UV irradiation responses in Giardia intestinalis

ARTICLE IN PRESS Experimental Parasitology ■■ (2015) ■■–■■

Contents lists available at ScienceDirect

Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

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UV irradiation responses in Giardia intestinalis Q1 Elin Einarsson a, Staffan Svärd a, Karin Troell b,* a b

Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden Department of Virology, Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden

H I G H L I G H T S

• • • •

UV radiation dose of 2 mJ/cm2 induces DSB in both trophozoites and cysts. Large differences in survival between Giardia cysts, trophozoites and encysting cells. DNA metabolism proteins are differentially expressed after UV treatment. Active DNA replication is linked to repair of UV-induced DNA lesions.

G R A P H I C A L

Vegetative growth

6 7 8 9 10 11

Full length article

M G1 G2 S

A B S T R A C T

RNA sequencing

4N

Trophozoite Active DNA replication

Pre-cyst Active DNA replication

Mature cyst No active replication

8N 2 mJ/cm2

Flow cytometry Differentiation

1 2 3 4 5

10 mJ/cm2

Phosphorylated histones

Pre-Cyst

Excystation Excyzoite Cyst 16N

Microscopy

50 mJ/cm2

100 mJ/cm2

DNA replication

24

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

A R T I C L E

I N F O

Article history: Received 23 December 2014 Received in revised form 20 March 2015 Accepted 22 March 2015 Available online Keywords: UV stress Giardia Trophozoites Cysts Inactivation DNA replication

A B S T R A C T

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm2 kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm2 and 50 mJ/cm2 respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water. © 2015 Elsevier Inc. All rights reserved.

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1. Introduction Giardia intestinalis, a flagellated unicellular parasite, infects the small intestine and cause watery diarrhea. It infects both humans and livestock and certain genetic variants of the parasite are

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* Corresponding author. Fax +18309162. E-mail address: [email protected] (K. Troell).

considered to have zoonotic potential (Ryan and Caccio, 2013). The parasite is spread worldwide and regarded as the most common cause of protozoan diarrhea (Caccio and Sprong, 2011). Giardia is mainly transmitted via water and food and these transmission routes result in both sporadic cases and outbreaks. Giardia has been associated with numerous waterborne outbreaks over the last decades (Karanis et al., 2007; Marshall et al., 1997). Over 30% of the reported outbreaks were associated with drinking water systems contaminated, or presumably contaminated, with Giardia (Karanis

http://dx.doi.org/10.1016/j.exppara.2015.03.024 0014-4894/© 2015 Elsevier Inc. All rights reserved.

