Characterization of assemblage specific genes in Giardia intestinalis
Franziska Pietsch
Degree project in biology, Master of science (2 years), 2010 Examensarbete i biologi 45 hp till masterexamen, 2010
Biology Education Centre and Department of Cell and Molecular Biology, Uppsala University
Supervisor: Staffan Svärd
1 Table of Contents
1. INTRODUCTION ... 2
1.1 Key features of Giardia intesinalis trophozoites and cysts ... 2
1.2 Clinical impact, symptoms and pathogenicity of G. intestinalis... 2
1.3 Life cycle of G. intestinalis ... 3
1.4 Genotypes of G. intestinalis and assemblage specific genes... 4
1.5 Aim of this study ... 5
2. RESULTS ... 6
2.1 Selection of assemblage specific genes by bioinformatics analysis ... 6
2. 2 Expression check of the selected assemblage specific sequences and preparation of vector constructs ... 6
2.3 Encystation of trophozoites in different encystation media... 7
2. 4 Transfection of Giardia trophozoites and localization of assemblage specific genes ... 9
3. DISCUSSION ... 14
4. MATERIALS AND METHODS... 19
4.1 Bioinformatics... 19
4.2 Oligonucleotides ... 19
4.3 Expression check... 19
4.4 Preparation of vector systems for localizations studies ... 20
4.4.1 Cloning into expression systems... 20
4.4.2 Mini‐Maxi Preparation ... 20
4.5 Sequencing of plasmids ... 21
4.6 Cell culture ... 21
4.7 DNA extraction ... 21
4.8 RNA isolation ... 22
4.9 Transfection of Giardia trophozoites by electroporation ... 22
4.10 Encystation ... 23
4.11 Watertreatment of cysts ... 23
4.12 Cytoskeleton preparations... 23
4.13 Paraformaldehyde (PFA) and Methanol/Acetone Fixation ... 24
4.14 Localization of specific proteins/Immunostaining of HA‐tag transfectants... 24
5. ACKNOWLEDGMENTS ... 25
6. REFERENCES... 26
7. APPENDIX ... 28
1. INTRODUCTION
1.1 Key features of Giardia intesinalis trophozoites and cysts
The intestinal eukaryotic parasite Giardia intestinalis (also referred to as Giardia lamblia or Giardia duodenalis) is a binucleated flagellate belonging to the order of diplomonadida. It is further classified as a diplomonad, whose members are found in anaerobic or microaerophilic environments
1.
The parasite passes through two life cycle stages: an infectious cyst and a disease causing trophozoite. Cysts are oval shaped and approximately 8‐12 µm long and 7‐10 µm wide. They are able to survive in water and moist environments for several months, due to their strong cyst wall, that protects the cells from hypotonic lysis
2. The major components of the cyst wall are N‐acetylgalactosamine and three cyst wall proteins (CWP1, CWP2, and CWP3)
1,3. The vegetative growing trophozoites are pear‐shaped and measure 12‐15 µm in length and 5‐9 µm in width. Their two nuclei are symmetrically placed on either side of the midline.
Though eukaryotic, Giardia trophozoites lack mitochondria, peroxisomes and a typical Golgi apparatus. The cytoskeleton includes one or two median bodies of unknown function, a ventral adhesive disk and four pairs of flagella (anterior, caudal, posterior, and ventral), which emerge from basal bodies
1,3.
1.2 Clinical impact, symptoms and pathogenicity of G. intestinalis
G. intestinalis is a major cause of diarrheal disease worldwide with approximately 280 million symptomatic human infections (giardiasis) every year
1. The human prevalence rate of giardiasis is 2‐7% in industrial countries, while in most developing countries, 20‐30% of the human population are carriers of giardial parasites
3. Furthermore, G. intestinalis is a potential zoonotic pathogen and in addition to humans, more than 40 mammalian species, including dogs, cats, cattle etc. are susceptible to infections with G. intestinalis
4,5. The parasite spreads via the fecal‐oral route and infections are most often caused by ingestion of contaminated water, where the infectious dose can be as low as ten cysts
3,6. Symptoms of giardiasis appear within one to two weeks after infection and include watery diarrhea, epigastric pain, nausea, vomiting and weight loss, with a stronger clinical impact in small children, undernourished individuals and immunodeficient patients
1,3. Chronic infections are common, particularly in developing countries, where giardiasis is endemic and can result in malabsorption, reduced growth, and developmental retardation in children
7. Persistent infections with G. intestinalis have also been linked to irritable bowel syndrome and arthritis
8
. However, Giardia infections are most often effectively treated with nitroimidazoles,
metronidazole and tinidazole in particular
4. In addition, about half of the infections are
asymptomatic and often resolve spontaneously
1. Thus, the clinical manifestation of
giardiasis is highly variable and its severity dependents on the interplay between virulence of
the parasite, and the developmental, nutritional and immunological status of the host
9.
Although G. intesitinalis is the most studied diplomonad and an important pathogen in both,
developing and industrialized countries, there is still very limited insight into how the
parasite causes disease. G. intestinalis is not invasive, hardly causes mucosal inflammation
and until recently no secreted toxins were known
8,10. Up to now only very few pathogenicity
3
factors have been described in Giardia spp. and the most frequently mentioned virulence factors include (i) the adhesive disk, by which the trophozoites suck to the mucosa of the host’s upper intestine
11, (ii) the four flagella pairs, which serve for motility and attachment, and (iii) the variant‐specific surface proteins (VSPs) that allow antigenic variation and thereby help the parasite to avoid the host’s immune system
12.
1.3 Life cycle of G. intestinalis
The life cycle of G. intestinalis can be divided into a cyst and a trophozoite stage (Figure 1).
