrane protein (HCMp) dataset queried with regular expressions modeled after HCNCp; ≥400 amino acids, and ≥ 20 CxxC or CxC motifs minus the CRGKA motif. CXC motifs are rare in VSP’s, but repeated 8 times in HCNCp. TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) [177] was used to identify only those HCNCp-like proteins with a single transmembrane domain. We found an additional 60 large HCMp’s that fulfilled these criteria. HCMp’s are rarely found in other parasites or model organisms with the exception of the intestinal parasite Entamoeba histolytica (6 HCMP’s) and the free-living ciliated protozoan Tetrahymena thermophila (30 HCMp’s), which is capable of switching from commensal to pathogenic modes of survival. If the genome size is considered, based on predicted ORFs, T. thermophila has 5 times fewer HCMp than Giardia. Interestingly, after Paper II was published, a genome survey of the closely related diplomonad Spironucleus salmonicida, which infects cultivated salmon, was reported [178]. The predicted amino acid sequences of 149 Spironucleus genes containing more than 10% cysteine, with multiple CXXC, but no CRGKA and GGCY motifs. Less than 10% of the cysteine-rich proteins in Spironucleus showed highest 19

sequence similarities to Giardia, but were most similar to proteins of the ciliate T. thermophila. This suggests that cysteine-rich proteins in diplomonads vary greatly and that the gene families appear to have expanded independently in the Giardia and Spironucleus lineages [178]. Although most members of the genus are parasites of fish, birds and mice, essentially nothing is known about their virulence mechanisms [178]. Spironucleus vortens has been cultivated in vitro and grows in Giardia culture medium at a remarkably wide range of temperatures (5 –340 C). Cysts isolated from fecal samples of Spironucleus muris have been excysted, but the in vitro lifecyle has not been completed in any Spironucleus genus. The finding that HCMp/HCNCp-like proteins exist in a wider range of diplomonad protozoans, strengthens Giardia’s unique position as model differentiation organism and more importantly, for understanding how to complete lifecycles in other diplomonad protozoans of economic and medical importance. The ability to infer novel motifs that define a putative protein family is not trivial and requires the combination of multiple alignment methods with human pattern-recognition skills [179]. Biological motifs are more than just conserved nucleotide or amino acid strings, but have underlying structural properties among the seemingly diverse consensus sequences. We hypothesized that the 60 HCMp’s identified using the sequence structural “rules” of the cyst wall protein HCNCp (i.e. ≥400 amino acids, ≥ 20 CXXC or CXC motifs minus the CRGKA motif), possess either functionally or spatially equivalent features that can be detected. We first used the open-source probabilistic modeling software HMMER [180]. An HMM (Hidden Markov model) transforms the information contained within multiple sequence alignments into a position-specific scoring system, that is used for sensitive database searching. HMMER (http://hmmer.janelia.org/) software was used to build custom HMM’s, or to use professionally curated HMM’s (Pfam; http://pfam.janelia.org/ [181]). Custom and curated HMM’s were used to search for common themes or subgroups within the giardial HCMp dataset. We found 6 E. histolytica HCMp’s annotated as transmembrane kinases (TMK). The TMK’s have been postulated to be involved in sensing and responding to extracellular stimuli [182] in E. histolytica. We used the non-kinase extracellular domains to build a custom TMK HMM. We used PFAM’ search for HCNCPs resembling VSP (PF03302) and epidermal growth factor (EGF-like) (PF00008) from the PFAM database. A common feature of the EGFlike repeats (39-40 aa) used to build PF0008 is that they are found in the extracellular domain of membrane-bound proteins or in secreted proteins. Together these findings suggest that the HCMp’s have extracellular domains, which are possibly involved in transducing environmental stimuli. MEME (Multiple Em for Motif Elicitation)/MAST (Motif Alignment and Search Tool) (http://meme.sdsc.edu/meme/meme.html) [183, 184] software was then used to look for related groups within the HCMp dataset. MEME /MAST software is especially useful for discovering patterns where little or nothing is known in advance. We found a total of nine groups, with groups 1 and 2 containing most of the HCMp’s (25 proteins), but with diverse motif structure within each group, with HCNCp in group 1. In contrast, the rest of the groups (groups 3-6, TMK-like, VSP-like and EGF-like) were remarkably similar to each other within each classification. The importance and function of these 9 groups are presently undetermined, but suggest distinct roles and functions bases on sequence similarity, with the possibility of other HCNCp’s existing. Proprotein convertase 6B (PC6B) is thought to be involved with the endoproteolytic processing of precursor proteins [185], and contains the repeated cysteine-rich domain: Cx2Cx3Cx2Cx5-7Cx2Cx10-15Cx3C. This domain is thought to involve 20

