FACULTY OF SCIENCE AND TECHNOLOGY

MASTER THESIS

Curriculum: Biological Chemistry Autumn semester, 2020

Open

Author: Kaneez Fatima

(Author signature)

Tutor: Prof. Dr. Mark van der Giezen

Master thesis title: Entamoeba invadens; a study about the role of PLD1 and PLD2
in encystation

Keywords: Phospholipase D1 and 2 Number of pages: 68

Entamoeba histolytica + appendices/other: 2
Entamoeba invadens
Transfection
Stavanger, 4.12.2020
Cysts formation
date/year
 ACKNOWLEDGMENTS

There were a number of people who helped me during the time period of my thesis, none who
helped more than my supervisor Professor Dr. Mark van der Giezen. Thank you for giving me the
opportunity of being your student. It was a learning experience and one of the most important
lesson I learnt during the process was that things do not always go according to the plan and one
has to come up with solutions on the go. At the time, not getting proper results was stressful but
looking back, it was an opportunity for improvement and finding solutions to very interesting
problems. Dr. Mark always had my back and helped throughout the process.

I would like to pay my heartiest regards and compliments to Dr. Dugassa Nemie-Feyissa for his
continuous motivation and guidance and for trust in my abilities. Thank you for always helping
and offering your guidance. I would also like to thank Dr. Marina Alexeeva, who is such an
outstanding and humble person, who was always there to welcome me in the hour of need, who
always paid her attention towards my problems regarding research work and guided me by her
remarkable knowledge. Thanks to Tia Renee Tidwell (PhD scholar) for helping me with
Fluorescence Microscopy.

Last but not the least, this piece of my hard work is dedicated to my family: my parents (Syed
Muhammad Sibtain Gillani and Kalsoom Sibtain), and my brothers who always had faith in my
abilities and always stood beside me in every moment of my life.

Sincerely,

Kaneez fatima

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 ABSTRACT
One of the most common problem in the developing world is infectious diseases caused by poor
living conditions and one such condition is called amoebiasis. The causative agent of which is
Entamoeba histolytica; a microaerophilic, parasitic amoebozoan species, which causes intestinal
and extraintestinal amoebiasis in humans. Although there are certain drugs available for treating
this disease, more research is needed to come up with novel drugs. Furthermore, basic research is
needed to understand the different stages of its life cycle and possible therapies during its dormant
stages. One such stage is the encystation of its cell, a crucial stage which basically involves the
transformation of motile trophozoite into resistant cyst. It is extremely difficult to treat cysts, and
a growing body of research has shown that phospholipase D (PLD) activity is significantly
increased in Entamoeba histolytica during encystation, indicating that it plays a critical role in cyst
formation. PLD is a widely distributed enzyme in all branches of life that hydrolyzes
phosphatidylcholine. It is believed to play an important role in the regulation of cell physiology by
extracellular signals such as hormones, neurotransmitters, growth factors, and cytokines. Due to
our inability to effectively induce encystation in vitro in Entamoeba histolytica, our knowledge
about the cyst form remains limited. This also hampers our ability to develop cyst-specific
diagnostic tools. This thesis is about role of PLD during cyst formation in Entamoeba. In this study
we attempted to investigate a potential role of PLD during encystation in the laboratory model
organism Entamoeba invadens as encystation is not experimentally achievable in human parasite
Entamoeba histolytica. The research included amplification of PLD1 and PLD2 genes by
extraction of genomic DNA and cloning of the genes into standard cloning vector pGEM-T-Easy
and then in the Entamoeba-specific vector pEiNEO. Electroporation of the cloned Entamoeba
specific vectors was performed by transfecting the Entamoeba invadens trophozoite cells. On other
hand, encystation activity was also checked by introducing 0.6% n-butanol and t-butanol in 50%
LYG media (encystation medium) but it did not show any significant inhibition. Fluorescent
microscopy results showed no effect of n-butanol on cysts formation. Bioinformatics investigation
showed that the HKD active site motif of PLD was found conserved throughout Entamoeba
species as in animal, plants, and fungi.

