Bactericidal/permeability-increasing (BPI) - like proteins in Giardia intestinalis
Dimitra Peirasmaki
Degree project inapplied biotechnology, Master ofScience (2years), 2013 Examensarbete itillämpad bioteknik 45 hp tillmasterexamen, 2013
Abstract
G. intestinalis is the most studied diplomonad since it is the most common cause of waterborne enteric disease in humans and other mammals. However, there are still genes found in the genome of the parasite that have not been studied yet and could give important information on the how the parasite reacts in certain environments.
The purpose of this project was the study of some unidentified proteins found in the genome of G. intestinalis in order to identify and characterize them, taking into consideration that they might belong to the lipid-binding family proteins (LBP) called bactericidal/permeability proteins (BPI).
The results from this project show promising indications that the BPI-like proteins studied from G. intestinalis could belong to either the BPI or the LBP family of proteins. However, at this point nothing more can be said since there are no results that could prove that these proteins are in fact BPIs. In order to gain this type of information further experiments have to be performed.
Table of Contents
Abstract ... 1
1. Introduction ... 4
1.1 Giardia intestinalis ... 4
1.2 Life cycle of G. intestinalis and transmission of Giardiasis ... 5
1.3 Morphology of G. intestinalis ... 6
1.4 Human bactericidal/permeability-increasing protein ... 7
1.4.1 Structure of human BPI ... 7
1.4.2 The multiple activity of human BPI ... 8
1.4.2.1 Antimicrobial activity ... 8
1.4.2.2 Endotoxin neutralizing activity ... 8
1.4.2.3 Opsonic activity ... 8
1.5 Human lipopolysaccharide binding protein ... 9
1.6 Correlation and differences between BPI and LBP ... 9
1.7 Aim of the project ... 10
2. Materials and Methods ... 11
2.1 Construction of episomal vector for the transfection of Giardia intestinalis .... 11
2.1.1 Selection markers and C-terminal localization tag ... 11
2.1.2 Cloning of genes of interest into the PHA-5 vector ... 11
2.2 Construction of vector for protein purification ... 12
2.2.1 Selection markers and N-terminal localization tag ... 12
2.2.2 Cloning of genes of interest into the pGEX vector ... 12
2.3 DNA extraction from Giardia intestinalis ... 12
2.4 PCR amplification of genes ... 13
2.5 Ligation ... 14
2.6 Transformation of E. coli ... 14
2.7 Plasmid mini-preparation ... 14
2.8 Restriction digestion of DNA and PHA-5 vector (or pGEX vector) ... 14
2.9 Sequencing of cloned plasmids ... 15
2.10 Culture conditions for Giardia intestinalis ... 15
2.11 Plasmid big scale preparation for transfection of Giardia intestinalis ... 15
2.12 Transfection of Giardia intestinalis using electroporation ... 16
2.13 SDS-PAGE electrophoresis ... 16
2.14 Western Blotting ... 17
2.15 Fixation ... 18
2.16 Immunofluorescence ... 18
2.17 RNA purification ... 19
2.18 cDNA synthesis ... 19
2.19 RT-qPCR (quantitative Real Time Polymerase Chain Reaction) ... 20
2.19.1 Absolute Quantification (efficiency of reaction) ... 20
2.19.2 Relative Quantification ... 21
2.20 Protein purification ... 21
3. Results ... 23
3.1 Protein comparisons ... 24
3.2 Structural comparison of BPI-like proteins with human BPI and LBP ... 24
3.3 Electroporation of Giardia intestinalis ... 25
3.4 Immunofluorescence of tagged proteins from stable transfectants ... 25
3.5 Expression data of BPI-like proteins during encystation of G. intestinallis ... 28
3.5.1 Western Blots for protein level expression of tagged proteins ... 29
3.6 Interaction experiments between Giardia intestinalis and bacteria ... 29
3.6.1 RT-qPCR for RNA-level protein expression in Giardia intestinalis post interactions ... 30
3.6.2 Western Blots for protein level expression in Giardia intestinalis post interactions ... 32
3.6.3 Immunofluorescence for tagged proteins in G. intestinalis post interactions ... 33
3.6.4 Growth inhibition results for E. coli and B. subtilis post interaction with G. intestinalis... 39
3.7 Protein purification ... 41
3.7.1 SDS-PAGE gels ... 42
3.7.2 Western blot ... 44
4. Discussion... 45
5. References ... 51
6. Acknowledgements ... 54
7. Appendix ... 55
1. Introduction 1.1 Giardia intestinalis
Giardia intestinalis, also known as Giardia lamblia and Giardia duodenalis is a
"teardrop" or "pear" shaped flagellate cosmopolitan protozoan parasite of humans and belongs in the family Hexamitidae (order Diplomonadida) and the genus Giardia (Cavalier-Smith T., 1993; Cavalier-Smith T., 2003) (figure 1).
Figure 1. G. intestinalis evolution tree shows that the parasite belongs to domain: Eukaryota, supergroup: Excavata, clade: Fornicata, phylum: Sarcomastigophora, class: Zoomastigophora, order: Diplomonada, family: Hexamitidae, subfamily: Giardiinae and genus: Giardia (http://tolweb.org; Adam RD. 1991).
Van Leeuwenhoek, the inventor of microscope, was first to see Giardia through a home-made microscope in 1681, but a Czech scientist by the name of Lambl did a more extensive research in 1859 (Ford BJ. 2005).
Since 1859, 51 species of Giardia have been identified, 1 of which has been isolated from fish, 14 from birds, 4 from amphibians, 2 from reptiles, 2 from humans and 28 from other mammals (Luján HD, Svärd SG. 2011). It is clear that the specificity of the host varies among the different Giardia species; for example G. muris infects mainly rats and mice (Wolfe MS. 1992), G. agilis infects amphibians , G. ardeae and G.
psittaci infect birds (Ivanov AI. 2010), while G. intestinalis is one of the ten most important enteric parasites that affect humans worldwide. It is considered to be the most common intestinal pathogenic protozoa of humans, but at the same time it can affect other nonhuman species like beavers, cows, domestic dogs and cats (Ivanov AI.
2010).