the stabilization or intracellular localization of an endoproteinase [46]. A regular expression was constructed from the published consensus sequence and interestingly; HCNCp had 4 of these characteristic motifs. The bioinformatic methods and analysis software used are not only freely available, but also extremely useful, in helping to understand what is either presently experimentally intractable or too labor-intensive to pursue otherwise. 7.3 PAPER III The onset of differentiation in many cell types is associated with arrest of the cell cycle, suggesting that cell cycle proteins influence the transition into the differentiated state [75]. Environmental stresses directly affect the various stages of the cell cycle and the checkpoints [186]. Checkpoints are control mechanisms that ensure accurate cell division before progression into the next cell cycle phase, and can exist at every point in the cell cycle. A special checkpoint is called the restriction point, where a shift occurs between proliferative and quiescent states at a specific time in the cell cycle. In other cells this critical release event usually occurs in G1. This allows cells to retain viability by shifting to a minimal metabolism during differentiation or when conditions are suboptimal for growth [187]. In Paper III we describe development and validation of a synchronization method using aphidicolin and the use of that synchronization to determine a differentiation restriction point in Giardia.

Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNA replication. It blocks the cell cycle at early S-phase, and is a specific inhibitor of eukaryotic DNA polymerase A and D. We first determined that 5 mg ml-1 was the minimal dose of aphidicolin needed to induce cell growth arrest in a 24 h exponentially growing population. Flow cytometry analysis was then used to estimate the length of time in each different cell cycle stage. Most untreated trophozoites (70-80%) from an exponentially growing population were in G2/M/cytokinesis, as has previously been reported [76]. Based on flow cytometry analysis of untreated cells and a 6 h generation time (Reiner, unpublished) we estimated the time the cells were in each stage as: G1 (0.7h), S (1.3h) and G2/M/cytokinesis (4h). Successful synchronization is dependent upon release after arrest in G1/S and we did that by replacing the aphidicolin-containing medium with fresh growth medium. We arrested trophozoites for 6 h with 5 mg ml-1 aphidicolin in a stop-release synchronization experiment. After release many cells rapidly (≤ 0.5h) entered S phase and the DNA content increased from 4N (G1) to 8N (G2) between 1-2 h. By 3 h post-release a 4N subpopulation appeared, indicating that cell division had occurred. The relative size of the 4N peak increased until the 4 h time-point and decreased again as the synchronized population re-entered S phase. It is unlikely that aphidicolin caused much cellular damage, since virtually the entire G1-arrested population resumed replication, and synchrony was maintained during one whole cell cycle. The WB-C6 isolate belongs to assemblage A and is the strain used for genomic and transcriptomic studies, but does not infect mice. Assemblage B isolates have been used in an experimental human and [188], animal infection studies, and are found in natural outbreaks [189-191]. So we followed the same protocol using assemblage B (GSM83H7) with the experimentally determined longer 10 h generation time and the same 21