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et al., 2007). For drinking water outbreaks, deficiencies in water treatment processes are the most cited reasons (Karanis et al., 2007). Giardia has two stages in its life cycle to meet changes in the surrounding environment. The trophozoite is the replicating, diseasecausing stage and is found in the upper small intestine. The cyst is the infectious, dormant life form and spread of the parasite into the environment occurs through feces of humans and animals (Ankarklev et al., 2010). The cysts have a lower metabolic rate than the trophozoites (Paget et al., 1998) and are resistant to the surrounding environment, being able to survive for several weeks in cold water outside the host. The cysts are commonly found in raw sewage, wastewater and surface waters (Budu-Amoako et al., 2012a, 2012b; Lobo et al., 2009; Plutzer et al., 2008). Cysts are formed during the passage through the lower intestinal tract. During this process, encystation, the trophozoite is encapsulated in a cyst-wall and undergoes endoreplication, giving rise to a cell with four nuclei and sixteen genome copies (Bernander et al., 2001). The resulting cyst is ready to meet a hostile environment and to quickly colonize, once ingested by a new host. The parasite will meet many environmental factors once it is discharged from the host, ultraviolet (UV) light being one. This stress is present both in the environment through sun light as well UV radiation commonly used to treat drinking water. Ultraviolet disinfection has been introduced at many water and wastewater treatment plants as a microorganism reduction method (Hijnen et al., 2006). The use of UV has proven effective especially for waterborne protozoan parasites such as Giardia (Linden et al., 2002) and Cryptosporidium (Shin et al., 2001). The latter being less sensitive to chemical treatments with for example chlorine (Betancourt and Rose, 2004). Due to its maximum absorbance at 260 nm, DNA is considered the primary target of UV radiation (Rastogi et al., 2010). UV radiation is divided into the UV-C (240– 290 nm), UV-B (290–320 nm) and UV-A (320–400 nm) regions of which UV-C is the commonly used wavelength in water treatment due to its harmful effects on microorganisms. UV-C radiation of microorganisms may cause double strand breakage (DBS) or form dimers between adjacent bases in the DNA, the latter being the most prevalent photoreaction resulting from UV-C (Cadet et al., 2005). The type of DNA lesions that are induced is dependent on the wavelength of UV, the DNA sequence, and protein–DNA interactions. The biological effects of damage depend on the type of lesion induced, its genomic location and the developmental state of the injured cell. Formation of base dimers interferes with important cellular functions like DNA replication and transcription. This type of damage may be lethal but the level of sensitivity to UV radiation is highly species-specific. Many organisms have developed multiple strategies to avoid, or repair DNA lesions. If the lesions are repaired correctly, the DNA is restored to its original state and, after some delay, the cell proceeds in the cell cycle. Bacterial tolerance to UV exposure as well as mechanisms to repair DNA damage are well described (Hader and Sinha, 2005). In contrast, many studies have showed effective inactivation of cysts and oocysts (Craik et al., 2001; Hijnen et al., 2006; Linden et al., 2002). However, studies on Giardia inactivation have given conflicting results. Inactivation of the infectious cyst stage has been reported (Linden et al., 2002), while Li et al. (2008) showed that Giardia trophozoites can survive exposure to UV radiation up to 10 mJ/cm2. In recent years several studies have been performed studying transcriptional changes during different stress conditions in Giardia (for example DTT, drugs, differentiation, host-interaction and oxidative stress) (Birkeland et al., 2010; Morf et al., 2010; Muller et al., 2008; Raj et al., 2014; Ringqvist et al., 2011; Spycher et al., 2013). However, this is the first report on transcriptional profiling on Giardia after UV irradiation. Determining the changes in gene expression in both life stages of this parasite during UV exposure will help to understand the general stress response as well as the specific response to a naturally found stressor encountered by the parasite in the environment.

The objective of the present study was to explore the responses when Giardia is exposed to UV irradiation. We investigated the inactivation effect of UV radiation on Giardia in growing trophozoites, encysting cells and cysts by measuring the level of reactivation of radiated cells. RNA sequencing was used to identify genes participating in DNA damage response as a consequence of UV irradiation. Inactivation and reactivation was measured using in vitro excystation, flow cytometry and detecting DNA lesions by staining of phosphorylated histone H2AX. Our data show that there are minor transcriptional changes in Giardia due to UV radiation and that replicating cells recover better than dormant cysts after UV treatment. 2. Materials and methods 2.1. Reagents and cell cultivation All reagents were obtained from Sigma Chemical Co unless indicated otherwise. Giardia intestinalis trophozoites of strain WB clone C6 (ATCC no. 50803) were cultivated in TYI-S-33 medium with a pH of 7.0 prepared as in Jerlstrom-Hultqvist et al. (2010). To induce encystation a slightly modified high bile protocol was used (Kane et al., 1991). In brief, the growth medium was removed from cultures that were 70–80% confluent and encystation media with pH 7.8 containing 1.25 mg/ml of bovine bile was added. In vitro generated cysts were harvested 30 hours post-induction of encystation by centrifugation and kept at 4 °C in water for a minimum of 48 hours prior to experiments. 2.2. UV irradiation and post-UV cell viability Trophozoites were grown in UV transparent 15 ml polypropylene tubes (Sarstedt, cat. No. 62.554.502) and when the culture reached approximately 20% confluence the medium was removed and the cells were placed in a UV-crosslinker (UVC 500, Amersham Biosciences). The trophozoites were UV treated at irradiation doses 2, 5, 10, 20, 50 and 100 mJ/cm2 of 254 nm UV-C and the tubes were rotated 180° in order to irradiate most cells i.e. the tubes were irradiated twice. The cells were radiated for 10 s each time, i.e. totally 20 s per sample. After irradiation, pre-warmed freshly made TYIS-33 medium was added to the cells immediately. The confluence of the cultures was evaluated in terms of confluence 48 hours posttreatment and scored in intervals; + up to 20%, ++ 21–70%, +++ 71– 100%. The viability of irradiated water treated cysts was carried out by excystation followed by monitoring if the excyzoites were able to leave the cysts and if they were able to establish a culture of trophozoites. The same procedure was carried out for the cysts generated from UV treated encysting cultures (12 and 22 hours post induction). The cysts were kept in distilled water at 4 °C for a minimum of 2 days prior excystation as described in Boucher and Gillin (1990). The cultures were monitored daily and evaluated in terms of confluence. The confluence at day 7 post-irradiation was scored using the same intervals as for trophozoites (see discussion earlier). One week post-treatment all cultures with no detectable live cells were considered non-viable. Untreated cysts were used as controls to make sure that the excystation itself was successful and the cysts were regarded as non-viable if they were not able to establish a culture in the same time span as the controls. For RNA seq the irradiation dose of 2 mJ/cm2 was selected for both trophozoites and water resistant cysts. Trophozoites were grown to 80% confluency and thereafter UV treated at the selected dose as described earlier. The cells were allowed to recover for 3 hours in TYI-S-33 at 37 °C prior RNA extraction or fixation on slides for histone H2A staining. The cysts suspended in water were placed in the middle of a Petri dish and irradiated at the same settings as the