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 CONTENTS

1. Introduction ............................................................................................................................. 1

1.1 Introduction ...................................................................................................................... 1

1.2 E. invadens as model organism ........................................................................................ 1

1.3 Biology of E. invadens ..................................................................................................... 2

1.4 Life cycle of Entamoeba .................................................................................................. 2

1.5 Epidemiology of E. histolytica ......................................................................................... 6

1.6 Disease pathology ............................................................................................................ 6

1.7 Transmission, diagnosis, and treatment ........................................................................... 7

1.8 Entamoeba cyst ................................................................................................................ 8

1.9 Change in gene expression during encystation and excystation .................................... 11

1.10 Structure of PLD ......................................................................................................... 13

1.11 Role of PLD in encystation ........................................................................................ 16

1.12 Aims of this study ....................................................................................................... 17

2 Material and Methods – Bioinformatics Analysis ................................................................ 19

2.1 Sequence retrieval of PLD ............................................................................................. 19

2.2 Identification of conserved sequence ............................................................................. 20

2.3 Multiple sequence alignment and phylogenetic tree construction ................................. 20

2.4 Proteomics study of PLD ............................................................................................... 20

2.4.1 Homology modelling .............................................................................................. 20

2.4.2 Superimposition ...................................................................................................... 20

2.4.3 3D model of PLD .................................................................................................... 21

2.4.4 Validation of model (Ramachandran plot) ............................................................. 21

3 Material and Methods – Molecular Work ............................................................................. 22

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3.1 LYI-S media for culturing Entamoeba invadens ........................................................... 25

3.2 Culturing of E. invadens................................................................................................. 25

3.3 Trophozoite cell culture storage ..................................................................................... 26

3.4 Recombinant DNA technology steps ............................................................................. 26

3.4.1 Growing E. coli JM109 ........................................................................................... 26

3.4.2 Preparation of competent cells ................................................................................ 26

3.4.3 Transformation ........................................................................................................ 27

3.4.4 Plasmid isolation ..................................................................................................... 27

3.4.5 Vectors for cloning ................................................................................................. 28

3.4.6 pEiNEO-LUC restriction digestion......................................................................... 29

3.5 DNA extraction .............................................................................................................. 30

3.6 Polymerase chain reaction amplification ....................................................................... 31

3.6.1 Agarose gel electrophoresis .................................................................................... 32

3.6.2 Purification of PCR products .................................................................................. 33

3.7 Cloning of purified PCR products into vectors .............................................................. 33

3.7.1 Standard vector (pGEM-T-Eeasy) .......................................................................... 33

3.7.2 E. invadens vector (pEiNEO) ................................................................................. 34

3.8 Mini cultures for mini prep ............................................................................................ 35

3.9 NanoDrop One ............................................................................................................... 35

3.10 Subsequential restriction digestion ............................................................................. 35

3.11 Long term storage of bacterial clones......................................................................... 36

3.12 DNA sequencing......................................................................................................... 36

3.13 Transfection ................................................................................................................ 36

3.14 Encystation of E. invadens ......................................................................................... 37

3.15 Calcofluor staining and fluorescence microscopy ...................................................... 37

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 3.16 n-Butanol and t-Butanol activity on PLD ................................................................... 38

4 Results ................................................................................................................................... 39

4.1 Bioinformatics analysis .................................................................................................. 39

4.1.1 Conserved sequences in PLD.................................................................................. 39

4.1.2 Phylogenetic tree ..................................................................................................... 39

4.1.3 Proteomics work ..................................................................................................... 40

4.1.4 3D model of E. invadens PLD ................................................................................ 40

4.1.5 Superimposition of PLD1 protein models .............................................................. 40

4.1.6 Superimposition of PLD2 protein models .............................................................. 41

4.1.7 Validation of model (Ramachandran plot) ............................................................. 42

4.2 Molecular Work ............................................................................................................. 43

4.2.1 Isolation and PCR amplification of E. invadens genomic DNA ............................ 43

4.2.2 Determination of DNA concentration using NanoDrop One ................................. 44

4.2.3 DNA fragments on gel after restriction digestion ................................................... 45

4.2.4 Sequencing results .................................................................................................. 47