aphidicolin concentration (5 mg ml-1) to synchronize and successfully release from G1/S arrest. This demonstrates the utility of this synchronization method for both major human genotypes. We next wanted to evaluate both synchrony and gene expression in aphidicolin-treated populations, so we bioinformatically identified orthologs of cell cycle genes in Giardia to find candidates for analysis by real-time quantitative PCR (QPCR). We identified the total number of Giardia cell cycle orthologs (Reiner, Svard, Gillin, unpublished data) using reciprocal BLAST (E-value ≤ e-6) best-match analysis of 4 model organism (human, plant, budding yeast and fission yeast). This cell cycle ortholog dataset was then used to identify Giardia orthologs of 2,100 professionally curated transcriptionally regulated cell cycle genes (http://www.cbs.dtu.dk/cellcycle/) from the same model organisms [192]. The authors of this seminal study classified the transcriptionally regulated cell cycle genes into approximately 800 orthologous groups. Strikingly, only 5 orthologous groups of cell cycle genes were periodically expressed in all 4 model organisms; the mitotic cyclins, three groups of histone proteins (H2A, H2B and H4) and subunits of the ribonucleotide-diphosphate reductase (RNR) complex [192]. We identified a total of 217 putative transcriptionally regulated genes in Giardia, but only 8 from the four orthologous core cell cycle groups, since Giardia lacks the RNR pathway [193]. The unidirectional progression from one cell cycle phase to the next is due to the irreversible nature of mitotic cyclin destruction. The transition from G2 phase to mitosis is controlled by cyclin dependent kinase Cdk1 and cyclin B1 [194]. Cyclin B is normally up-regulated in the late G2 stage of the cell cycle, promotes G2/M transition, and is then targeted for ubiquitination and proteasomal degradation during mitosis [195]. We bioinformatically identified and confirmed a Giardia cyclin B ortholog (gCycB; GL50803_3977), using phylogenetic analysis (protein maximum likelihood alignment) comparing it to cyclins in 38 other species. We used glycyl tRNA synthetase (GL50803_9011), which showed constant expression throughout the cell cycle, as an endogenous control for real-time QPCR analysis. By comparing the ratio of gCycB to glycyl tRNA synthetase after release from aphidicolin arrest, we found that gCycB expression gradually increased reaching a maximum at 3 h corresponding to G2, and decreased again with entry into G1/cytokinesis after 4 h. The three putative transcriptionally regulated cell cycle histone genes: H2A (GL50803_14256), H2B (GL50803_121045) and H4 (GL50803_135001 were upregulated early after release, similar to the large number of early S-phase cells, observed at 0.5 and 5.5 h in the flow cytometry analysis. This confirms that periodic cell cycle regulation is conserved in, and is part of, Giardia’s relatively simple cell cycle machinery. Using a comparative bioinformatic approach, we now putatively have a dataset of Giardia’s transcriptionally regulated cell cycle genes. It will be quite informative to see how many of our predictions are correct (or even close), after our synchronized microarray study is completed. (Troell, Reiner, Svard unpublished). Previous studies suggested that commitment to encystation occurs via exit from a putative cell cycle restriction point in G2 [76]. To test the hypothesis that encystation efficiency in a synchronized population should be highest immediately after the restriction point is passed, we used an easily measured marker for encystation, the biosynthesis of ESV’s, the prerequisite for Giardia cyst wall formation. The highest production of ESVs was observed when encystation was initiated approximately 3 h post 22

release from aphidicolin arrest. This corresponds to mid/late G2 phase and suggests that the encystation restriction point is located in the G2 stage of the giardial cell cycle. This is an important result, not only biologically but also technically. First, because it strengthens the initial report that Giardia makes the final decision to either complete its cell cycle or exit into dormancy late in G2 [76]. Secondly, it provides the necessary tools for further analyses of this important differentiation. 7.4 PAPER IV In other cells the basal body is central to the cell cycle and differentiation [42, 48, 49, 57]. In Paper IV we integrate the data obtained using high-throughput organelle and affinity proteomics with genomic and transcriptional data obtained during differentiation using bioinformatic tools to build a basal body interolog network. We tested the hypothesis that the basal bodies are the cellular control center during both differentiations.