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trophozoites. The cysts were collected and kept in water at 4° for 3 hours to recover prior RNA extraction, fixation on slides for histone H2A staining or excystation. Control trophozoites and cysts without irradiation were exposed to the same handling as treated cells.

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national genomics infrastructure (Uppsala, Sweden). Total polyadenylated RNA was converted into double-stranded cDNA by random hexamer oligonucleotides using TruSeq RNA preparation kit (Illumina Inc.). The libraries were sequenced as 100 nt non pairedend on the Illumina HiSeq 2000 instrument.

2.3. Immunofluorescence of phosphorylated histone H2A 2.6. Transcription analysis To visualize the occurrence of damaged DNA in cells and cysts, an antibody against phosphorylated Ser139 in human histone H2AX (AR-0149, LP BIO) was used. This antibody has earlier been shown to detect DNA damage in Giardia (Hofstetrova et al., 2010). Trophozoites were placed on wells of poly-L-lysine coated slides (ThermoFischer) and allowed to attach before fixation with methanol (at −20 °C for 5 minutes) followed by permeabilization with acetone (at −20 °C for 5 minutes). The slides were then allowed to air dry before 15 μl droplets of PBS were added to the cells for rehydration and then blocked with 5% bovine serum albumin (BSA) dissolved in PBS for 60 minutes. Thereafter the primary antibody detecting phosphorylated Ser139 in human histone H2AX was added as 15 μl droplets in the dilution 1:250 in 2 % BSA/0.1% Triton-X100/PBS for 90 minutes. After washing the wells six times with PBS, the secondary antibody anti-rabbit FITC (Sigma) diluted 1:200 in 2% BSA/ 0.1% Triton-X100/PBS was added and left to incubate for 60 minutes. Thereafter the washing procedure was repeated and the slides were mounted in Vectashield containing DAPI (Cat. no. H1-100, Vector Laboratories). The slides were viewed in a Zeiss Axioplan 2 fluorescence microscope and images were processed using software AxioVision LE release 4.8.2.0. For each condition, the affected (one or multiple nuclei stained) and unaffected cells and cysts were calculated for at least 200 cells or cysts. 2.4. Flow cytometry To determine the effect of UV irradiation on cell cycle progression we used an asynchronous cell population, irradiated at 2 mJ/ cm2. The cell-cycle progression was monitored using flow cytometry. Flow cytometry analysis of trophozoites was performed after fixation and staining according to Reiner et al., 2008. Briefly, cells harvested at 80% confluence were concentrated by centrifugation at 900 × g for 5 min. The pellet was re-suspended in 50 μl TYI-S33 culture medium and mixed with 150 μl cell fixative (1% Triton X-100, 40 mM citric acid, 20 mM dibasic sodium phosphate and 200 mM sucrose, pH 3.0). After fixation at room temperature for 5 min, 350 μl diluent buffer (125 mM MgCl2 in PBS, pH 7.4) was added and the samples were stored at 4 °C until use. Fixed cells were concentrated by centrifugation at 900 × g for 3 min and washed once in PBS. After pelleting by centrifugation, cells were re-suspended in 500 μl PBS containing 2.5 μg RNase A (Boehringer Mannheim, Cat. No. 109 16) and incubated for 30 min at 37 °C. RNase-treated cells were concentrated by centrifugation and re-suspended in 75 μl Tris– MgCl2 and 75 μl solution containing mithramycin A and ethidium bromide (Skarstad et al., 1996). The samples were analyzed in an A40 Analyzer Flow Cytometer (Apogee flow systems, Hemel Hempstead, UK) counting between 2000 and 10,000 cells in each sample.