The cyst represents the resistant, transmissive form of the parasite. A robust outer cyst wall and two inner membranes enclose the parasite, which at this stage holds four tetraploid nuclei, a fragmented adhesive disc, and disassembled flagella. Cysts can therefore survive outside the host’s intestine for a prolonged period of time and are highly resistant to the environment
1,13. Metabolism in giardial cysts is down regulated, hence placing the parasite into a dormant state. An intestinal infection is initiated with ingestion of non‐motile cysts present in contaminated water or food, or by interpersonal contact
14. While the cyst passes through the acidic milieu of the host’s stomach, excystation is triggered by stomach acids.
The cyst becomes metabolically active and passes on to the upper intestine, where it bursts and gives rise to a short‐lived excyzoite, which initiate the infection. The excyzoite subsequently undergoes a transformation into the disease‐causing trophozoite stage of the parasite, associated with an increase in metabolism and gene expression, segregation of organelles, upregulation of proteins associated with motility and assembly of the ventral adhesive disk. During this transformation procedure, the excyzoite divides twice without DNA replication, hence generating four motile trophozoites within 15 minutes
1,15. The released trophozoites proliferate in the small intestine, where they bind to the enterocytes and finally establish an infection
15(Figure 1). To complete its life cycle and be able to be transmitted to yet another host, the parasite has to transform from the trophozoite stage back into the infective cyst form. This long process of cyst formation can be divided into three distinct parts: (i) reception of the stimulus for encystation and the consequent upregulation of encystation‐specific gene expression, (ii) synthesis and intracellular transport of cyst wall components, (iii) assembly of the extracellular cyst wall
14,16. Encystation is triggered by external, host‐specific environmental factors, such as basic pH, high concentration of bile, and low levels of cholesterol encountered when the trophozoites pass on to the lower part of the small intestine
1. Early in encystation, large encystation‐specific vesicles (ESVs) are formed for transportation of cyst wall proteins (CWPs) to build the cyst wall. Furthermore, the eight flagella are internalized and the trophozoite loses its ability to attach to the intestinal epithelium as the adhesive disk fragments into four distinct parts.
The differentiating parasite gradually rounds up and undergoes DNA replication but does not
divide, leading to a cell with two tetraploid nuclei (Figure 1). During late encystation, these
nuclei separate, the DNA is replicated once more, and the cell enters a hypometabolic
dormancy, thus finally generating a cyst containing four tetraploid nuclei
1.
Figure 1: Life cycle of Giardia intestinalis After ingestion of the giardial cysts, excystation is initiated in the acidic environment of the host’s stomach and a short‐lived excyzoite is released. In the subsequent rapid course of differentiation, the excyzoite gives rise to four vegetative trophozoites, which colonize the upper small intestine via attachment to epithelial cells, proliferate and establish an infection. When the parasite migrates further down into the lower part of the intestine, encystation starts by internalization of the flagella and disassembly of the ventral disk; both organelles are subsequently kept in the cytoplasm. Further, synthesis of cyst wall proteins leads to the formation of encystation specific vesicles. While the cyst wall is build, the parasite rounds up and two cycles of DNA replication without cell division finally lead to a cyst with four nuclei.
Cysts are excreted with the feces. Picture adapted from Ankarklev et al. 2010
1.4 Genotypes of G. intestinalis and assemblage specific genes
G. intestinalis, which was previously thought of as a single species, is now described as a group of different variants. This is due to a growing number of studies showing that G.
intestinalis should be regarded as a species complex, whose genetically divergent members show little morphologic variation
9. The data supporting the discrimination of G. intestinalis into distinct genotypes relies on both, genomic and phenotypic analyses. Today eight distinct genotypes (A to H), also called assemblages, have been assigned. This A to H classification is mainly based on genotyping of several enzymes and highly expressed genes (glutamate dehydrogenase, beta‐giardin, elongation factor‐1 alpha, triose phosphate‐isomerase and small sub‐unit rRNA), without considering further genomic differences. In addition, there is a considerable amount of data concerning phenotypic differences, affecting amongst others, metabolism and biochemistry, growth rate (in vivo and in vitro), pH preference, drug sensitivity, and clinical features of different Giardia isolates
9.
Almost all cases of human Giardia infections are associated with assemblage A or B and according to recent epidemiological statistics, assemblage B isolates seem to be most frequent in human infections worldwide
1,9,17. Both assemblages are also found in other mammals
1. The remaining genotypes, grouped into assemblages C to G, are likely to be host‐specific, as assemblages C and D have been identified in dogs, cats, coyotes and wolves, assemblage E in cattle, sheep, goats, pigs, water buffaloes and mouflons, assemblage F in cats, and isolates belonging to assemblages G and H in rats and aquatic animals, respectively
9
.
5
The genomes of three different isolates belonging to assemblage A (WB), B (GS), and E (P15) have been sequenced
18. Using comparative genomics, a well‐conserved core of genes coding for 4557 proteins could be identified for the Giardia species complex. Surprisingly, the average amino acid identity in coding regions of assemblage A and B added up to only 78%. The protein identity between GS and P15 was 81% while WB and P15 shared 90%
identical proteins. The core proteome is estimated to be around 91% of the total genes, coding mainly for structural and housekeeping genes, but there is also significant genome variation between Giardia isolates, leaving room for antigenic variation, host‐specificity functions and possibly mechanisms of assemblage‐specific pathogenicity. Thus, the remaining 9% of the genome include the more variable part of the genome that contains conserved positional orthologs between P15, WB and GS as well as unique assemblage‐
specific genes. Most of those isolate‐specific genes are located in nonsyntenic regions of the genome and are categorized as hypothetical proteins. The majority of assemblage‐specific genes were found in the P15 isolate, namely 35, while in GS 31 unique genes were discovered and only five unique sequences were identified in the WB isolate. For some of the assemblage‐specific genes BLAST searches indicated several conserved regions originally from organisms other than Giardia, but none apart from one unique gene (one P15 specific gene codes for a bacterial acetyl transferase) has a sequence similarity to known sequences and thus their functions remained unknown. As already pointed out in the context of protein identity, assemblage E is more closely related to assemblage A than assemblage A is to B.