DNA sequence analysis has shown that there is a large number of different assemblages (genotypes) in G. intesinalis (from A to H). Briefly, assemblages A and B infect humans, while assemblages C and D infect dogs, E hoofed animals, F cats, G rodents and H seals (Jerlström-Hultqvist J. et al. 2010).
G. intestinalis is responsible for a parasite infection called "giardiasis" (or commonly known as "Beaver Fever") (Amar CF. et al. 2002).
1.2 Life cycle of G. intestinalis and transmission of Giardiasis
Giardiasis is caused by ingesting the parasite G. intestinalis and can be divided into two phases: acute and chronic.
The acute phase is usually a short-lived period and is characterized by flatulence, abdominal distension with cramps and diarrhea, while in chronic giardiasis, malaise, weight loss and other features of malabsorption are very often among the symptoms (Ford BJ, 2005).
G. intestinalis has a simple life cycle (figure 2) which consists of two different stages:
the cyst stage (infective stage), which is basically a
"resting stage", and the trophozoite stage (flagellated form) (Dawson SC. et al.
2010). When Giardia is in the cyst form, which resembles the bacterial endospore, has the ability to survive in hostile environments for really long periods of time. The cysts have been known to survive for months in cold water (Irshad M. et al. 2006).
After a cyst is orally ingested, via contaminated food or water, it excysts in the small intestine of the host, due to the high concentration of bile salts and the acidic environment of the stomach that trigger the excystation, and forms two trophozoites (Thompson RCA. 2008).
Each trophozoite divides by binary fission in the small intestine and is responsible for the symptoms of giardiasis. Some of the trophozoites are induced to encyst while they pass towards colon, where the pH is more basic and the concentration of bile salts lower. The life cycle of the parasite is completed after 72 hours post infection, when the cysts are passed in the feces; that way they can be possibly ingested by another host (Luján HD, Svärd SG. 2011).
Giardiasis can be diagnosed by finding cysts or trophozoites in the feces while nitroimidazoles and benzimidazoles, such as metronidazole (15mg/kg/day for 5 days), tinidazole (50mg/kg for one single dose) and furazolidone (8mg/kg/day for 10 days), are the main drugs used to treat human infections (Adam RD. 1991; Ivanov AI. 2010).
Figure 2. Schematic view showing the life cycle of G.
intestinalis. After the cyst is ingested, it excysts in the small intestine, due to the gastric acid during their passage through the host's stomach which triggers it and forms two trophozoites. The trophozoites attach to the intestinal epithilium with their adhesive disc and divide by binary fission with a generation time of 6-12 hours in vitro. Some of the trophozoites are induced to encyst while they pass towards the lower part of the intestine (Ankarklev J. et al.
2010).
1.3 Morphology of G. intestinalis
Both life cycle stages of G. intestinalis have a very distinct appearance.
The trophozoites have an average length of 10-20 μm and a 5-15 μm width (Touz MC. 2006). They appear in a
“teardrop” shape and they have two nuclei at the anterior end (figure 3). They also have four pairs of flagella which arise from the basal bodies clustered between the two nuclei (Ankarklev J. et al. 2010).
The broad anterior end of the trophozoites contains a concave area which covers half the ventral surface. This area includes the adhesive or
sucking disc that allows the parasite to attach to the surface of the host’s small intestine. The trophozoites also contain an axostyle (the structure at the base of the flagella) (which consists of two axonemes) or dark transverse rod, which may be a supportive element (Elmendorf HG. et al. 2003). There are also two curved median or parabasal bodies cross the axoneme and when it is observed under a microscope at an oblique angle they give the parasite a “smile” (Adam RD. 2001).
The cysts have an average length of 8-19 μm and a 7-10 μm width. They typically have an oval shape and contain 4 nuclei and remnants of the flagella and the axostyle (Touz MC.
2006) (figure 4). The 4 nuclei are usually located on one end (Ankarklev J. et al. 2010).
The cysts have a characteristic, rigid outer cyst wall which is composed of proteins and carbohydrate. This extracellular cyst wall is of an extreme importance for the survival of the cysts and the parasite in general, since it allows the parasite to persevere in fresh water, resist the stomach acidic environment of the host, and "travel" all the way through till the gut where it will excyst (Touz MC. 2006).
Figure 3. Trophozoites have an average length of 15μm and a width of 10μm and have a flattened teardrop shape. (A) DIC microscopy view and (B) vental view of a trophozoite shows:
N=nucleus, vd=vental disc, ba=bare area, afl = anterior flagella, pfl = posterolateraral flagella, cfl = caudal flagella, and vfl = ventral flagella (Dawson SC. et al. 2010).
Figure 4. A typical Giardia cyst has an average length of 8-19 μm and a 7-10 μm width (Touz MC. 2006).
1.4 Human bactericidal/ permeability-increasing protein
The human immune system is the main shield that protects the human body against diseases. The skin and the mucosa create the first lane of defense of the human body against any kind of microbial factors. However, these physical barriers are quite sensitive and as a result susceptible to many forms of injuries, which can make it easy for possible microbial factors to enter and cause infection (Gubern C. et al. 2006;
Schultz H. et. al. 2001). The innate immune system is responsible to rapidly respond to that kind of incursion in order to prevent a further invasion and consequently an infection. This kind of response from the innate immune system includes the phagocytosis by the neutrophils and the macrophages, which also produce nitric oxide (NO) which is toxic for the microbial factors and eventually it kills them (Gubern C.
et al. 2006; Levy O. 2000). It has been shown that the granules of neutrophils produce a large number of antimicrobial proteins which play an important part in the way the innate immune system reacts and how affective it can become. One of these endogenous antimicrobial proteins is a protein called "bactericidal/permeability- increasing protein" (BPI) and constitutes the 0.5-1% of the total protein the neutrophils produce (Elsbach P. 1998; Gubern C. et al. 2006).
1.4.1 Structure of human BPI
The human BPI is a single chain cationic protein with a molecular weight around 55kDa and a boomerang-like shape (figure 5). The BPI consists of 2 domains which are nearly superimposable (Elsbach P. 1998).
Each domain contains a phosphatidylcholane molecule which encourages the belief that the apolar pockets in each domain represent the sites where the BPI binds to lipids and possibly to the lipid A portion of lipopolysaccharides (LPS).