We aligned reads against the reference genome sequence using the BWA algorithm (version 0.6) (Li and Durbin, 2010) under default settings. The resulting alignments were filtered to remove lowquality mappings (map score <15). The cuffdiff program from the cufflinks package (version 1.3) (Roberts et al., 2011) was used to compute normalized expression values and determine significant expression changes against the available reference annotations for all our sample comparisons, correcting for multi-mapped reads (-u) and sequence biases (-b). Genome sequences and annotations were obtained from GiardiaDB (release 2.4) (Aurrecoechea et al., 2009). All data can be found in Supplementary Files S1 and S2. Genes with a FPKM value below 10 were considered to be non-significantly expressed and were therefore excluded from Supplementary Tables S1–S4 in Supplementary File S2. A large number of small genes encoding short peptides were also removed from Supplementary Tables S1–S4 due to the uncertainty of true expression of these hypothetical proteins. This is due to that they are only found in the genome of WB and the majority of them are present in highly variable regions in the genome and/or on scaffold ends. The DAVID algorithm (da Huang et al., 2009) was used to detect enriched functional groups within the differentially expressed genes in UV irradiated trophozoites and cysts as described in Faso et al. (2013). All clusters with enrichment score ≥1.3 are shown in Supplementary File S3. 2.7. Real time RT-PCR analysis Real-time RT-PCR was used to verify RNA seq data using cDNA as template generated from the samples used for RNA seq as well as two additional biological replicates. Total RNA was isolated and purified followed by DNase treated as previously described. cDNA synthesis was performed using hexamer oligos (Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit) according to manufacturer’s instructions. Relative gene expression of genes was performed using SYBR Green (Thermo Scientific Maxima SYBR Green/ ROX qPCR master mix) in an Applied Biosystems 7500 Sequence Detection system according to manufacturer’s instructions (Applied Biosystems Inc, Foster City, CA). Primers were designed to amplify four up-regulated genes along with three down-regulated histones, see Supplementary File S2, Table S6. All samples were run in quadruplicates and relative quantification was calculated using the ΔΔCt method as in Pfaffl (2001). Microsoft Excel was used for all calculations. 3. Results 3.1. Viability and DNA damage after UV irradiation

2.5. RNA purification and RNA sequencing Cells radiated with 2 mJ/cm2 were collected for RNA sequencing as well as controls. Only trophozoites and water resistant cysts were included, i.e. not pre-cysts. Total RNA was isolated using a standard TRIzol protocol and a homogenizer (Ultra-Turrax, IKA) was used to disrupt cells and cysts. After purification the samples were DNaseI (Thermo Fisher) treated. The concentration and the integrity of the purified RNA were analyzed on Agilent 2100 Bioanalyzer (Agilent) using the Eukaryote Total RNA Nano assay prior to library preparation and RNA sequencing at the SNP/SEQ facility of the SciLifeLab

Giardia intestinalis cysts are exposed to UV light during the transmission from one host to another in the environment. UV treatment is also being used to inactivate Giardia cysts in drinking water (Hijnen et al., 2006; Linden et al., 2002), whereas trophozoites only grow in the intestine of the infected host devoid of any UV exposure (Ankarklev et al., 2010). In order to study the cellular and molecular effects of UV treatment on Giardia we exposed trophozoites (strain WB-C6), encysting cells (12 and 22 hrs postinduction of encystation) and mature cysts produced in vitro to different UV-C radiation doses (see Section 2). Viability of cells after

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Table 1 Growth and establishment of in vitro cultures after exposure to UV irradiation.

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UV dose (mJ/cm2)

0

2

5

10

20

50

100

4 5 6 7

Trophozoites 12 hr encystinga 22 hr encystinga Mature cystsa

+++ +++ +++ +++

++ ++ ++ +

++ ++ ++ +

++ ++ ++ −b

+ ++ ++ −

+ ++ ++ −

+ N.D N.D −

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Q5

The cell cultures were monitored in regard of confluence and scored in intervals; + up to 20%, ++ 21–70%, +++ 71–100%. – denotes no living cells. a Cysts was excysted and in vitro cultures were subsequently established. b Excyzoites were visible after the excystation process but were unable to establish a culture.