P15 and GS share a set of 20 specific genes, while P15 and WB share only seven protein‐
coding genes that are not present in GS, while WB and GS have only one common protein‐
coding gene that is restricted to these two isolates
18.
Although these comparative genomics analyses have revealed differences in genes involved in survival of the parasite during the infection process, and while some of the genomic differences between assemblage A and B can explain a few biological and clinical differences between the two assemblages, still no genetic factors directing host specificity or adaptivity have yet been identified
9.
1.5 Aim of this study
The overall goal of this study was to verify and characterize newly identified assemblage‐
specific genes in the three distinct genotypes A, B and E of Giardia intestinalis. The methods
employed were analyses of gene expression control and localization of specific proteins
during different life stages of the parasite. In addition, the efficiency of different encystation
protocols was tested on the three giardial isolates WB, GS and P15.
2. RESULTS
2.1 Selection of assemblage specific genes by bioinformatics analysis
Earlier work had revealed a set of 102 putative genes predicted to be specific to one or two of the so far sequenced genotypes of G. intestinalis
18. In order to find the most promising candidates for subsequent characterization studies, a bioinformatics analysis was performed of the genotypes A, B and E. In total, 46 of the 102 specific sequences were chosen considering annotations and contig‐integrity (see appendix). Of those, 16 belonged to assemblage B (isolate GS), four to assemblage A (isolate WB) and 26 to assemblage E (isolate P15).
More than 50% of the selected open reading frames (25 ORFs in total) displayed strong hits for domains or signatures and/or existence in other organisms in the BLAST searches performed at the NCBI non‐redundant database. However, all but one of the selected ORFs was annotated as a hypothetical protein. The only exception was the P15‐specific gene, GLP15_874, which was already known to code for a bacterial acetyl transferase. Another three genes from assemblage E (GLP15_2898, GLP15_1606, GLP15_2740) could be grouped together, since they all contained an AAA‐4 domain, had a common ortholog in assemblage A (GL50581_952), and the latter two sequences displayed 91% amino acid identity. We further identified an island consisting of 18 adjacent putative genes in the P15 genome. For most of those sequences, nonsyntenic regions could also be found in the GS genome, but none of them was present in isolate WB. In contrast to at least some of the sequences chosen from the P15 and WB isolates, all 16 genes selected in GS were found to be unique to this assemblage. However, the percentage of sequences predicted to have their origin in an organism other than Giardia was highest for the genes selected from the GS strain.
2. 2 Expression check of the selected assemblage specific sequences and preparation of vector constructs
To determine which of the selected ORFs were expressed in Giardia trophozoites and which were either pseudogenes or silenced in the genome, a gene expression test was made using cDNA synthesized from each of the three different assemblages. The PCR revealed expression of only one ORF out of the four ORFs examined from assemblage A, due to failure of the remaining three primer pairs used. For assemblage B, seven out of 16 ORFs tested could be shown to be expressed. In assemblage E, five ORFs out of 26 tested were found to be expressed (Table 1). The expression level (weak/strong) was judged according to the intensity of the individual bands on the agarose gel. Of the 13 genes expressed, phylogenetic comparisons identified seven genes and suggested that of these four originated from bacteria, one from a protozoan eukaryote, one from a virus, and one from a higher eukaryotic organism. Hence most of the proposed specific genes that were expressed in the studied Giardia trophozoites were most likely taken up from bacteria.
7
Table 1: Expressed unique genes Assemblage specific genes that were found to be expressed in trophozoites of the P15, WB, and GS isolates, respectively. Expression levels are indicated on the outer right. Gray rows mark ORFs that were not used for subsequent localization studies
ORF Annotation Organism with best hit
Domain/ signature Expression cDNA GLP15_874 Acetyltransferase Anaerostipes
caccae
Acetyltransterase GNAT‐superf.
Weak
GLP15_906 Hypothetical Weak
GLP15_907 Hypothetical Weak
GLP15_1606 Hypothetical AAA‐4 domain Strong
GLP15_2740 Hypothetical AAA‐4 domain Strong
GL50803_10192 Hypothetical Collinsella aerofaciens
Weak
GL50581_5657 Hypthetical Weak
GL50581_5676 Hypothetical SMC domain Weak
GL50581_4508 Hypothetical Entamoeba dispar Methyltransferase TRM13 superf.
Weak GL50581_2613 Hypothetical Desulfatibacillum
alkenivorans
Lactamase B superf.
protein
Weak GL50581_3192 Hypothetical Bryantella
formatexigens
4‐
carboxymuconolactone decarboxylase
Strong
GL50581_3333 Replicas‐
associated
Faba bean necrotic yellow virus
Viral replication N‐
terminal domain
Weak GL50581_3321 Hypothetical Mannheimia
succiniciproducens
Conserved hypothetical protein
Weak
In addition, a PCR was run on clinical samples of assemblage A and assemblage B with the primer pairs for the expressed GS specific genes GL50581_5676, GL50581_2613 and GL50581_3192. While the GS positive control could be seen on the gel, no band of the right size appeared in any of the other genome samples (data not shown).
For subsequent localization studies of the expressed genes, vector constructs were prepared in E.coli. For five of the vector constructs the preparation could not be completed successfully, so that a set of nine genes (Table 1) was used for further characterization studies.