The 2 domains of the protein consist from barrels as well as β-sheet forms. BPI also contains a disulfide bond, necessary for the correct formation of the dimer (Lennartsson A. et al. 2005). The BPI has a high concentration of basic (mainly lysine) residues in the amino-terminal half of the molecule.
Figure 5. (A) Ribbon diagram of human BPI showing its boomerang-like shape. The NH2-terminal domain (light blue) and the COOH-terminal domain (darker blue) are shown, while at the same time is illustrating the two phosphatidylcholane molecules (red) and the disulfide bond (yellow). (B) A 70° rotating view of the (A) diagram is shown. (Beamer LJ. et al. 1997)
1.4.2 The multiple activity of human BPI
The BPI has multiple actions; the N-terminal of the protein is responsible for both the antimicrobial and endotoxin-neutralizing properties of the protein while the C- terminal gives the protein the ability to opsonize Gram-negative bacteria (GNB) (Levy O. 2000).
1.4.2.1 Antimicrobial activity
The BPI possesses a cytotoxic activity against the GNB and that is the reason why it is consider to be a "natural antibiotic". BPI selectively targets GNB due to the fact that the protein has the ability to bind to LPS, a phosphorylated glycolipid, and more specifically to the lipid A portion of the LPS which is a region in polysaccharides of great importance since it is responsible for the endotoxic properties of LPS (Niemetz et al. 1977; Schultz et al. 2007). The outer membrane of the GNB is rich in LPS which makes it easy for the protein to bind. Another factor that contributes to this kind of activity of the protein is the high concentration of basic residues in the N- terminal of the molecule. It is this cationicity of the molecule that is responsible for its targeting against the negatively charged bacterial envelope of the GNB. The positively charged residues that exist in the N-terminal of the BPI bind to the negatively charged LPS disturbing the cations that normally stabilize the outer membrane of the bacteria. Also, it is believed that hydrophobic interactions of the BPI's apolar lipid-binding pockets with the LPS's acyl-chains take place which is considered to contribute to the disruption of the outer membrane of the GNB (Levy O.
2000). It has been shown that the main effects of BPI against the GNB is the inhibition of their growth rate, the increase in the permeability of their membrane, the inhibition of cell division and the activation of bacterial phospholipid (PL) hydrolysis as well, which can be strengthen by the presence of antimicrobial peptides that belong to the cathelicidin and defensin families. In order for BPI to cause a more aggressive result and kill the bacteria, it has to enter their inner membrane (Elsbach P. 1998;
Levy O. 2000).
1.4.2.2 Endotoxin neutralizing activity
BPI, as it was described previously, is notable for its ability to bind with a great specificity to LPS and for that reason it acts as a recognition molecule for the immune system. At the same time it has been shown that when tested in vitro the molecule is able to neutralize the endotoxin (LPS) in different kind of biologic fluids and as a result to decrease the inflammatory effects of LPS and GNB (Levy O. 2000; Schultz et al. 2001).
1.4.2.3 Opsonic activity
The C-terminal of the molecule displays an opsonic activity after it has been observed that when bacteria are exposed to high concentrations of BPI (10-100 nM) are more susceptible to phagocytosis from the neutrophils (Levy O. 2000; Schultz et al. 2001).
1.5 Human lipopolysaccharide-binding protein
The lipopolysaccharide- binding protein (LBP) is a plasma protein that belongs to the family of lipid- binding proteins and is produced by hepatocytes during the acute phase response of the innate immune system and is secreted into the bloodstream (Beamer et al.
1998; Elsbach P. 1998;
Krasity BC. 2011). LBP binds to LPS and after it amplifies the signal of it, delivers it to CD14-LPS receptor which exists on the surface of the macrophages and other cells of the immune system (Beamer et al. 1998; Elsbach P. 1998). That way, LBP plays an important role in the acute mobilization of neutrophils to the infected sites of a tissue. From a structural point of view, LBP demonstrates a boomerang-like shape in the N-terminal of which the LPS-binding site occurs (Gonzalez M. 2007) (Figure 6).
1.6 Correlation and differences between BPI and LBP
Both BPI and LBP are members of a family of lipid-binding proteins and for that reason they share many common characteristics but on the other hand they exhibit some differences too, with the most important to be related to their function.
Both proteins are some of the most important components of the innate immune system since they are involved in the defense against bacterial pathogens. These two proteins share a common architecture and conserved residues, most of which are apolar, something that is related to their ability to bind to LPS. When it comes to structure both proteins have a characteristic boomerang-like shape, where the N- terminal possesses the LPS binding activity, and a conserved disulfide bond. The two proteins share a 45% sequence identity (Gonzalez M. 2007; Lennartsson A. et al.
2005). An alignment of the BPI and LBP sequences has shown an obvious evolutionary relationship between the proteins (Beamer et al. 1998).
Both proteins present a high-affinity for the LPS-binding, however the affinity of LBP is almost 70-fold lower than of BPI, something that can be explained from the fact that there is a higher number of basic residues that occupy the N-terminal in the human BPI than in LBP. That difference in the affinity of the LPS-binding can justify why the LBP does not demonstrates any antimicrobial activity as BPI does (Elsbach P. 1998; Gonzalez M. 2007). Thus, these two proteins have completely opposite functions; LBP is a plasma protein that induces the inflammatory immune response to LPS, whereas BPI neutralizes the toxic effects of LPS (Beamer LJ. et al. 1997;
Figure 6. Prediction of the structure of human LBP (PDB:
P18428) showing its boomerang-like shape. The NH2-terminal domain (blue) and the COOH-terminal domain (yellow- orange).
Beamer LJ. et al. 1998).Finally, human BPI has a higher pI (approximately 9.4) than human LBP (approximately 6.3) (Krasity BC. 2011).
In general and despite the differences that occur between the two proteins it is believed that they share a common ancestor which was a single-domain protein whose gene was duplicated, though the two-domain structure is common (Krasity BC. 2011).
1.7 Aim of the project
The purpose of this project was to study BPI-like proteins found in the genome of Giardia intestinalis. During this project, G. intestinalis was transfected with these BPI-like proteins in order for them to be tested, by performing interaction experiments between G. intestinalis and bacteria, to study their expression levels and how they may change during those kind of interactions and take more general information about these proteins by studying where they localize in the parasite giving us a better view of their purpose for the survival of the parasite and probably of their function in the cell. Since there is the possibility that these proteins could belong to the lipid-binding protein family, the purification of these proteins was essential in order to have a better understanding of their function.