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UV treatment was measured by growth (trophozoites) or excystation followed by growth (irradiated encysting cells and cysts). Trophozoites survived UV treatment up to 100 mJ/cm2 (Table 1) but a slight reduction in growth rate was seen already at 2 mJ/cm2, in line with an earlier study showing a lag phase in growth after UV treatment at 1 mJ/cm2 (Li et al., 2008). Cysts were unable to establish a trophozoite culture after an UV exposure of 10 mJ/cm2, even though excyzoites were present after completed excystation. No excystation of in vitro generated cysts was seen after treatment exceeding 20 mJ/cm2 (Table 1). This shows that trophozoites are more resistant to UV treatment than cyst but the growth rate is affected already at low levels of UV irradiation. The classical nucleotide excision repair (NER) proteins cannot be found in the Giardia genome (Franzen et al., 2009; Jerlstrom-Hultqvist et al., 2010; Morrison et al., 2007). The NER system removes UV lesions efficiently in cells that are not replicating their genome (i.e. G1 and G2 or non-cycling cells) (Novarina et al., 2011). Repair of UV-induced DNA damage has been linked to active DNA

replication in other eukaryotes, especially when NER is not active, via the post replication repair process and proteins that are shared between DNA replication and UV damage repair (Crevel et al., 2012; Novarina et al., 2011). It is possible that similar mechanisms exists in Giardia. Trophozoites grow vegetatively and replicate the genomic DNA while mature cysts are dormant and do not replicate the DNA (Bernander et al., 2001). Thus, differences in DNA replication activities is one possible explanation to differences in ability to repair UV damage. We first studied if UV treatment at 2 mJ/cm2 has any effect on DNA replication in trophozoites since an effect was seen on cell growth. Flow cytometry analysis showed that trophozoites arrested in the G1 and S phases of the cell cycle after UV treatment at 2 mJ/cm2 (Fig. 1B and C). After 24 hrs most cells were found in S and G2 (Fig. 1D) and normal distribution with a pre-dominant G2 cell population was seen again 48 hrs after UV treatment (data not shown). This UV-induced cell cycle arrest can explain the earlier observed lag phase in growth rate (Table 1). Encysting trophozoites replicate their DNA in the end of encystation (Bernander et al., 2001) and in order to further test the hypothesis of a link to DNA replication we exposed late encysting cells (12 and 22 hrs) to UV light. Encysting cells survived treatment up to 50 mJ/cm2 (or higher) (Table 1) but the excystation frequency was lower for cells exposed to high irradiation levels during encystation and consequently a delay to establish culture was observed (data not shown). In conclusion, both trophozoites and encysting cells are affected by UV treatment but replicating Giardia parasites are more resistant to UV treatment. UV treatment produces oxidized bases and single- and doublestrand breaks (DSB) in the genomic DNA of living organisms (Georgakilas et al., 2013; Rastogi et al., 2010). To study if UV treatment induces DSB in Giardia, which can result in cell growth arrest

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Fig. 1. Flow cytometry analysis of DNA replication in UV treated trophozoites. Trophozoites were treated with 2 mJ/cm2 of UV light and transferred back to growth medium. Non-irradiated trophozoites were used as control (A). Irradiated cells were fixed for flow-cytometry analysis after 3 (B), 6 (C) and 24 hrs (D). Trophozoites show a predominant G2 peak whereas cells 3 and 6 hrs after UV treatment show and increase in the G1 and S peaks. After 24 hrs cells can be found in the S and G2 stages of the cell cycle, indicating restart of DNA replication.

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Fig. 2. Cells stained with an antibody against phosphorylated histone H2A to detect DSB after UV irradiation. Trophozoites were exposed to UV irradiation at 2 mJ/cm2 and allowed to recover for 3 hours prior harvest of cells for immunofluorescence labeling. Very few of the control cells (A) had positive signals and if so, the signal was often present only in one nucleus. In contrast, UV exposed cells were labeled in both nuclei, a marker for DSBs (B). Cysts were placed in Petri dish and treated with the same irradiation dosage as trophozoites prior fixation for immunofluorescence. Positive signal was seen to a greater extent for the irradiated cysts (D) compared to the control cysts (C). Nuclei were stained with DAPI. Scale bars 10 μm.