4.2.5 Encystation .............................................................................................................. 48

5 Discussion ............................................................................................................................. 50

5.1 Genomic DNA isolation ................................................................................................. 50

5.2 Amplification of genes ................................................................................................... 51

5.3 Transformation in E. coli JM109 ................................................................................... 51

5.4 Transfection in Entamoeba invadens ............................................................................. 52

5.5 PLD and its role in Encystation ..................................................................................... 52

5.6 Bioinformatics of PLD ................................................................................................... 53

6 Conclusion ............................................................................................................................ 56

7 References ............................................................................................................................. 57

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8 Appendices ............................................................................................................................ 66

8.1 Appendix A: Chemicals and media ................................................................................ 66

8.2 Appendix B: Sequence alignment of cloned pGEM-T-Easy and PLD1/PLD2 ............. 71

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 LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

BLAST Basic local alignment search tool

Bp Base pairs

BSA Bovine serum albumin

DNA Deoxyribonucleic acid

dNTPs Deoxynucleoside triphosphates

G418 Geneticin 418 (antibiotic)

Hrs Hours

IPTG Isopropyl‐beta‐D‐thiogalactopyranoside

Min Minutes

PBS Phosphate buffer saline

PCR Polymerase chain reaction

PLD1 Phospholipase D1

PLD2 Phospholipase D2

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

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 LIST OF TABLES

Table 1. Proteins up regulated during encystation in E. invadens with little (one or two papers) or
no published literature................................................................................................................... 17
Table 2. List of sequence retrieval of PLD ................................................................................... 19
Table 3. LYI-S medium composition ........................................................................................... 25
Table 4. Reaction mixture for the double digestion of pEiNEO-LUC plasmid............................ 30
Table 5. PCR conditions using DreamTaq Polymerase for DNA template amplification ........... 31
Table 6. Specific primers used for gene amplification (uppercase indicates perfect match with
target gene; lowercase indicates newly introduced sequence with introduced restriction sites
indicates by italics). ...................................................................................................................... 32
Table 7. Setup of ligation reaction for pGEM-T-Easy vector and PLD1/PLD2 .......................... 34
Table 8. Setup of ligation reaction mixture for pEiNEO vector and PLD1/PLD2 ....................... 34
Table 9. Reaction mixture for restriction digestion ...................................................................... 35
Table 10. 50% LYG media composition ...................................................................................... 37
Table 11. Ramachandran plot statistics for constructed PLD model ............................................ 43
Table 12. Concentration of pGEM-T-Easy vector+PLD1/PLD2 insert using Nanodrop ONE ... 45
Table 13. Concentration of pEiNEO vector+PLD1/PLD2 insert using Nanodrop ONE ............. 45
Table 14. Cysts chitin observation by using bright field and fluorescence microscope ............... 49
Table 15. Encystation percentage (%) of E. invadens at different time intervals ......................... 49