2. Materials and Methods
2.1 Construction of episomal vector for the transfection of Giardia intestinalis
2.1.1 Selection markers and C-terminal localization tag The plasmid that was used
for the transfection of G.
intestinalis is an already existing vector (PHA-5 vector) created from members in the lab and is typically been used for the transformation of this parasite (figure 7).
The vector carries the puromycin N-acetyl- transferase (PAC) gene which has been cloned between the NcoI and XhoI sites and was used as the selection marker during the transfection.
Also, the vector contains a
3xHA tag which was used as a "target" during the localization experiments.
2.1.2 Cloning the genes of interest into the PHA-5 vector
The genes selected for cloning and tagging into the PHA-5 vector were extracted from genomic DNA of G. intestinalis (see Appendix for used primers) and amplified by PCR. The PCR products were analyzed on a 0.7% agarose gel dissolved in 1xTAE buffer which confirmed that their size was the correct one. The products were later purified using the "GeneJET PCR Purification Kit (250)" (Thermo Scientific) and digested with the restriction enzymes MluI and NotI. The digested products were purified once again and ligated into the vector for transformation.
Figure 7. The pPAC-3xHA-C (PHA-5) vector, used for the transfection of G. intestinalis, containing the PAC gene and 3xHA tag.
2.2 Construction of vector for protein purification
2.2.1 Selection markers and N-terminal localization tag The plasmid that was used
for the transformation of E. coli (BL21) is an already existing vector (pGEX vector) bought from GE Heathcare and is typically used for the transformation and expression of protein in E.
coli in order to be purified.
The vector carries the ampicilin (AmpR) gene which was used as the selection marker during the transformation and also, contains a GST tag which was used as a
"target" during the protein purification experiments (figure 8).
Moreover, the vector contains the lac operon which was used for the over-expression of the proteins that were purified. For the induction of the operon Isopropyl-β-D-thio- galactoside (IPTG) was used.
2.2.2 Cloning the genes of interest into the pGEX vector
The genes selected for cloning and tagging into the pGEX vector were extracted from genomic DNA of G. intestinalis (see Appendix for used primers) and amplified by PCR. The PCR products were analyzed on a 0.7% agarose gel dissolved in 1xTAE buffer which confirmed that their size was the correct one. The products were later purified using the "GeneJET PCR Purification Kit (250)" (Thermo Scientific) and digested with the restriction enzymes BamHI and NotI. The digested products were purified once again and ligated into the vector for transformation.
2.3 DNA extraction from Giardia intestinalis
Cells from a confluent tube of G. intestinalis were harvest by centrifuge at 2500rpm for 5 minutes. The supernatant was removed and the cell pellet was washed in 1ml of cold PBS to remove any media components by centrifuge at 2500rpm for 5 minutes once again. The pellet was resuspended in 500µl of lysis buffer including 50mM
Figure 8. The pGEX vector, used for the transformation of E. coli, containing the AmpR gene and GST tag.
incubated at 56°C for at least 1 hour and mixed occasionally by vortexing. 20µl RNase A were added and the mixture was incubated for 20 minutes at room temperature. To the tube we added 275µl of Phenol (pH 8), 275µl of Chisam (24:1 chloroform:isoamyl alcohol) and mixed carefully by vortexing for 20 seconds. The sample was centrifuged for 10 minutes at 13000rpm. The aqueous phase of the tube (upper phase) was removed and an equal volume of Chisam was added in it. After vortexing for 20 seconds and centrifuging the tube for 10 minutes at 13000rpm the aqueous phase was precipitated with an equal volume of isopropanol and followed by a 10 minute incubation at room temperature. The tube was centrifuged for 10 minutes at 13000rpm, the supernatant was removed and the pellet was washed with 1ml of 70% cold ethanol and then centrifuged for 5 minutes at 13000rpm at 4°C. The supernatant was removed and the pellet was air-dried and dissolved ddH2O (20-300µl depending on the amount of start material). The final concentration of genomic DNA was measured by Nanodrop
2.4 PCR amplification of genes
All designed primers were dissolved to 200µM and for each one of them a 1:10 dilution was made to get the concentration 20µM. A primer mix of the forward and the reverse primer from the 20µM tube was made, which gave a primer mix of 10µM, that we used in the final PCR reaction. For the cloning the proof-reading polymerase, Phusion Hot-Start II High-Fidelity polymerase (Finnzymes), was used for the amplification of the genes. The mastermix (final volume of 40µl) for the one PCR reaction included:
FOR 1 REACTION ADDED
8µl of 5x Phusion® HF Reaction Buffer (Finnzymes) 4µl of dNTPs (stock 2mM)
0.6µl of Phusion Hot-Start II DNA polymerase (Thermo Scientific) 2µl of the primermix (10µM)
4µl of DNA template 21.4µl of dH2O
The general PCR conditions used were:
PCR CONDITIONS
Initialization step: 98°C for 30 seconds Denaturation step: 98°C for 10 seconds
Annealing step: 59°C for 30 seconds Elongation step: 72°C for 45 seconds
Final elongation: 72°C for 10 minutes Final hold: 4°C for ∞
x 35 cycles
After the end of the PCR program, 5µl of the products were visualized on a 1%
agarose gel dissolved in 1xTAE buffer. The agarose gel ran at 100V for 30-40 minutes after ethidium bromide 0.5μg/ml was added to the liquid agarose. The PCR products were purified, digested with the required digestion enzymes and stored at -20
°C.
2.5 Ligation
The ligation mixture used for the ligation of the purified DNA insert into the vector included:
FOR 1 REACTION ADDED: 20-100ng of linear vector DNA
1:1 to 5:1 (molar ratio over vector) of insert DNA 1.5µl of 10x Buffer for T4 DNA ligase (Fermentas) 0.5µl of T4 ligase (Fermentas)
0.75µl of 10mM ATP
The mixture was incubated overnight at 16°C.
2.6 Transformation of E. coli
The transformation of competent E. coli cells (DH5α) was performed with heat-shock.