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and repair, we used an antibody that specifically label phosphorylated Histone H2AX, a marker for DSBs in Giardia genomic DNA (Hofstetrova et al., 2010). Phosphorylation of Histone H2AX was present in 70% of the irradiated (2 mJ/cm2) trophozoites compared to only 11% in the non-irradiated trophozoite control samples. Both nuclei were stained in most irradiated trophozoites, whereas only one of the nuclei was stained in the control cells (Fig. 2), as also observed after drug treatment with aphidicolin by Hofstetrova et al. (2010) cysts were UV treated 30% were positively stained compared to only 5% of the control cysts. The positively stained cysts were often stained in all four nuclei (Fig. 2). This shows that a UV irradiation dose of 2 mJ/cm2 was sufficient to induce DSB in trophozoites and cysts and that both cell types respond by phosphorylating Histone H2AX. The DSBs may be the cause of the cell cycle arrest seen in Fig. 1 and also explains the observed lag phase in culture establishment (Table 1). 3.2. Transcription analysis of UV treated cells Entamoeba histolytica and Cryptosporidium parvum, pathogenic intestinal protozoan parasites, have both been shown to induce gene expression changes following UV irradiation (Weber et al., 2009; Zhang et al., 2012). Here we studied if also G. intestinalis can respond to UV irradiation with gene expression changes. Trophozoites and cysts were exposed to 2 mJ/cm2 of UV irradiation, recovered in growth medium for 3 hours, followed by total RNA extraction and

RNA sequencing. The overall expression analysis revealed few genes with large gene expression differences (5 genes >5-fold up or down) between UV irradiated and control trophozoites and cysts (Supplementary File S1). To study changes in the pattern of gene expression as a response to UV irradiation we selected a cutoff value at ≥1.5-fold change between irradiated and control cells, in line with earlier studies of Cryptosporidium oocysts (Zhang et al., 2012). We found that 108 and 202 genes in trophozoites and cysts, respectively had a significant (≥1.5-fold) differential expression due to UV treatment. In trophozoites, 36 genes were up-regulated and 72 downregulated whereas in cysts 73 genes were up-regulated and 129 down-regulated (Supplementary File S1). Six genes were selected for relative quantification by RT-qPCR to verify the RNA seq data and included two biological replicates of the experiments as well as the cDNA generated from the RNA used for RNA sequencing. We observed a general agreement between the RNA seq and qRT-PCR data when comparing the fold changes in expression (Table 2). We performed a cluster analysis of the RNA seq data in order to identify cellular processes that are changed due to UV treatment. Several stress/heat-shock proteins were up-regulated in radiated trophozoites (Supplementary File S3) but the majority of the 10 most up-regulated genes are hypothetical proteins (Supplementary File S2, Table S1). Several kinesins, motor proteins involved in vesicle transport along microtubules, and Nek kinases were up-regulated (Supplementary File S3). A small set of genes with homology to proteins involved in DNA repair and/or DNA

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Table 2 Validation of RNA seq data by real-time qRT-PCR for selected G. intestinalis genes. The RNA seq data were validated using quantitative real-time RT-PCR using cDNA generated from the sequenced RNA (replicate 1) as well as from two biological replicate experiments. The table shows a comparison of the fold changes of gene expression 3 hours after UV irradiation of trophozoites for RNA seq and two biological replicate experiments. Gene

RNA Replicate 1 Replicate 2 Replicate 3 seq

50803_15148 Chaperonin dnaJ 1.8 50803_15380 CDC8 1.7 50803_16412 Heat-shock protein 1.8 50803_88765 Cytosolic Hsp70 1.5 50803_14256 Histone H2A −2.5 50803_14212 Histone H3 −2.5 50803_135001 Histone H4 −2.0