Giardia cytoskeletons including basal bodies were isolated from non-synchronized cultures of mostly interphase (2-4% in mitosis) trophozoites by detergent extraction. After sonication and Dnase treatment, the cytoskeletons were pelleted and then separated using sucrose density gradient (20-65%) centrifugation and fractions collected. The fractions with the highest concentration of basal bodies, identified using centrin antibody, were analyzed by MudPIT. Individual peptides were matched to proteins in the Giardia genome. Three independent analyses detected a total of 23,402 peptides, corresponding to 310 proteins (identified by at least two peptides) and 358 proteins by a single peptide signal. Giardia protein homologs were identified (BLASTp; E-value ≤ e-10) and validated using published data. They include: Giardia (7 basal body proteins), centrosome, centriole or basal body proteins from other organisms (133), human spindle proteins (13), flagella/cilia (21 proteins) and proteomic and immunolocalization studies (24 proteins). There were 98 proteins identified without any sequence similarity and considered unique to Giardia. We confirmed that 7 of these Giardia-specific proteins are indeed in the basal body by epitope-tagging and immunolocalization. This validates our purification and analyses. Of these, 3 localized only to the basal bodies while the remainder also localized to flagella and/or paraflagellar rods and the median body. The localization of basal body proteins to these mysterious organelles provides valuable insights into their role in giardial behavior. Post-genomic Giardia biology involves converting genome-scale and high-throughput transcriptional and proteomic data into systems-level insights. But working at this scale requires biologically plausible simplifying assumptions, and a robust verification strategy, especially with a divergent protozoan like Giardia. In network inference models, three broad types of validation are used: experimental, literature-driven curation and computational cross-validation [196]. Due to the paucity of giardial genetic approaches, only the latter is a viable option. But with a completed genome, a differentiation transcriptome and basal body organelle proteome, To understand the functional relationships of Giardia’s basal body proteome, in our further analyses, we 23

included: (1) basal body-enriched proteins identified by ≥ 2 peptides), (2) proteins detected by only single peptides that were cross-validated to other basal body proteomes, (3) immunologically (ELISA or IFA) validated (4) and known Giardia basal body proteins. We first identified human orthologs of these giardial basal body proteins using phylogenomic analysis. The use of functional orthology allowed us to construct a high confidence protein-protein interaction network (613 proteins and 3,023 interaction edges). To generate a higher level organizational model, the interaction edges in the orthologous interactome, were clustered into functional categories followed by manual refinement. The multiple human homologs of each Giardia protein is collapsed into a corresponding single Giardia node, and the overall interaction edges are retained, to generate a Giardia interolog network model (interactome based on functional homologs). The resulting network shows a striking multipartite organization with clusters showing two major levels of organization. At the lower level there are eight protein clusters with high clustering coefficients (61/93 edges among 49 nodes) having strongly correlated GO (gene ontology) functional annotation reminiscent of molecular machines: signal recognition (CaM, Ras GTPases), signal transduction (NEK, CDK [cyclin-dependent kinase], PKA, protein phosphatases), and effector complexes (cytoskeletal motor proteins, and protein biosynthesis apparatus). The higher level of organization represents communication channels or functional interdependencies among specialized clusters. Thus, the interolog model reveals a hitherto unappreciated level of complexity in the function of Giardia basal bodies. The Giardia interolog model is a minimal model, as it includes only those proteins with human homologs, and the absence of specific proteins in the interolog model does not exclude their functional role in the basal body. While the interolog model is static, Giardia is a highly dynamic organism that responds appropriately to a variety of external signals. Both encystation and excystation entail global changes in cell shape, motility, division and metabolism that must be coordinated. To address whether the expression of genes encoding basal body proteins changes during differentiation, we measured their mRNA expression levels during growth, encystation and excystation by SAGE. Forty-seven percent (175/369 genes) of the basal body proteins had a SAGE tag. There were 118 transcripts that significantly changed during differentiation (encystation/excystation) of these 61% were upregulated in encystation and 31% during excystation. When SAGE expression data was overlaid onto the basal body interolog network, the model predicts that during encystation protein biosynthesis is upregulated and during excystation signaling and cytoskeletal genes are upregulated. This hypothetical network prediction is quite satisfying because it is congruent with the biology of differentiation. Specifically, new pathways for cyst wall protein synthesis and transport are induced. We have shown that Giardia basal body signaling proteins are required for and are upregulated in cysts and during excystation. The increase in cytoskeletal proteins reflects their need for cytokinesis and motility during excystation. The model therefore supports the centrality of basal bodies in regulating differentiation. CaM localizes exclusively to the giardial basal bodies (Paper 1). To independently corroborate the interolog model, we used CaM as the bait in an affinity purification experiment. Fifty-four proteins from cellular extracts bound to CaM-conjugated Sepharose in a calcium dependent manner (EGTA elutable), of these, 10 were found in 24