The transformation of competent cells of E. coli was performed by thawing the cells on ice from -80°C and aliquoting 100μl per reaction. To 100µl of competent E. coli cells (DH5α), 3-5µl of the ligation mix, containing the vector and DNA insert (~10- 100ng DNA vector/100µl), was added. Next, the cells sit on ice for 15-30 minutes and were heat shocked for 1 minute at 42°C. The cells were placed on ice again for 2 minutes before 900 µl of LB were added to the tube and incubated at 37°C and shaking for 1 hour. Thereafter, the cells were plated out on selective LA plates supplemented with 50µg/ml ampicilin. Finally, the plates were incubated overnight (approximately 16 hours) at 37°C.
2.7 Plasmid mini-preparation
A single colony of the transformed E. coli was picked and inoculated in 5ml LB media containing 50µg/ml ampicilin at 37°C for approximately 16 hours and shaken at 200rpm for aeration. The plasmid from the overnight culture was extracted using the GeneJET plasmid miniprep kit (Thermo Scientific) according to the manufacturer's instructions.
2.8 Restriction digestion of DNA and PHA-5 vector or (pGEX vector)
The mixture of the digestion of the vector pPAC-3xHA-C (PHA-5) with the "Fast Digest" enzymes MluI (or BamHI for pGEX vector) and NotI contained:
FOR 1 REACTION ADDED:
1µg of template (PCR product or plasmid)
3µl of 10x Fast Digest Green Buffer (Thermo Scientific)
1µl (1u) Fast Digest MluI (or BamHI for pGEX vector) (Thermo Scientific) 1µl (1u) Fast Digest NotI (Thermo Scientific)
Water, nuclease-free to 30µl (final concentration 30µl) The reaction mixture was incubated for 1 hour at 37°C
For the plasmid an additional digestion was performed by adding 1µl (1u) of the FastAP™ thermosensitive Alkalane Phosphatase (1u/µl) (Fermentas) in order to dephosphorylate the vector and avoid self ligation of it. Once again, the mixture was incubated for 1 hour at 37°C. The digested product was visualized on agarose gel (0.7% for the PCR products and 1% for the plasmid). Thereafter, the PCR products were purified with GeneJET PCR Purification Kit (250) (Thermo Scientific) as mentioned above, while the plasmid was gel purified to remove the DNA insert using the "QIAquick® Gel Extraction Kit (250)" (QIAGEN).
2.9 Sequencing of cloned plasmids
To verify the correct sequences of the cloned plasmids, samples of the plasmids were sent for sequencing at Uppsala Genome Center. All the samples contained 450ng of plasmid together with efficient sequencing primers (4pmol/tube) and water to a final volume of 18µl.
2.10 Culture conditions for Giardia intestinalis
All G. intestinalis cultures were cultivated in polystyrene screw cap tubes (Nunc) in 10 ml of TYDK media at 37°C.
250 ml of basal media contained: 7.5g Peptone, 2.5g Glucose, 0.5g NaCl, 0.05g L- ascorbic acid, 0.25g K2HPO4, 0.15g KH2PO4, 0.5g L-Cysteine and 2.5ml Ferric ammonium citrate solution (2.2mg/ml). The pH was set to 6.8 using 5M NaOH and thereafter the media was filter sterilized using 0.45μm filter-units (Corning). Next, 25ml filter sterilized bile (12.5mg/ml) was added to the sterile media. To complete the media, 10% bovine serum (heat inactivated) was added to it. The basal media was stored at 4°C.
2.11 Plasmid big scale preparation for transfection of Giardia intestinalis
The plasmid preparation were scaled up in order to obtain a sufficient amount of plasmid (around 20-30µg) for the transfection of G. intestinalis. As in the mini- preparations (see section 2.7), a single colony of the transformed E. coli was picked and inoculated in 30ml LB media containing 50µg/ml ampicilin at 37°C for approximately 16 hours and shaken at 200rpm for aeration. The plasmid from the overnight culture was extracted using the GeneJET plasmid miniprep kit (Thermo Scientific) by making a few modifications in order to obtain a bigger volume of plasmid.
2.12 Transfection of Giardia intestinalis using electroporation
A nearly confluent tube of G. intestinalis (approximately 1×107 cells) placed on ice for 15 minutes so that the Giardia cells will detach from the tube and the cells were collected by centrifuge the tube at 2500rpm for 5 minutes. The pellet was resuspended in 300µl of TYDK and placed to a 4mm gap electroporation "Gene Pulser® Cuvette"
(BIO-RAD) and placed on ice. To the cuvette, 20µg of the episomal plasmid was added, mixed with pipette and electroporated immediately.
The settings used for the electroporator "Gene Pulser" (BIO-RAD) were: pulse in 350V, capacitance in 960µF and resistance in 800Ω.
After the electroporation the cuvettes were incubated on ice for 10 minutes and later the electroporated cells were transferred to culture tubes with 10 ml of warm TYDK media. The electroporated cells were grown at 37°C for 24 hours before 50μg/ml of puromycin, which was the selection drug, was added. Every week, fresh media and selective drug was given to the transfected cells until they become confluent, allowing the cells to be passed as wild type cells. In order to avoid contamination during the cell cultivation, 100μg/ml of gentamicin was added to the culture tubes. The transformed stains of G. intestinalis were frozen in 1ml of TYDK media containing 10% of DMSO (Dimethyl Sulfoxide) (Sigma-Aldrich) and they were stored in -80 °C as a stock.
2.13 SDS-PAGE electrophoresis
In order to prepare the sample for western blotting, the cultured cells of transformed E. coli or transfected G. intestinalis were centrifuged at 3000rpm for 5 minutes at 4°C. The supernatant was discarded and the cell pellet was washed in cold PBS and centrifuged again at 3000rpm for 5 minutes at 4°C as before (the washing was repeated 2 times). The final cell pellet was resuspended in 1ml of RIPA buffer containing: 1% NP-40, 0.1% SDS, 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.5%
Sodium Deoxycholate and 1mM EDTA, mixed with protease inhibitor cocktail (Roche). The cell suspension was lysed by mixing gently and placing on ice for 15 minutes. Next, the samples were centrifuged at 13000rpm for 5 minutes at 4°C and the supernatant was transferred to a new vial.