2.3 Encystation of trophozoites in different encystation media
In order to perform localization studies not only in the vegetative growth stage of the
trophozoites, but also in the infectious, growth arrested cyst form of the parasites, an
encystation protocol for the assemblages GS and P15 had to be established. Therefore,
different media compositions were prepared. The medium base was supplemented with
either porcine or bovine bile in different concentrations. Further, either lactic acid (5mM), or
CaCl
2(2.5mM), or sodium butyrate (15mM or 30mM) was added to the encystation medium.
It was also tested if encystation was more efficient when the trophozoites were grown in pre‐encystation medium before induction of encystation. Table 2 summarizes the observed encystation efficiencies in wild‐type trophozoites.
Encystation of WB, performed in the standardized way as described below, resulted in a high yield of cysts, though WB cysts could also be acquired in most of the other encystation media tested.
The number of cysts in encysting GS cell cultures was generally lower than in WB. Here, the encystation efficiency was up to 50% encysting cells, if the trophozoites were grown in pre‐
encystation medium before encystation was triggered with encystation medium (containing either lactic acid and bovine bile or calcium and bovine bile). However, the highest cyst yield in GS was obtained by a two step starvation protocol that involved addition of delipidated serum (LPDS) as described below. Hardly any cysts were formed when GS trophozoites were directly incubated in encystation medium supplemented with a high bile concentration and only few cysts were found when the encystation medium was supplemented with butyrate.
P15, which was originally isolated from a piglet, encysted best if encystation medium containing 1.5% to 2% porcine bile was added directly or if the cells were incubated in pre‐
encystation medium first and then exposed to encystation medium containing 1.5% porcine bile and lactic acid. Addition of more than 3% porcine bile to the encystation medium led to cell lysis in all assemblages. Few cysts of P15 could be obtained if encystation was triggered with encystation medium containing porcine bile supplemented with calcium after incubation in pre‐encystation medium. However, the encystation rate in P15 was the lowest and slowest of the three isolates in all media tested. Encystation in P15 took at least 24 hours longer than in WB and GS, since the highest number of P15 cysts were present three days after encystation was induced.
It was observed that the trophozoites of all assemblages seemed to tolerate high concentrations of bile better, when the pH was more basic (pH 7.6 vs. pH 8).
When the integrity of the water‐treated cysts was checked via fluorescence microscopy,
between one third and two thirds of the cysts were stained with the cyst wall antibody and
DAPI. In addition, no staining of the nuclei with propidium iodide, which only penetrates cell
walls of dead cells was seen, thus the cysts displayed features of viable cells.
9
Table 2: Encystation efficiency of different encystation protocols in the isolates WB, GS, and P15. The encystation efficiency was divided into four grates: 1: good, 2: ok, 3: poor, 4: no encystation. Bovine and porcine bile per se were added to encystation medium pH 7.8, supplemented with 10% serum. Prior to addition of delipitaded serum (LPDS) trophozoites were grown in pre‐encystation medium lacking bile O/N (2 step starvation); LPDS was added to plain encystation medium pH 7.8. CaCl2 and lactic acid were added to
encystation medium pH 7.8 supplemented with 2% porcine bile and 10% serum. Sodium butyrate was added to encystation medium pH 7.8 supplemented with 10% serum and 5% bovine bile for assemblages WB and GS or 1.5% porcine bile for assemblage P15.
Bovine bile %
Porcine bile % LPDS %
2 5 7,5 10 15 1,5 2 2,5 3 5 5 8 10
WB 1 1 1 1 3 4 2 2
GS 3 2 3 4 2 1 1
P15 3 4 4 4 4 1 1 3 4 4 4 4
Lactic acid 5mM Sodium butyrate mM
CaCl2 2,5mM
Direct After pre‐encystation 15 30
WB 2 2 2 3 4
GS 2 1 2 3 4
P15 3 2 4 3 3
2. 4 Transfection of Giardia trophozoites and localization of assemblage specific genes
In order to localize the expressed genes in Giardia trophozoites and cysts, trophozoites of each assemblage were transfected with vector constructs carrying each of the nine genes.
After construction each cloned gene was checked for correctness by DNA sequencing. Stable transfectants could only be obtained for three of the constructs, giving one WB transfectant WB_10192 and two GS transfectants; namely GS_5676 and GS_2613. The remaining six constructs were introduced into the trophozoites without selection, hence resulting in transient transfectants.
Transfectants were fixed in both, paraformaldehyde (PFA) and methanol/acetone. During PFA fixation, proteins and DNA are cross linked with very little protein extraction, whereas methanol permeabilizes the cell while precipitating the proteins and might thus lead to withdrawal of proteins soluble in the cytoplasm. Comparison and correlation of the corresponding images of both fixation methods allowed a more precise evaluation of localization and expression level of the various proteins.
WB trophozoites transfected with a plasmid carrying the WB specific gene GL50803_10192 revealed a very variable expression level among the cells. Strong gene expression according to the staining intensity displayed was found in about one out of 40 trophozoites (Figure 2).
Neither the PFA‐fixed nor the methanol/acetone‐fixed cells showed a specific localization
pattern. Instead, the protein localized to the entire cytoplasm in diffuse foci when the
expression level was low and appeared uniformly distributed when the staining reached very
high intensities. The same was true for samples taken of transfected WB cells at different
time points after induction of encystation (data not shown).
Figure 2: Localization of GL50803_10192 in WB trophozoites WB10192 transfectant cells stained with anti‐HA (green). Nuclei stained with DAPI (blue). Cells were fixed in methanol/acetone (A,C) or 4% PFA (B,D) and processed for immunofluorescence. Exposure time A and C 1000 ms, 40‐times magnification. Exposure time B and D 50 ms, 100‐times magnification.