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 LIST OF FIGURES

Figure 1. Life cycle of E. histolytica (Image taken from CDC, 2010) ........................................... 4
Figure 2. Stereographic illustration of the crystal structure of phospholipase D (PLD) from
Streptomyces sp. Overall view of 3D-structure. ........................................................................... 14
Figure 3. The stereographic illustration of the PLD in Streptomyces sp PMF. The view from the
outer membrane. ........................................................................................................................... 15
Figure 4. gDNA isolation, PCR amplification & cloning into pGEM-T-Easy standard vector. .. 22
Figure 5. Cloning into pEiNEO (E. invadens vector) ................................................................... 23
Figure 6. Cysts formation activity in three different 50% LYG media such as wild type, n-butanol
& t-butanol .................................................................................................................................... 24
Figure 7. Commercially available Promega pGEM-T-Easy vector map Taken from SnapGene. 28
Figure 8. E. invadens specific vector pEiNEO-LUC .................................................................... 29
Figure 9. PLD phylogenetic tree generated from Phylogeny.fr and editied in Figtree ............... 39
Figure 10. 3D model of E. invadens PLD, superfamily domain PLN02866. ............................... 40
Figure 11. Superimposition of PLD1 template (6ohr.1 chain A) in light blue and E. invadens PLD1
model in golden............................................................................................................................. 40
Figure 12. Superimposition of E. invadens PLD in orange and E. histolytica constructed protein
model in dark blue. ....................................................................................................................... 41
Figure 13. Superimposition of E. invadens PLD2 model in orange and 6ohm.1 template in purple.
....................................................................................................................................................... 41
Figure 14. Superimposition of E. invadens PLD2 model in forest green and E. histolytica in yellow.
....................................................................................................................................................... 42
Figure 15. Ramachandran Plot ...................................................................................................... 42
Figure 16. PCR amplification from E. invadens genomic DNA .................................................. 44
Figure 18. pGEM-T-Easy vector cloned with PLD1 gene constructed by using Snapgene. ........ 47
Figure 19. pGEM-T-Easy vector cloned with PLD2 gene constructed by using Snapgene ......... 48
Figure 20. Cysts formation at different time interval. .................................................................. 49
Figure 21. Alignment representation of active domain motif site (HKD) #1 ............................... 54
Figure 22. Alignment representation of Active domain motif site (HKD) #2 .............................. 54
Figure 23. Bioline hyperladder 1 .................................................................................................. 70

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Figure 24. Alignment representation of cloned pGEM-T-Easy+PLD1 along with reference gene
and primers.................................................................................................................................... 73
Figure 25. Alignment representation of cloned pGEM-T-Easy+PLD2 along with reference gene
and primers.................................................................................................................................... 75

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 1. INTRODUCTION
1.1 Introduction
Entamoeba species are unicellular eukaryotes that parasitize all classes of vertebrates and some

invertebrates. In 1969, the WHO expert committee adopted the term “Amoebiasis” which is a life

threatening infection of the gastrointestinal tract of human beings and defined it as the state of

harboring the pathogenic protozoan Entamoeba histolytica with or without clinical signs and

symptoms (Organization, 1969). In the past, many people infected with E. histolytica developed

no symptoms and some infections would clear without any treatment. This fact led to the

origination of two hypotheses; one was proposed by Emil Brumpt in 1925 according to which,

there are two species, one is virulent and invasive while the other one is morphologically identical

but harmless. The second hypothesis stated that E. histolytica is a commensal organism of the

human colon having potential to turn into a pathogenic organism (Aguirre-Beltran et al., 1999).

Only three species of Entamoeba have been proven to cause disease and sometimes death in their

hosts: Entamoeba histolytica, a parasite of humans, Entamoeba nuttalli, a parasite of nonhuman

primates, and Entamoeba invadens, a parasite of reptiles. In humans, six species of Entamoeba

have been reported out of which only Entamoeba histolytica is pathogenic and the remaining five

(E. dispar, E. moshkovskii (unclear pathogenicity), E. coli, E. hartmanni, and E. polecki) are

commensal (Ngui et al., 2012; Gomes et al., 2014).

1.2 E. invadens as model organism

E. histolytica is the main species related to diarrhea and enteritis in human beings, E. invadens on

the other hand is the causative agent of amoebiasis in reptiles, including snakes and lizards, and

has similar life cycle to E. histolytica. E. histolytica is endemic in many countries globally and is

thought to be a foremost parasitic cause of human morbidity and mortality. Likewise, E.

1
invadens can be responsible for substantial losses of reptiles in zoological collections. Research

has focused on usefulness of E. invadens as a model organism for studying the process

of Entamoeba cyst formation (Mi-ichi et al., 2016; Morf & Singh, 2012).

1.3 Biology of E. invadens

E. invadens like E. histolytica exists in two forms; an invasive and motile trophozoite and an

infective cyst (Naiyer et al., 2019).

❖ The trophozoite varies in size from 10 to 60 µm, although very large forms of up to 90

µm have also been observed occasionally in case of E. histolytica. While E. invadens

trophozoites size varies from 15-25 µm. Actively moving trophozoites produce broad and

finger-like pseudopods and contain a spherical nucleus surrounded by nuclear membrane

(Bhattacharya et al., 2000; Ali IK et al., 2012).