A protein assay (BIO-RAD) of the supernatant was performed according to the manufacturer's instructions, were a protein concentration of 3-5µg/µl was the preferred for PAGE. In the protein samples, 2x SDS-PAGE sample buffer was added to yield a 1x sample buffer concentration and they were boiled for 10 minutes.
Thereafter, 10μl of the boiled samples were loaded on the SDS-PAGE gel.
FOR A 10%SDS-AGE GEL: Stacking (upper) gel
3.4 ml Water
0.63 ml Upper Tris buffer (1/4) pH= 6.8 50 ul SDS
50 ul APS
0.83 ml Acrylamide buffer (30%) 5 ul TEMED
5 ml
Separating (lower) gel
4 ml Water
2.5 ml Upper Tris buffer (1/4) pH= 8.8 100 ul SDS
100 ul APS
3.3 ml Acrylamide buffer (30%) 4 ul TEMED
10 ml
After the boiled samples and the PageRuler™ prestained protein ladder (ThermoScientific) been loaded, the gel ran at 100V between 30-50 minutes in 1X Running buffer.
2.14 Western Blotting
After the proteins were separated, the SDS-PAGE gel was transferred to Polyvinylidene Flouride (PVDF) membrane (Pall Life Sciences) by placing the membrane on top of the gel stacked between pads and filter-papers. The stack was placed in 1x Transfer Buffer and the transfer was performed at 35mV overnight at 4°C with continuous stirring. The membrane was blocked using 3% non-fat dry milk in phosphate buffered salane (PBS) containing 0.1% Polyoxyethylenesorbitan monolaurate, Tween20 (Sigma-Aldrich) to reduce unspecific binding of the antibodies. The membrane was blocked for one hour followed by washing three times for five minutes each in PBS-T. The membrane was incubated with Anti-HA antibody (product no. H 9658, Sigma-Aldrich) as the primary antibody which was diluted 1:10000 in PBS with 1% BSA and 0.1% Tween20 for two hours. Thereafter the membrane was washed as previously followed by an hour incubation with the secondary antibody, Anti-mouse coupled with horseradish peroxidase (HRP) (product no. P0161, Dako) diluted 1:10000 in 3% non-fat milk dissolved in PBS with 0.1%
Tween20. After one hour of incubation the membrane was washed as before with PBS
containing 0.1% Tween20. Blots were developed using ECL Plus detection system (Amersham ECL Plus detection kit, GE Healthcare) according to the manufacturer's instructions.
2.15 Fixation
In order to prepare the samples for the fixation, one confluent tube of Giardia was used and put on ice for 10 minutes and centrifuged at 2500rpm for 5 minutes. The cell pellet was washed in PBS and centrifuged again at 2500rpm for 5 minutes. Next, the cell pellet was resuspended in 1ml of ice-cold HBS-glucose buffer (Hepes Buffered Salane; HBS) to wash away media components and centrifuged again at 2500rpm for 5 min. Thereafter, 15µl drops of cells were placed on poly-L-lysine coated
"Diagnostic Microscope Slides" (Thermo Scientific) with 10 wells. The cells were allowed to attach to the surface of the slide at 37°C for 5 minutes in a humidity chamber.
The G. intestinalis cells were fixed according to the paraformaldehyde (PFA) fixation protocol.
Following the paraformaldehyde (PFA) fixation protocol, 15µl of 4% PFA was added to the droplets and incubated at 37°C for 20 minutes. The fixative was removed using vacuum suction and 15µl of 0.1M Glycine which was dissolved in PBS was added to the wells to quench any remaining traces of fixative. The fixative was removed once again using vacuum suction and the wells were washed 5 times with PBS before 15µl of 0.1% Triton-X dissolved in PBS were added and incubated for 30 minutes at 37°C.
Once again, the fixative was removed using vacuum suction and the wells were washed 5 times with PBS. Finally, 15µl of 2% BSA dissolved in PBS and 0.05%
Triton-X (blocking buffer) were added and the slide was incubated over-night at 4°C in a humidity pan.
2.16 Immunofluorescence
After the overnight incubation, the blocking solution was removed by vacuum suction and 15µl of anti-HA direct monoclonal antibody (Alexa Fluor labeled MonoHA) diluted 1:250 times in the blocking buffer were added and incubated for 2 hours at room temperature. Next, the antibody was removed using vacuum suction and the wells were washed 5 times with PBS before 15µl of the secondary anti-mouse antibody conjugated to Alexa 488 diluted 1:200 times in the blocking buffer were added and incubated for 1 hour at room temperature. The antibody was removed using vacuum suction and the wells were washed 5 times with PBS. In the final step of the fixation, 3µl of mounting media Vectashield containing the DNA stain 4',6'- diamidino-2-phenyldole (DAPI) were added and a cover slip was placed over the wells and sealed with nail varnish. The slide was stored at 4°C or -20°C in darkness.
The transfected cells were examined with a Zeiss Axioplan2 fluorescence microscope and the images were processed using the software Axiovision Rel. 4.8.
2.17 RNA purification
In order to prepare the samples for RNA purification, cells from a confluent tube of G. intestinalis were centrifuged at 2500rpm for 5 minutes and the pellet was washed in 1 ml of cold PBS. The cells were pelleted again by centrifuge at 2500rpm for 5 minutes and the pellet was resuspended in 1 ml of Trizol® Reagent (Invitrogen). The cells left to stand for 5 minutes in room temperature and then placed on ice. Next, 0.2ml of chloroform per 1ml of Trizol Reagent were added and after the sample tubes were cap securely, they mixed by vortexing for 15 seconds and incubated at room temperature for 2-3 minutes. Thereafter, the samples were centrifuged at 13000rpm for 15 minutes at 2-8°C. The centrifugation caused the mixture to separate into a lower red, phenol-chloroform phase and a colorless upper aqueous phase (RNA remains exclusively in the aqueous phase). The upper aqueous phase was transferred carefully without disturbing the inter-phase into fresh tube and precipitated by mixing with 0.5ml of isopropyl alcohol per 1ml of Trizol Reagent. The samples were incubated at 15-30°C for 10 minutes and centrifuged at 13000rpm for 10 minutes at 2- 4°C. The RNA precipitate, often invisible before centrifugation, formed a gel-like pellet on the side and bottom of the tube. The supernatant was completely removed and the RNA pellet was washed twice with 1ml of 75% cold ethanol by vortexing and centrifuge at 13000rpm for 5 minutes at 2-8°C.TheRNA pellet was air-dried for 5-10 minutes and dissolved in 20-30µl of DEPC-treated water. The final concentration of the RNA was measured by Nanodrop and the tubes containing the RNA were stored in -20°C.