our basal body proteome and validated by comparative proteomic analysis. In support of the interolog model, these 10 proteins represented five major functional groups, with 4 of them known to immunolocalize to Giardia basal bodies. The lack of direct interaction edges between most of the purified proteins and CaM in the interolog model suggests that CaM-binding by these proteins may be indirect. Alternatively, in Giardia these proteins might bind calmodulin through uncharacterized motifs. Ribosomal proteins are especially prominent in basal body proteomic analyses in other organisms and are often considered contaminants. However, the multiple interactions between these proteins and proteins of other functional groups, and our finding that several such proteins co-purify with calmodulin, suggest that they may be legitimate basal body components. The dynamic nature and complex composition of Giardia’s basal bodies captured in the interolog model further strengthens the hypothesis in Paper I suggesting that basal bodies are a communication center regulating Giardia differentiation (Fig. 1). These findings are also consistent with previous inhibitor studies of basal body proteins during excystation. Together these findings demonstrate an unappreciated level of sophistication of basal body signaling during protozoan differentiation and flagellar biogenesis and potentially serve as a generalized model of cellular dormancy and awakening.

Figure 1. The basal body interolog network. The network will be used for hypothesis generation and experimental verification. The functions of the basal bodies during encystation and excystation are predicted using SAGE data. Ongoing experiments will use synchronized populations for isolating basal bodies for proteomics and cell cycle microarrays.

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8 CONCLUSIONS Systems biology studies complex interactions in biological systems, from the perspective of integration rather than reduction. It involves discovering emergent properties that arise from global interactions in a biological system. From the first time I heard about it, this subject has fascinated me, and it has been a true joy during my doctoral studies to help “assemble” a Giardia network model. The challenge has been to acquire enough skills to computationally interpret the biological “wiring” of a supposedly simple cell like Giardia, which in actuality contains an unappreciated depth of complexity. Fortunately, during the last 5 years, there has been an explosion of Giardia-specific “omic” data, and expert collaborators, willing to attempt such an ambitious undertaking. The increasing integration of well-planned empirical research, with the sophistication and speed inherent to in silico research, will continue to provide a deep well to draw from, for future datadriven Giardia research. In this thesis the roles of Ca2+ signaling, homeostasis and transduction during excystation were examined and a cellular control center hypothesized (Paper I), and suggested (Paper IV), to be localized to Giardia’s 8 basal bodies. The role of Ca2+ signaling and the function of the basal bodies throughout the cell cycle remains unexplored, but with a working synchronization method (Paper III), investigations are now feasible. The discovery of a differentiation-specific cysteine-rich protein family (Paper II) at first appeared at first to be “off the beaten” thesis track, but that, interestingly, remains to be demonstrated. We will soon know if there are any cell cycle-specific members of the HCMp family, after the results of the recently completed synchronized microarray experiments are analyzed (Troell, Reiner, Svard, unpublished). At the very least, one novel invariant cyst protein was discovered, which curiously localizes to the cyst body. Further studies are needed, using fluorescently-tagged HCNCp during excystation, to be able to definitively understand if it is truly an excyzoite-specific protein. Other future studies needed concern the role of the HCMp homologs from other species. Interestingly, one of those HCMp containing species Spironucleus salmonicida, is Giardia’s close relatives and can be cultivated at 40 C (Svard, Andersson unpublished), and is an excellent candidate for future in vitro differentiation studies. The understanding of Giardia’s encystation restriction point (Paper III) is in its infancy. We used ESV’s as our marker for encystation, but synchronizing cyst formation, could possibly be used for increasing the presently low levels of excyzoites seen during in vitro excystation. In addition, since excystation itself is a natural synchronization, further studies using flow cytometry could be implemented to determine where excyzoites reenter the cell cycle after awakening from dormancy. The temporal phase of the cell cycle where data is collected from is a key determinant of protein interactions [197]. One drawback of our basal body interolog model (Paper IV) is using interphase trophozoites for the isolating basal bodies. At least one Giardia signaling protein, aurora kinase localizes to the basal bodies during mitosis [52], and analyzing basal bodies from synchronized trophozoites will increase the proteomic sensitivity of other important temporally expressed proteins. Following our initial 26