GS_2613 trophozoites were transfected with a plasmid comprising the sequence for a GS unique protein (GL50581_2613) belonging to the lactamase B superfamily. When examined with immunofluorescence microscopy, the cells were stained weakly, but evenly throughout the cytoplasm in a punctuate pattern. In some cells, faint flagellar staining could also be observed (data not shown).
The same dot‐like structure was also exhibited by all three transiently transfected GS trophozoites (GS_4508, GS_3192, GS_3321; data not shown).
GS trophozoites carrying the vector with the coding sequence for GL50581_5676 however displayed a very specific localized staining pattern. This GS‐specific sequence contains a Structural Maintenance of Chromosomes (SMC) domain and was predicted to localize to the nuclei. Unexpectedly the protein was found to localize to the flagellar pores of the anterior flagella and to the rim of the disk between them, thus giving the impression of a
“headphone” structure (Figure 3). As could be seen in the PFA‐stained trohpozoites, some cells displayed staining along the entire rim of the cell body (ventrolateral flange). Intense staining of vesicles throughout the cytoplasm could frequently be observed. It was further noticed that cells displaying a strong staining of the anterior rim also tended to have larger intensely staining foci in the anterior part of the cell.
11
Figure 3: Localization of GL50581_5676 in GS trophozoites GS5676 transfectant cells stained with anti‐HA (green). Nuclei stained with DAPI (blue). Cells were fixed in 4% PFA (A,D and C,F) or methanol/acetone (B,E) and processed for immunofluorescence. A and D shows PFA‐stained wild type trophzoites of the GS isolate (2000 ms exposure time, 100‐times magnification). Exposure time B and E 600 ms, 40‐times magnification.
Exposure time C and F 800 ms, 100‐times magnification.
To estimate if the GS‐specific protein GL50581_5676 was linked to the membrane or to the cytoplasm of the trohpozhoites, the transfectants were demembranated and examined via fluorescence microscopy. As shown in the left panel of Figure 4, the rim was lost and the cytoskeletons displayed staining at the axonemes where the anterior flagella exit the cell, at the rim of the entire cell body, and in diffused foci throughout the cytoplasm. Thus, the protein appeared to be both membrane and cytoskeleton associated.
In order to follow the localization of this GS_5676 protein in encysting cells and cysts, the GS
transfectants were kept in encystation medium and samples were taken at different time
points (six hours, 24 hours, and 30 hours after induction of encystation). After six hours of
encystation, the GL50581_5676 seemed to preferentially localize to the rim along the entire
cell body. After 24 and 30 hours of encystation, the protein was found to give a strong signal
in some cysts and was disassembled in most of the remaining trophzoite‐like cells (Figures 4
B, E, C, F).
Figure 4: Localization of GL50581_5676 in demembranated GS trophozoites and encysting GS transfectants GS_5676 transfectant cells stained with anti‐HA (green). Nuclei stained with DAPI (blue). Cells were fixed in 4%
PFA and processed for immunofluorescence. Exposure time was 1000 ms. A and D cytoskeleton prepared trophozoites, 100‐times magnification, B and E 24 h after initiation of encystation, C and F 30 h after initiation of encystation, both samples 40‐times magnified.
The transient transfected P15 trophozoites were inspected by fluorescence microscopy six hours after transfection. All trophozoites seemed to contain the plasmid and express the inserted protein with a uniform intensity. P15_874 trophozoites, which were transfected with a plasmid carrying an acetyl transferase unique to P15, exhibited a punctuate pattern, in which the dot‐like and circular structures did not seem to appear in the very anterior and posterior part of the cells, but apart from that could be found within the entire cytoplasm. A pronounced staining of the ventral flagella in all P15‐874 transfectants could also be observed (Figure 5, A, D).
A similar appearance was observed in P15_907 transfectants. In these cells however, the overall staining was of a lower intensity, and the dot‐like structures seemed to accumulate more to the posterior part of the trophozoites and the ventral flagella were not as strongly marked by the fluorescent labeling (Figure 5 B, E), indicating a slightly different localization and expression of P15_907 in comparison to P15_874.
P15 trophozoites that were transfected with a vector carrying the P15 specific gene
GLP15_1606, a sequence containing an AAA‐4 domain, displayed similar features to the
other two P15 transfectants. Here, the diffuse foci seemed to be most pronounced, while
the staining of the ventral flagella, as seen in the P15_874 and P15_907, was missing or very
faint in many cells (Figure 5 C, F).
13
Figure 5: Localization of GLP15_874, GLP15_907 and GLP15_1606 in P15 trophozoites P15 transfectant cells stained with anti‐HA (green). Nuclei stained with DAPI (blue). Cells were fixed in 4% PFA 6 h after transfection and processed for immunofluorescence. Exposure time was 1000 ms, magnification was 100‐times. A and D P15_874, B and E P15_907 and C and F P15_1606
3. DISCUSSION
Comparative genomics of the two human Giardia isolates GS and WB and the non‐human isolate P15 resulted in the identification of a set of 102 putative assemblage‐specific genes
18
, of which some might be linked to host specificity and assemblage‐specific symptoms and thus give some insight into how the parasite causes disease. Because very little is known about virulence factors in Giardia the characterization of assemblage‐specific genes might improve understanding of giardial infections.
The aim of this project was to evaluate these putative assemblage‐specific genes, characterize their protein products by localization and thus prepare a basis for subsequent development of diagnostic and epidemiologic tools for giardiasis.
WB is the most studied Giardia isolate and knowledge of its genome is the most complete.
Therefore, the majority of unique sequences were expected to be found in the WB genome.