2.18 cDNA synthesis
The cDNA synthesis was performed using the "RevertAid H Minus First Strand cDNA Synthesis Kit" (Thermo Scientific).
To prepare the samples for the cDNA synthesis first removal of the genomic DNA from the RNA samples was necessary in order to avoid any contamination.
FOR 1 REACTION ADDED
1µg of RNA
1µl of 10x Reaction Buffer with MgCl2 (Thermo Scientific) 1µl (1u) of DNase I, RNase-free (1u/µl) (Thermo Scientific).
Water, nuclease-free to 10µl (final concentration of 10µl) The samples were firstly incubated at 37°C for 30 minutes.
To the samples 1µl of 50mM of EDTA (Fermentas) was added and incubated at 65°C for 10 minutes.
The first strand of cDNA was synthesized by RT-PCR.
FOR 1 REACTION ADDED IN THE INDICATED ORDER
5µg of total RNA
1µl of Random Hexamer Primer (0.2µg/μl) 4 μl of 5x Reaction Buffer
1 μl of RiboLock RNase Inhibitor (20u/μl) 10 of mM dNTP mix
1μl of RevertAid H Minus Reverse Transcriptase (200u/μl) (final concentration of 20μl)
The reaction samples were mixed gently and centrifuged before put them in the PCR machine.
For the random hexamer primed synthesis the reaction samples were incubated for 5 minutes at 25°C followed by 60 minutes at 42°C. The reaction was terminated by heating at 70°C for 5 minutes. The final cDNA was stored at -20°C.
2.19 RT-qPCR (quantitative Real Time Polymerase Chain Reaction)
The first step of the qPCR was to for the absolute quantification was to make a melting curve (association curve) which was used to test the efficiency of the designed primers (see Appendix for used primers).
2.19.1Absolute Quantification (efficiency of reaction)
In order to prepare the samples for the absolute quantification, dilution of standard cDNA from wild type Giardia was used, where the cDNA was diluted 1:10, 1:100, 1:1000 and 1:10000 times. The forward and reverse designed primers (100µM) were mixed to create a mix stock of 10 µM, which was diluted to a primer mix stock of 1µM. Next, 2.5 µl of the primer mix (1µM) were mixed with 12.5 µl of "Maxima SYBR Green/ROX qPCR Master Mix" (Thermo Scientific) per well. Those 15 µl of the master mix were added into the MicroAmp™ 96-well plate (AB Applied Biosystems) by reverse pipetting and 10 µl of DNA per well were added later. All samples were in quadruplicates for optimal results. The a 96-well plate was sealed and placed in the qPCR machine (7300 Real Time PCR system) (AB Applied Biosystems) in the correct orientation for analysis.
The thermal profile used was:
Procedure Temperature Time
Stage 1: Initial 50°C 2 minutes
Stage 2: Hot Start 95°C 10 minutes Stage 3:
Annealing 95°C 60°C 15 seconds 1 minute X 40 Stage 4: Melting
curve 95°C 15 seconds
60°C 30 seconds
95°C 15 seconds
2.19.2 Relative Quantification
In order to prepare the samples for the absolute quantification, dilution of standard cDNA from wild type Giardia was used, where the cDNA was diluted 1:50 times.
The forward and reverse designed primers (100µM) were mixed to create a mix stock of 10 µM, which was diluted to a primer mix stock of 1µM. Next, 2.5 µl of the primer mix (1µM) were mixed with 12.5 µl of "Maxima SYBR Green/ROX qPCR Master Mix" (Thermo Scientific) per well. Those 15 µl of the master mix were added into the MicroAmp™ 96-well plate (AB Applied Biosystems) by reverse pipetting and 10 µl of DNA per well were added later. All samples were in quadruplicates for optimal results. The a 96-well plate was sealed and placed in the qPCR machine (7300 Real Time PCR system) (AB Applied Biosystems) in the correct orientation for analysation.
The thermal profile used was:
Stages Temperature Time
Stage 1: Initial 50°C 2 minutes
Stage 2: Hot Start 95°C 10 minutes Stage 3:
Annealing 95°C 60°C 15 seconds 1 minute X 40
2.20 Protein purification
For the protein purification of the GST tagged protein, overnight culture of E. coli (BL21) transformed with the pGEX fusion construct had to set up containing 100 µg/ml ampicilin as a selection drug. The next day, the overnight culture was diluted 1:10 times in fresh LB medium containing 100 µg/ml ampicilin. The cells grew at 37°C till they reach an OD 600 =0.6-0.8 which represents the mid-log phase. When the OD600 reached the desired level, the bacterial culture was induced by adding 0.5mM isopropyl-β-D-thio-galactoside (IPTG) and allow them to grow for an additional 4 hours at 28°C. After the 4 hour incubation, the cells were harvested at 4°C at 4500 rpm for 25 minutes. Due to problems of solubilization of the protein, in this step of the protein purification, the "Rapid GST inclusion body solubilization and renaturation kit" (Cell Biolabs) was used according to the manufacturer's instructions.
After the bacterial cell lysis and the inclusion body solubilization and renaturation, 50µl of Gluthione Sepharose™ 4B beads (50%) (GE Healthcare) was added per 1ml of cell extract containing the GST fusion protein. The beads were incubated overnight at 4°C with end-over-end rotation. The beads were washed three times with 1xPBS and one time in the elution buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA and 1mM DTT) in order to equilibrate them. After the washing, the beads were incubated overnight at 4°C with end-over-end rotation in the elution buffer containing the enzyme PreScission™ protease (GE Healthcare) (20µl/1ml of elution buffer).