approach of predicting basal body function during differentiation using SAGE data overlaid onto the basal body network (Paper IV), experiments are in progress, using synchronized cells and Giardia microarrays to globally identify cell cycle-specific proteins at each phase of the Giardia cell cycle (Troell, Reiner, Svard unpublished). We can then use the cell cycle transcriptome data overlaid onto our current model to predict key cell cycle proteins for further verification using QPCR. Equally important as the phase of the cell cycle, is the physical state of the cytoskeleton, in restricting potential interactions in cellular networks [165]. It could be that the tripartite structure seen in the interolog model reflects the interphase localization of basal bodies to the cytoskeleton. Using basal body proteins from synchronized populations at all stages of the cell and lifecycle will be quite informative for understanding cytoskeletal remodeling and for refining our network model. We are also in the process of constructing a Giardia cell cycle network model from microarray data to eventually look at Giardia’s special cell cycles during differentiation. The activation of signal transduction pathways causes transcription factors to induce global expression programs that direct cells along distinct developmental pathways [197]. Another global approach would be to use a genome-wide binding assays that combines chromatin immunoprecipitation (ChIP) with DNA microarray technology [145]. These could be used to look for transcription factors controlling Giardia’s dramatic responses to physiomimetic stimuli. It is hoped that, understanding the relation of the cell cycle to differentiation at a network level, in a “simple” protozoan like Giardia, may shed deeper insights into the mechanisms of cell cycle arrest and awakening from dormancy in other complex eukaryotic cells.

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9 ACKNOWLEDGEMENTS I want to express my deepest gratitude and thankfulness to all those (listed below) who have been there to make this dream a reality, whether they knew it or not: ---------------------------------------------------------------------------------------------------------To My Mentors: Fran – To my other job interviewer--If it’s true “iron sharpens iron”, then I should be a razor blade by now. Too little time and not enough space to express my gratitude for the three decades of hard work, good science and lifelong friendship. Staffan - For initiating, facilitating and making me realize I could do this. For the humor, the enthusiasm, deep creativity and classic Swedish hospitality. I will always be grateful! ---------------------------------------------------------------------------------------------------------To My Teachers: Daniel Burrows- my first science teacher, Ralph Scorpio- taught me the only biochemistry I ever learned. Michael Blum and John Teske- who taught me pathology NIH : Roland Faulkner, Louis Diamond-Thanks for giving a rookie a chance, David KeisterWho put the DK in TY, Eugene Weinbach- biochemist extraordinaire, Michael Gottlieb-never without a smile, Allen Sher, Ted Mattern, Alan Cheever, To my Bioinformatics Instructors Andrew McArthur –someday I’m going to write some Perl || Mitch Sogin-hope this isn’t too folksy? Hilary Morrison-thanks for the genome, Michael Cipriano- my computer hero. Gene Owens, Mihai Pop, John Quackenbush , Gary Owens, Alex Bateman UCSD: To My Giardia Colleagues: Past: Shayne “The Clash” Boucher, Steve Aley-thanks for not telling about New Orleans. Siddhartha Das-who can eat the hottest food and still laugh, Gary “G-Man” MezarrosMr. Ca2+ Present: Barb “Babs” Davids-now I can go to Bronx again, Tineke “BelgianProt” Lauwaet-back to the networks, Charles Davis-who taught me how its done, Herndon “Big Bro” Douglas, Mike Gault, Scott Herdmann, ChooMan, Ken Hirata, Lynette Corbeil, Rachel Schreier, Don Guiney David Bailey, ChooMan, Cassie, Julia and Cynthia. To My People: SouthBay-Too many true believers to mention --I love you all!! Yard 5- “This ones for you!” James Robins-who showed me the way back! To My American Family: Aunt Sylvia & Uncle Mac-who asked me the toughest Giardia questions ever? Rosella Reiner- My lifelong angel Karl & Anna Houmann-- will you still talk to me, even if its not a Danish degree? Lars “RoadTrip” Houmann, Julie R., KeeKee, Cammie, and Peter “McGyver” Houmann- you guys rock! Brian, Margo, Reese, Allie Page- When are we going to check out that new cabin? 28