❖ The cyst is round or slightly oval bodies having diameter of 10 to 16 µm in E. histolytica.

The cyst of E. invadens has diameter of 11-20 µm. Initially it contains one nucleus but after

maturation, it has four nuclei (Bhattacharya et al., 2000).

1.4 Life cycle of Entamoeba

The life cycle of E. invadens is identical to E. histolytica and has four stages; the trophozoite, pre-

cyst, cyst and metacyst (Arredondo JL et al., 2014). Infection with E. histolytica begins by

ingesting fecally contaminated water or food containing E. histolytica cysts. The infective cyst of

the parasite survives the stomach and intestinal passages. In the bowel lumen, excystation occurs

leading to aggregation of motile and potentially invasive trophozoites in the intestinal mucin layer.

Trophozoites can form new cysts leading to self-limited and asymptomatic infection. The

trophozoite of E. histolytica first converts into pre-cyst form with single nucleus. This form

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matures into tetra-nucleated cyst as it moves down and out of the colon as shown in Figure 1

(Leiva, 2009; Brunette et al., 2009; Garmie et al., 2016).

Cysts are usually found in formed stool while trophozoites are found in diarrheal stool. In the large

intestine, the trophozoites multiply by binary fission and produce cysts, whereas both stages

(trophozoite and cyst) are passed in feces. The cyst can survive days to weeks in the external

environment due to the protective wall around them and are responsible for transmission

(Papadakis et al., 2016). On the other hand, trophozoites passed in the stool are destroyed rapidly

once outside the body and would not survive the gastric environment if ingested. In most cases,

trophozoites remain confined to the intestinal lumen of non-invasive infection making individuals

asymptomatic carriers and pass cysts in their stool. In some patients, the trophozoites invade the

intestinal mucosa (intestinal disease), or, through the bloodstream, extra-intestinal sites such as the

liver, brain, and lungs (extra-intestinal disease), with resultant pathologic manifestations (Brunette

et al., 2009; Hemphill et al., 2019).

In case of E. invadens, the infective cysts are passed in the feces and are stable in the environment.

Transmission occurs by ingestion of infective cysts present in the feces. The main infective form

is cyst. It releases the motile trophozoites after ingestion by the host. The trophozoites invade the

intestine and liver and spread hematogenously through the portal vein. Proteolytic enzymes

released by the parasite disrupt intestinal mucosa and the epithelial barrier facilitating tissue

penetration (Chia et al., 2009).

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 Figure 1. Life cycle of E. histolytica (Image taken from CDC, 2010)

Life cycle of E. histolytica (CDC, 2010) where cysts and trophozoites are passed in feces. Ingestion of mature
cysts from fecally contaminated food, water or hands leads to Entamoeba histolytica infection. Trophozoites are
released in the small intestine via excystation which migrate to the large intestine. Trophozoites may remain
confined to the intestinal lumen (A: noninvasive infection) with individuals continuing to pass cysts in their
stool (asymptomatic carriers). Trophozoites can invade the intestinal mucosa (B: intestinal disease), or blood
vessels, reaching extra intestinal sites such as the liver, brain, and lungs (C: extra intestinal disease).

Amoebic pathogenesis causes host mucosal barrier depletion, colonic lumen adherence and

cytotoxicity and colonic epithelium invasion (Espinosa-Cantellano & Martinez-Palomo, 2000).

This parasitic damage leads to colitis and disseminated disease in some cases. The development

of disease depends on both host and parasitic genotypes (Garmie, 2016). Parasitic transmission
4
depends on the host environmental factors. The link between parasite, host, and environmental

factors in the virulence of E. histolytica has been highlighted by Chelsea Marie and William A

Petri. When E. histolytica progresses to virulence, the destruction of the colonic environment can

lead to degradation of protective mucus, disrupted epithelial barrier function, deregulated ion

transport, local and systemic inflammation, impaired nutrient absorption, and disruption of the

microbiota (Marie et al., 2014).