After the overnight incubation, the beads were centrifuged at 4°C at 13000rpm for 5 minutes and the supernatant (which contains the purified GST fusion protein) was
collected. Thereafter the beads were washed again one more time with the elution buffer and after one more centrifugation at 4°C at 13000rpm for 5 minutes, the supernatant was collected again.
In order to concentrated the collected supernatant and as a result the GST purified fusion protein, the Vivaspin® 6 Centrifugal Concentrator columns (GE Healthcare) were used according to the manufacturer's instructions.
3. Results
In the G. intestinalis genome there are at least 19 proteins that have been found to contain a BPI domain (table 1). The proteins can be found in 3 different assemblages (A, B and E) corresponding to 4 different isolates (WB, AS, GS and P15). This project focuses in the WB isolate which belongs to assemblage A, since it affects mainly humans and also is the most well-studied isolate from Staffan's Svärd group, where the project took place.
Isolate Annotation Locus tag Assemblage
WB
Hypothetical protein GL50803_102575 Assemblage A Hypothetical protein GL50803_111973 Assemblage A Hypothetical protein GL50803_112630 Assemblage A Hypothetical protein GL50803_112914 Assemblage A Hypothetical protein GL50803_112938 Assemblage A Hypothetical protein GL50803_113130 Assemblage A Hypothetical protein GL50803_113165 Assemblage A Hypothetical protein GL50803_16293 Assemblage A
AS Hypothetical protein AS175 Assemblage A
P15
Hypothetical protein GLP15_230 Assemblage E Hypothetical protein GLP15_712 Assemblage E Hypothetical protein GLP15_2478 Assemblage E Hypothetical protein GLP15_2725 Assemblage E Hypothetical protein GLP15_3045 Assemblage E Hypothetical protein GLP15_3522 Assemblage E Hypothetical protein GLP15_4118 Assemblage E Hypothetical protein GLP15_5002 Assemblage E GS Hypothetical protein GL50581_1015 Assemblage B Hypothetical protein GL50581_890 Assemblage B
Table 1. The 19 BPI-like proteins found in G. intestinalis categorized by assemblage and isolate.
3.1 Protein comparisons
In the beginning of the project multiple alignments of these BPI-like porteins were performed in order to get a better understanding of the different assemblages and the different isolates which could possibly give us a first idea of the relation between them.
Figure 9. Phylogenic analysis based on the amino acid sequences of 16 BPI-like proteins found G.
intestinalis genome where the WB isolate is demonstrated with red, P15 isolate with green and GS isolate with blue.
Figure 10. Phylogenic analysis based on the nucleotide sequences of 16 BPI-like proteins found G.
intestinalis genome where the WB isolate is demonstrated with red, P15 isolate with green and GS isolate with blue.
Figure 11. Phylogenic analysis based on the promoter region (-150 +0) sequences of 16 BPI-like proteins found G. intestinalis genome where the WB isolate is demonstrated with red, P15 isolate with green and GS isolate with blue.
From the phylogenic analysis above, is clear that the genes that belong in the same isolate share many similarities (figure 9 and 10), while there is also the possibility of recombination during evolution between the isolates WB and P15 (figure 11).
3.2 Structural comparison of BPI-like proteins with human BPI and LBP
Using the protein structure homology-modeling server SWISS-MODEL (http://swissmodel.expasy.org) for the structure prediction of some of the BPI-like proteins of interest in G. intestinalis and also for the human BPI and LBP protein, the following structures were obtained (figure 12). In the following figure examples of two BPI-like proteins are shown and order to obtain these structures the protein sequences had to be given to the server. These protein sequences were obtained from
the Protein Data Bank (PDB) (http://www.rcsb.org) for the human BPI, the Universal Protein Resource (UniProt/Swiss-Prot) (http://www.uniprot.org) for the human LBP and the Giardia Genomics Resource (GiardiaDB) (http://giardiadb.org) for the BPI- like proteins from G. intestinalis.
Figure 12. Structure prediction of human BPI (PDB ID: 1BP1, Chain: A), human LBP (Swiss- Prot ID: P18428) and the two BPI-like proteins GL50803_113130 and GL50803_111973 which belong to assemblage A and to the WB isolate. Note that the N-terminal of the proteins is colored blue while the C-terminal is colored yellow-orange. For the protein GL50803_111973 the sequence identity with the human BPI is 13.737% and for the GL50803_113130 is 12.727%.
From figure 12 it can be seen the structure similarities between human BPI and LBP with the BPI-like proteins and especially the similarities that occur between the N- terminal of all proteins.
3.3 Electroporation of Giardia intestinalis
After the electroporation of G. intestinalis cells was performed there were surviving cells, but in order to make sure that the plasmid transfected into the parasites was still present and that a stable transfectant was established, a period of approximately three weeks of selection had to pass. During this project, 7 stable transfectants were established. Each transfectant was electroporated with one of the 7 BPI-like proteins that belong to assemblage A and isolate WB (see table 1).
3.4 Immunofluorescence of tagged proteins from stable transfectants
After the establishment of stable transfectants the next step of this project was to find where the transfected proteins localize in the parasite. In order to be able to do that, the proteins that were selected and transfected at the C-terminal were also tagged by the epitope tag 3xHA. All immunofluorescence pictures (figure 13,14, 15 and 16), for 4 out of the 7 transfectants, were taken by a fluorescent microscope, where the tagged proteins are stained with the direct anti-HA antibody in green while the nuclei were
stained with DAPI in blue. All pictures were taken with the 100x oil-immersion objective.
An ER pattern was observed where BPI-like proteins localize.
Figure 13. Localization of the HA-tagged GL50803_102575 protein using fluorescence microscopy. Left panel shows the phase contrast while the right panel shows the merged of localized HA-tagged protein in green and DAPI stained nuclei in blue. Picture A represents the phase contrast for picture B and picture C represents the phase contrast for picture D.
Figure 14. Localization of the HA-tagged GL50803_112630 protein using fluorescence microscopy. Left panel shows the phase contrast while the right panel shows the merged of localized HA-tagged protein in green and DAPI stained nuclei in blue.
Figure 15. Localization of the HA-tagged GL50803_113130 protein using fluorescence microscopy. Left panel shows the phase contrast while the right panel shows the merged of localized HA-tagged protein in green and DAPI stained nuclei in blue.