Thad & Amy, Russ & Jen, Eric & Lisa, Bruce “EasyRider” Hanscom , Ouida Hanscom, Big Al & Carol Mae – Is it warm enough to visit yet? Mike & Chris, -Love you all, even if you are Patriot fans To my Swedish family: Tord and Eva- for your hospitality, graciousness and Midsommer memories Eva, Kalle and Johan–for my very first memories of Uppsala. Thank-you for taking me in so warmly. When you coming back to visit? Anna, Moa and Vera- for my very first memories of Stockholm. Next time the steak will be perfect. Daniel-Thank-you for all the crazy times, for the smiles, the soccer riots, the songs and of course, the science-you truly have the gift of landing on your feet and define for me swedishness. To the Blue Mountain Boys: Steve “Little Buddy” Weider—thanx for getting me my NIH job. Randy”TR7” Gray, Richard “StrongMan” Kramer For getting me thru the tuff years: Sharon Reed- For Lauren and Gail--can never thank you enough. Jim & Annette Neal -for being there thru it all. Ed & Karen Bryan--Ed, so glad I never had to go toe-to-toe with you. Scottie- for that time at Coasters! Terri & Doug “Pushing Tin” Cohee –for getting me off the street and on my feet. Sheryl & Jim “the Captain” Miller- for teaching me how to sail, on more than just the water. Henry & Syrini De Silva-for all the spicy hot food, warm hearts and deep smiles. Leo -for taking care of Lauren & Cynthia -for taking care of me. My California Bike Crew: Mark “Mr.Clean” White, Joseph “MLK” Lewis, and Burt “Barrack” Norris Mark-thanks for the constant support and friendship. We’re going up (:-------------------------------------------------------------------------------------------To the “Bad Boyz of Giardia” (in order of service) 1) Per Hagblom#- One tuff ‘Ol Boy 2) Staffan Svard#- Loves wide-mouth Mickies, Manchester United—prefers Sauce Béarnaise. 3) Daniel Palm#- Olympic torch protester (2004), Hammarby hooligan and Béarnaise sauce aficionado Katarina Roxstrom-Lindquist*, Rolf “G2/M” Bernander *, Johan “Crosscheck” Ankarklev* Karin “InSynch” Troell*, Hanna Skarin*, Uffe Ribacke*-your finally working with the right parasite. *

Eligible to become members in good standing after: 1) Finishing one tour of duty in San Diego. 2) Valid receipt from either Phil’s BBQ, Bronx Pizza or El Cuervo. 3) Preparing the perfect Béarnaise sauce. -------------------------------------------------------------------------------------------------

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To my parents: Harold and Ellen- Thank-you from the bottom of my heart for never giving up on me. And for the unconditional love- could not even imagine where I would be without it. I love you! To my children: Jeremy, Sonja, Lauren, Alex, Julie otherwise known as: SurfKing, Jimbo, BabyDoll, Dinkman and Cameron Diaz and their other-halves: Matt “Yes sir” Anderson and Andrew Strauja Dreams really do come true ---Never give up! I love you all. Big Al –now it’s your turn to shine, never look back. You’re the one they can’t beat! BabyDoll-I am so proud of you and happy for the place you’re at. You are truly an original! -------------------------------To my New York Lady: Gail Ellen “Hanscom” Reiner--From a summer kiss 35 ago-- until now, you were always on my mind, in my heart, and of my soul. Could not have done this without you. All my love forever!

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