Surprisingly, the bioinformatics analyses revealed the opposite. In fact, six and seven times as many assemblage specific genes were found in the GS strain and the P15 isolate, respectively
18. There are two possible scenarios to explain this finding: either P15 and GS have picked up more genes during evolution than WB, or has WB lost some of its unique genes. Since WB has been grown in laboratories for more than two decades now, some of the genes linked to in vivo conditions such as those required for host‐pathogen interactions might have been lost during the time of in vitro cultivation. In contrast, the P15 isolate has been grown in the laboratory for a very short time, and thus is expected to reflect closely a wild‐type Giardia isolate
18and it in fact harbors the highest amount of unique genes. On the other hand, seven out of the ten unique genes selected for this study that have been found to be expressed in Giardia trophozoites are indicated to be originally from organisms other than Giardia, first and foremost from bacteria. Furthermore, Giardia spp. have both, bacterial and archaeal features and a significant amount of data suggests that those properties were most likely gained via lateral gene transfer
1,18,19. Hence, uptake of foreign DNA appears to happen occasionally in Giardia spp. and it seems likely that interplay between both trends has led to the unequal amount of unique genes that are found in the different isolates today.
According to the bioinformatics, the set of unique genes can be classified into different
groups: genes with supposed bacterial origin, genes with assumed viral ancestry, genes that
are autonomous, and those that form a gene family. One of those gene families found in P15
consisting of five genes, which had a single ortholog in GS, indicating possible redundancy of
the unique genes. Yet another class of unique genes was found when we identified an island
consisting of 13 adjoined ORFs. Surprisingly, none of these genes could be shown to be
expressed in the trophozoite stage of the parasite. The same was true for about two thirds
of the assemblage‐specific genes whose expression was checked in this study. Since all of
those genes were annotated as hypothetical proteins, some of them might actually be
silenced in the genome or be pseudo‐genes. Those genes could, however, also only be
expressed under certain conditions, such as in encysting cells, in cysts, or during interaction
with epithelial cells. Future expression studies with cDNA obtained during different life cycle
stages or during co‐culturing with Giardia host cells might reveal expression of those unique
genes under particular circumstances.
15
In order to be able to study the unique genes in cysts and encysting cells of all isolates, an efficient encystation protocol was needed for each assemblage. Encystation in Giardia has been studied intensively
14,16,20,21,22. However, the duration of the encystation process and initiation conditions seem to vary a lot between various isolates and laboratories
13and different encystation protocols have to be tested repeatedly to evaluate their efficiency.
Hence, the data of the encystation efficiency of the different encystation media and encystation protocols presented in this study have to be regarded as preliminary observations, because some of the media compositions were only tested once or twice and the cyst yields obtained with the same encystation protocols were variational. The high variation in duration of cyst formation and best induction condition of encystation can partly be explained by spontaneous generation of new variant‐specific surface proteins (VSPs), of which some are resistant to high bile concentrations and a basic pH (the requirements of the standard encystation protocol), while others are very sensitive to these settings, resulting in rupture of the trophozoites. New VSPs are generated about every six to 14 generations
12, hence producing trophozoites that react differently to the same encystation conditions. The variation in susceptibility to the same encystation conditions suggests a distinctive phenotypic difference between the various isolates. The physiological state in the lower small intestine, where encystation is claimed to be induced by host‐specific factors
1is consistent with a high concentration of primary bile salts, fatty acids, and a slightly alkaline pH
20. Besides those factors, bacterial metabolites might take part in the stimulation of encystation. Both, lactic acid and butyrate, which were added to encystation media in this study to test their effect in the context of encystation, are metabolic products of the intestinal microflora in humans
23,24. Lactic acid has previously been shown to induce encystation in WB
20and butyrate was argued to be linked to increased virulence expression in enterohaemorrhagic Escherichia coli
23. Especially addition of lactic acid gave a good cyst yield in the two human isolates and some cysts were also produced in the non‐human P15 strain. Since lactic acid and CaCl
2led to formation of some cysts
13(13) in all three isolates tested, further improvement of an encystation protocol containing one of those substances might be useful.
The last characterization step that was performed on the selected assemblage‐specific genes was an attempt at their localization in Giardia trophozoites and cysts. Unfortunately only three transfectants were stable in these experiments and the remaining proteins were only be localized in transiently transfected trophozoites. All ten genes tested were introduced only into their original assemblage.
It is known that circular transfected plasmid DNA is maintained as multimeric episomes in the WB isolate
25. In contrast, circular plasmids introduced into the GS isolate are integrated into the genome by homologous recombination, resulting in the insertion of the transfected DNA
25. This phenomenon might reflect differences in the structure or recognition of DNA replication origins between the WB and GS isolate and analogical assemblage A and B
25,26. However, it is not known if circular transfected plasmid DNA is integrated or is maintained as an episome in the P15 isolate, since GS and WB have been the only two strains studied in any molecular detail up to date
9.
The only gene of the WB isolate that was found to be expressed in WB trophozoites
(GL50803_10192) has an ortholog in GS. It is not present in the non‐human isolate P15,
hence it could possibly play a role in human‐specific infection of the giardial parasite
18.
BLAST searches indicated a strong relation to the actinobacterium Collinsella aerofaciens,
which resides in the human intestine, suggesting the possibility that the gene may have been taken up by horizontal transfer in the gut.
There was a strong disparity between fluorescence intensity among the transfected WB trophozoites and cysts (Figure 2). This difference suggested the possibility that either the copy number of the plasmid varied between the cells or that expression of this gene was regulated in some way. One possibility is that the gene is only expressed in cells that are triggered somehow, while it is silenced or down regulated in most of the other cells.
Cells expressing the GS‐specific genes GL50581_5676 and GL50581_2613, respectively were stable in transfection and could easily be grown in vivo. However, cells transfected with the vector carrying the GS_2613 gene had a much slower growth rate than the other stable transfectants. The reason for this is unknown but, because GS_2613 carries a domain of the lactamase B superfamily, it could be speculated that it might be a gene whose expression is regulated by environmental factors. The staining pattern displayed by GS_2613 transfected cells was non‐specific and low, suggesting a low expression level of the transfected GS_2613 protein in the trophozoites.