1.1 1.0 1.1 1.2 −2.5 −2.5 −2.5

1.2 1.3 1.3 1.2 −1.1 −1.4 −2.0

1.0 1.2 1.0 1.2 −1.4 −1.3 −1.1

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replication were slightly (1.2- to 1.7 -fold) up-regulated in trophozoites (Supplementary File S2, Table S5) but they were unchanged or down-regulated in UV treated cysts (Supplementary File S1). It remains to be seen if these proteins have a role in DNA repair in Giardia. Nine out of the ten most down-regulated genes in UV exposed trophozoites were histones, representing all histones except cenH3, which instead is up-regulated (Supplementary File S2, Table S2 and S5). Variable surface-proteins (VSPs) protect the parasite against the adaptive immune system and Giardia isolates have 150–250 VSP genes but only one is present on the cell surface (Ankarklev et al., 2010). In the starting population of trophozoites many VSPs are expressed but several are down-regulated after UV treatment (Supplementary Files S1 and S3). This suggests a connection between VSP expression and DNA repair/replication. Among the genes that were most up-regulated in UV irradiated cysts were found to encode ribosomal RNAs (Supplementary File S2, Table S3). Since only polyA tailed transcripts should be present in the RNA Seq library this is either the result of contaminating cDNAs or a true increase of rRNA polyadenylation in cysts after UV irradiation. The majority of the regulated genes were small, hypothetical proteins without known function (Supplementary File S3). Among the up-regulated genes, there is a putative NADPH oxidoreductase (GL50803_17151) and a msrA-like gene (GL50803_4946) that potentially could be involved with protection against oxidative stress caused by UV irradiation. Several VSP genes are up-regulated in cysts after UV treatment (Supplementary File S1), further strengthening the link between UV treatment and changes in VSP expression. The downregulated genes in cysts are mainly hypothetical genes (Supplementary File S2, Table S4) but also a 5S rRNA gene, proteases and VSP genes (Supplementary File S1).

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4. Discussion Exposure to UV light is a natural stressor that the G. intestinalis cysts encounter outside its host during transmission. It is important to gain knowledge about how the parasite responds to challenges in its natural environment to understand the parasite’s basic biology and to understand how the disease can be limited. Hence, we used 2 mJ/cm2 UV irradiation treatment of cells for transcription analysis of the UV stress response. This dose was chosen based on the observation that all cell types where affected but not killed. Radiated cells were allowed a recovery time (3 hours) after which the RNA was isolated. The chosen recovery time was based on a similar study in E. histolytica where it was shown that the transcriptional response was most evident after 3 hours (Weber et al., 2009). In addition, our chosen recovery time falls in between those used in a study on UV-C treatment on Cryptosporidium oocyst in

which significant transcriptional regulation at both 0.5 hours and 5 hours post-radiation (Zhang et al., 2012) was shown. Our transcriptional analysis showed that 108 genes in trophozoites and 202 genes in cysts changed their expression upon UV-C irradiation more than 1.5-fold and 85 genes showed more than 2-fold changes. We could detect up-regulation of genes coding for heat shock proteins and cell cycle associated genes, this was also confirmed by real time RT-qPCR. Several heat shock proteins are among the most upregulated genes in Giardia upon UV exposure (Supplementary File S1). Earlier studies in human cells, bacteria and eukaryotic microbes have shown that heat shock proteins are induced in response to UV treatment (Fulgentini et al., 2015; Trautinger, 2001). UV radiation of cells can lead to the production of reactive oxygen species (ROS), capable of changing membrane and protein structure (Fulgentini et al., 2015; Trautinger, 2001), and this has been suggested to induce heat shock proteins and chaperones. Hence, the UV radiation could also influence the survival of cells by affecting the protein structure. There was a small up-regulation of selected DNA repair/ replication associated genes in the transcription analysis of radiated trophozoites (Supplementary File S2, Table S5) and we could verify that the trophozoites arrested in the G1/S cell cycle stages using flow cytometry (Fig. 1). Interestingly, all histones except cenH3 were repressed in radiated trophozoites (File S1) and the expression of histones is tightly linked to DNA synthesis during the S-phase of the Giardia cell cycle (Reiner et al., 2008). A down-regulation of histone expression suggests a reduced population of replicating cells and it would imply that the cells are arrested in G1 or early in the S phase due to DNA damage. Eukaryotic cells respond to DNA damage by activating checkpoint pathways to preserve the integrity of the genome (Zhou and Elledge, 2000). It has been shown for human cells that ionizing radiation induces down-regulation of histones on the mRNA level in addition to an inhibition of DNA synthesis (Su et al., 2004). Radiated cells arrested at a checkpoint in G1, which indicate that this checkpoint regulates both histone expression and DNA synthesis (Su et al., 2004). This arrest gives the cells an opportunity to repair the DNA damages induced and continue proliferation and it is possible that something similar happens when Giardia is treated with UV-C. The response to UV-C treatment of three other protozoan parasites, Entamoeba histolytica, Cryptosporidium parvum and Toxoplasma gondii, has recently been published (Ware et al., 2010; Weber et al., 2009; Zhang et al., 2012). In E. histolytica, trophozoites subjected to UV-C treatment were studied using microarrays and the expression of genes encoding Fe—S clusters-containing proteins and DNA repair proteins were reported to be up-regulated, whereas genes associated to the cytoskeleton were repressed (Weber et al., 2009). The mechanism of DNA repair by homologous recombination has also been studied in this parasite by inducing double stranded DNA breaks by UV-C treatment (Lopez-Casamichana et al., 2008). The RAD52 epistasis group of genes was shown to participate in DNA damaged response and the DNA damage was repaired by the homologous recombination pathway (Lopez-Casamichana et al., 2008). This is the major pathway used by lower eukaryotes, including protozoan parasites, to repair double stranded DNA damages (Bhattacharyya et al., 2004). Putative meiosis genes were detected in the genome of G. intestinalis (Ramesh et al., 2005) but much remains to be studied about the mechanism of homologous recombination in this parasite. Further studies of the proteins listed in Table S5 might reveal how Giardia deals with DNA damage. It has been reported that human cells submitted to UV stress in general show low alternation in transcription levels (Rieger and Chu, 2004). This was also observed for E. histolytica (Weber et al., 2009) and C. parvum (Zhang et al., 2012) and now also in this study. The question is if this low alteration in transcription will be reflected on the protein synthesis in the cell.