The GS_5676 protein contains a domain of Structural Maintenance of Chromosomes (SMC).
SMC proteins mainly function in chromosome organization and GS_5676 was thus expected to localize to the nuclei. Surprisingly it instead located in the anterior rim of the trophozoites and also accumulated at the flagellar pores, where the anterior flagella leave the cell body.
Those two structures are located close to the ventral adhesive disk of the parasite and very likely to be in tight contact to the epithelial cells of the host, when the trophozoites colonize the small intestine and attach to the mucosa. Thus GS_5676 might play a role in interaction with and attachment to the host’s cells. In addition, the staining of GS_5676 protein displayed lots of dot‐like structures throughout the cytoplasm, which might be vesicle organelles transporting the protein to the anterior part of the cell. In encysting cells (six h after induction of encystation), GS_5676 was found to localize along the whole disk and along the whole cell body. A similar pattern has been noted in trophozoites overexpressing the giardial ornithine carbamoyl transferase (OCT)
27. After 24 h and 30 h of encystation, the GS_5676 protein appeared to be disassembled and was highly expressed in some of the cysts, showing a dynamic character (Figure 4 B,E,C,F). In demembranated trophozoites only the axonemes and punctuated structures were still present, while the rim was gone, hence indicating that the GS_5676 protein is both, membrane‐ and cytoskeleton‐associated (Figure 4 A, D).
The remaining six proteins (P15_874, P15_907, P15_1606, GS_3321, GS_3192, GS_4508)
were only localized in transiently transfected trophozoites. All of them exhibited a similar
punctuated staining throughout the entire cell body, independent of whether the
trophozoites belonged to the GS or P15 strain. Staining of the punctuated dot‐like structures
was more pronounced in some transfectants than in others and the staining of the flagella
also differed between the various transfectants (Figure 5). In contrast to the two stable
transfectants WB_10192 and GS_5676, all cells of each transiently transfected cultures
displayed a rather uniform staining intensity (Figure 5). However, when the fluorescence
images of the transiently transfected cells were compared to those of the wild‐type of the
same isolate, that had been exposed to the light for twice as long, the transiently transfected
cells seemed much brighter and more punctuate (GS wt cells Figure 3 A,D; P15 transiently
transfected cells Figure 5).
17
Unfortunately, we were not able to stably introduce the transfection vector carrying one of the P15‐specific genes into P15 trophozoites and even P15 cells transfected with the empty plasmid grew slowly and hardly reached confluence when kept under selection pressure.
This is very different to the situation with WB cells, which grew almost at a normal rate when carrying an empty vector. It would be advisable to test a stepwise increase in the concentration of the selective antibiotic in order to obtain stable transfectants for the P15 isolate. To verify the achievement of the transient transfections, a known, highly expressed protein of the P15 assemblage should be cloned into the same vector and serve as positive control for the transfection. It could further be tested if the transfection vector can be stably integrated into the P15 chromosome and thus lead to a steady expression of the plasmid.
Another approach towards generating stable transfectants in P15 would be to alter the transfection vector, to adapt it to the genetics of P15, for example by exchanging the promoters and the 3’UTRs of the resistance cassette. By doing so, it could be ruled out that the expression of the resistance marker was too low in the transfected cells. Another possibility to explain the inability to stably transfect P15 might be that the slower growth rate of P15 trophozoites relative to those from GS and WB cells prevented the transfected P15 trophozoites to grow up to a sufficient amount of cells, which expressed the plasmid and could withstand the selection pressure. Possibly, a high copy number of the plasmid would be needed in each cell to make sure that the plasmid would not get lost upon cell division and that the transfected culture would be able to establish a resistant colony under selection conditions.
Future localization experiments could also include a co‐localization of the unique proteins with the cyst wall proteins (CWPs) and/or the encystation‐specific vesicle (ESVs) as well as confocal microscopy, which would allow a closer insight into the specific localizations.
With the outline of this study, an overall new approach for development of diagnostic and epidemiologic tools has been suggested and performed. The bioinformatics comparison revealed assemblage specific sequences
17,18, of which some could be shown to be expressed in Giardia trophozoites and some also in giardial cysts. Hence, the expression of the putative genes identified in the comparative genomics has been verified and several of the unique proteins have subsequently been localized in their original isolate. Those unique proteins are promising candidates for use as marker proteins for genotyping in epidemiological studies and in clinical settings. Ideally, candidate genes should be specific to only one assemblage, but present in all isolates of the considered genotype and expressed in all life stages of the parasite.
In order to check the expression of some of the expressed GS‐specific genes (GS_5676, GS_2613, GS_3192) in assemblage B isolates other than GS and rule out the presence of those genes in assemblage A isolates, a PCR using primers of the three assemblage A specific genes mentioned above was ran on gDNA of different clinical Giardia samples belonging to assemblage A and B. Unfortunately, none of the clinical isolates gave a positive PCR result.
That would suggest the absence of these genes in the clinical isolates of both, assemblage A
and B. However, those clinical DNA samples were not from pure cultures, but also contained
a lot of foreign DNA, and a nested PCR should be performed as it is usually done when
clinical samples are examined. Further, polymorphism in the genomes of the various isolates
leads to slight differences in their DNA sequence and the presence of SNPs in the primer
binding sides cannot be ruled out. Hence, the conditions for this PCR were suboptimal.
The next step towards an application of the assemblage specific proteins would be the development of specific antibodies against those proteins, which subsequently could be used to screen patient’s samples quickly and easily. This procedure could also be applied for other species, such as Entamoeba histolytica – E. dispar or Cryptosporidium parvum – C.
enteritis, for example.