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We also tested the viability after several different irradiation doses and UV treatment did not kill Giardia trophozoites, but the cysts could not survive irradiation doses that were higher than 5 mJ/cm2. This is in accordance with earlier studies that have shown that G. intestinalis cysts could not survive UV doses of 16 and 40 mJ/cm2 (Linden et al., 2002) and that cysts exposed to even the low dose (1 mJ/cm2) of medium pressure (MP) UV irradiation failed to establish an infection in gerbils (Shin et al., 2010). In contrary, a previous study reported that trophozoites exposed to irradiation doses of 10 mJ/cm2 were able to survive and replicate (Li et al., 2008). This is in accordance to the present study where trophozoites survived tested irradiation doses up to 100 mJ/cm2 and the cysts showed a much higher sensitivity to UV-C treatment. Since we hypothesized that the active replication machinery may aid the cell in escaping cell death due to damages caused by irradiation, we investigated if encysting cultures could survive UV treatment and still be able to excyst after completion of the encystation process. We irradiated encysting cultures at 12 and 22 hours post induction based on knowledge that the DNA replication takes place approximately 15–20 hours in our encysting cultures (Bernander et al., 2001). The cysts resulting from irradiated encysting cultures were able to excyst and establish a culture. In contrast, this was not observed in mature cysts where UV treatment effectively blocked ability to grow. Furthermore, when irradiated cysts (10 mJ/cm2) were put in excystation media we observed excyzoites exiting the cyst. However, these cells did not survive and could not establish a culture. This indicates that the UV treatment did not actually kill the cell but damaged its DNA. This strengthens the hypothesis that the replication machinery is important for survival of this parasite. Mature cysts have, unlike encysting cells, already finished its DNA replication and might not have had an active replication machinery at the time of irradiation. Therefore these cells could not detect and repair the DNA lesions. When the excyzoite later exits the cyst it rapidly needs to undergo two rounds of cytokinesis. The excyzoite cytokinesis takes place without DNA replication, resulting in four trophozoites, each with two 2N nuclei. Hence the excyzoite will arrest in the G2/M checkpoint if the DNA is not intact before cytokinesis and will therefore not proceed in the excystation process. 5. Conclusions To summarize, we analyzed the transcriptome of UV-C exposed Giardia trophozoites and cysts. The cysts are the infectious dormant life form that spreads via contaminated water and UV light is a natural stressor either in the environment or in water treatment processes. Trophozoites were also included in the study to be able to compare the stress response between the cysts and the replicating state of the parasite. The transcription analysis revealed small changes but we could detect an up-regulation of heat shock proteins and selected DNA repair/DNA replication proteins combined with down-regulation of histones in trophozoites. We could also show that an active DNA replication machinery is important for this parasite to survive UV-C. Acknowledgements We thank Marc Höppner and Mannfred Grabherr for their help with the transcription analysis. This project was supported by grants Q2 Q3 from the Swedish Research Council (VR and FORMAS), SVA ReQ4 search Fund and Ivar och Elsa Sandbergs Stipendiefond. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.exppara.2015.03.024.

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