Molecular categorization tools have been of great value in understanding the pathogenesis
and host range of Giardia isolates before
3and the identification and certification of
assemblage specific genes has now provided candidates for future studies in diagnostics and
epidemiology and might also reveal factors important for host specificity and specific
pathogenicity.
19
4. MATERIALS AND METHODS
When not indicated otherwise, chemicals and reagents were obtained from Sigma Chemical Co, USA. Cloning reagents were ordered from Fermentas Sweden, Helsingborg, if not stated otherwise.
4.1 Bioinformatics
A list (see appendix) of the assemblage specific genes and genes shared between two assemblages was provided
18and served as starting material for the bioinformatics analyses.
The open reading frames (ORFs) selected for further characterization were chosen considering available annotations including expression data when indicated, predicted features, and possible hits in other organisms, expressing a similar gene. Further selection was based on size and localization of the ORF in the genome and integrity of the contigs. The examinations were performed in GiardiaDB (http://giardiadb.org/giardiadb) and Giardia genomes ACT (PubMed ID: 15976072).
4.2 Oligonucleotides
Primers for expression check were designed in PremierBiosoft (http://www.premierbiosoft.com/netprimer/index.html ) and primers for localization studies were designed in ApE (http://biologylabs.utah.edu/jorgensen/wayned/ape) and RestrictionMapper (http://www.restrictionmapper.org/ ). All primers were ordered from Sigma‐Aldrich (see appendix).
The oligonucleotides were dissolved in ddH
2O to 20 µM. To obtain a 10 µM mix of each primer pair, forward and reverse primer of each ORF were pooled in a ratio of 1:1. Their function was tested on genomic DNA of the relative Giardia genotype.
4.3 Expression check
To check expression of the proposed genes, RNA was isolated from trophozoites of each assemblage and cDNA was generated using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s manual. Each reverse transcription reaction contained approximately 500 ng RNA. Samples were DNase treated using the TURBO DNA‐free Kit (Ambion, Austin, TX) according to the manufacturer’s instruction.
For PCR, 20 ng/µl of DNA, 1 µl of a 10 µM primer pair, and ddH
2O was added to PuReTaq
Ready‐To‐Go beads (GE Healthcare, Little Chalfont, UK) to a reaction volume of 25 µl. The
PCR program was set to 5 min denaturing period at 95°C followed by 30 cycles of 95°C for 30
sec, 56°C for 30 sec, and 72°C for 45 sec. Extension was run for 7 min at 72°C PCR products
were analyzed on a 1.5% agarose gel dissolved in 0.5 x TBE (1 l 10 x stock solution contained 108 g Tris base, 55 g Boric acid and 40 ml 0.5 M EDTA pH 8.0). A 1 kb ladder served for determination of the product sizes.
4.4 Preparation of vector systems for localizations studies 4.4.1 Cloning into expression systems
The vector pPHA‐5‐3 x HA was provided (see appendix).
Genes chosen for localization studies were amplified on genomic DNA of the particular Giardia genotype via PCR. The PCR reaction was set up with Phusion High‐Fidelity DNA Polymerase (Finnzymes, New England BioLabs, Espoo, Finland) according to the manufacturer’s instruction, applying 4 µl of template DNA. The PCR program was set as follows: 3 min denaturing period at 98°C 35 cycles of 15 sec at 98°C, 15 sec at 60°C, 1 min at 72°C, and final extension at 72°C for 10 min. PCR products were verified on and extracted from a 1% agarose gel in 1 x TAE (1 l of a 50 x stock solution contained 242 g Tris base, 57.1 ml acetic acid and 100 ml 0.5 M EDTA). Gel purification was done with the QIAquick Gel Extraction Kit (Qiagen, Sollentuna, Sweden) according to the manufacturer’s manual. The purified samples were double digested with the restriction enzymes MluI and NotI at 37°C for 2 h. Each digest reaction was set to a final volume of 30 µl and contained 200 ng PCR product, 1 µl of each enzyme, 3 µl 10 x FastDigest buffer and ddH
2O. The vector was cleaved with the same reaction mix. After 1 h incubation at 37°C 1 µl FastAP was added to the vector digest and the reaction incubated for another hour at 37°C. The digested products were column purified using the QIAquick PCR Purification Kit (Qiagen).
Ligation was performed with Ready‐To‐Go T4 DNA Ligase beads (Amersham Biosciences, Piscataway, NJ), adding 3 µl of the digested vector and 10 µl of digested insert. The final volume was adjusted with ddH
2O to 20 µl and the solution incubated at RT for 2‐5 h.
The ligation product was transformed into competent E.coli DH5α cells (2 µl ligation product were mixed with 100 µl of E.coli cells) via heat shock for 45 sec at 42°C. Transformed E.coli cells were plated on pre‐warmed LB plates supplemented with 50 µg/ml ampicillin. After O/N incubation at 37°C, 4 single colonies were picked from each plate and grown O/N in 2.5 ml LB medium supplemented with 50 µg/ml ampicillin on a shaker at 37°C. Plasmids were purified from 1.5 ml of O/N culture using the NucleoSpin Plasmid Kit (Macherey‐Nagel, Duren, Germany), resulting in a DNA concentration of approximately 200 ng/µl.
To check the quality of the plasmids, 5 µl of the purified plasmid were cleaved at RT with 0.5 µl of MluI and 0.5 µl of NotI for 1 h. Digest products were analysed on a 1% agarose gel in 1 x TAE. One positive plasmid sample of each construct was sent for sequencing and stored at ‐20°C.
4.4.2 MiniMaxi Preparation
In order to gain a high yield of plasmid for subsequent transfection of Giardia trophozoites,
big scale O/N cultures of E.coli carrying the desired vector were set up. Therefore, 2 1000 ml
21