Among the known amebic factors involved in pathogenesis are signaling pathways involving

heterotrimeric and Ras superfamily G proteins. The current knowledge of roles of heterotrimeric

G protein subunits, Ras, Rho and Rab GTPase families in pathogenesis of E. histolytica as well as

their downstream signaling effectors and nucleotide cycle regulators have been studied (Bosch et

al., 2013). Signaling of heterotrimeric G proteins likely regulates amebic motility and attachment

to host cell and killing host cell via activation of an RGS-RhoGEFs effector (Bosch et al., 2013).

Rho family GTPases and RhoGEFs and Rho Effectors modulate the dynamic actin cytoskeleton

of E. histolytica and other associated pathogenesis related cellular processes like migration,

phagocytosis, invasion and evasion of host responses by surface receptor capping (Labruyère &

Guillén, 2006; Meza et al., 2006; Bosch et al., 2013). Multiple roles have been performed by a

remarkably large family of 91 Rab GTPases in a complex amoebic vesicular trafficking system

which is prerequisite for phagocytosis and pinocytosis and secretion of virulence factors like

amoebapores and cysteine proteases (Christy & Petri, 2011; Bosch et al., 2013). A recent study of

G proteins signaling in E. histolytica has improved the understanding of parasitic pathogenesis

although much remains to be discovered. Yet this study highlighted possible targets such as

heterotrimeric G protein, Rab GTPase, Rho family GTPases, as well as RhoGEFs and Rho

effectors for pharmacological manipulation (Bosch et al., 2013).

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1.5 Epidemiology of E. histolytica
The prevalence of amoebic infection varies with the level of sanitation and is usually higher in

tropics and subtropics as compared to temperate climates (John, 2006). Amoebiasis is the third

most common parasitic cause of death after malaria and schistosomiasis (Organization, 1969;

Dhanalakshmi & Parija, 2016). The cyst is the transmissive form of E. histolytica and one infected

person can pass up to 45 million cysts daily (Herman et al., 2017). E. histolytica roughly infects

50 million people globally and about 10% of the world population is infected with higher rates

occurring in nations where sanitation is poor (Garmie, 2016).

1.6 Disease pathology

Amoebic colitis can result in mucosal thickening to discrete ulcer formation up to necrosis and

intestinal wall perforation (Hardin et al., 2007). The process always starts with adherence of the

trophozoites to the colonic mucus and epithelium. After adhesion of the amoebae, cytolysis is

mediated by the amoebapores, small proteins that are highly effective in lysing lipid bilayers

(Serrano-Luna et al., 2013). The attacked host cells undergo apoptosis (Bansal D et al., 2009;

Tazreiter, 2010). The third major group of pathogenicity factors are the cysteine proteinases. These

enzymes play an important role in tissue invasion, e.g. they were shown to disturb enteric

microvilli and thereby mediate a close contact between the amoebae and enteric cells (Ralston &

Petri, 2011). After tissue invasion the amoebae enter the host blood stream and there encounter the

complement system and host antibodies. These can be cleaved by excreted cysteine proteinases

(Nakada-Tsukui & Nozaki, 2016). Interestingly, the strict commensal Entamoeba dispar does not

express the cysteine proteinase 5 (CP5). The gene encoding CP5 is present, but highly degenerated

and therefore not expressed (Wilson IW et al., 2019). Most of the other pathogenicity factors are

present in E. dispar but are only weakly expressed, e.g. one study showed differences in the

6
structure and activity of pore forming peptides in E. dispar (Espinosa-Cantellano & Martinez-

Palomo, 2000; Ximénez et al., 2017). These discrepancies could to some extent explain the

differences in pathogenicity of the two closely related species, but more recent studies show that

many more factors seem to be involved in Entamoeba pathogenicity, for instance protection

against oxidative stress by peroxiredoxin (Nakada-Tsukui & Nozaki, 2016; Tazreiter, 2010).

Moreover, pathogenicity also depends on the host response during the development of the disease.

When trophozoites finally reach the liver, probably via the portal circulation, they cause typical

abscesses consisting of areas of dead hepatocytes, liquefied cells, and cellular debris (Prakash &

Oliver, 2019; Rigothier et al., 2002). The often surprisingly small number of amoebae relative to

the size of the abscess could hint to an ability to elicit apoptosis in hepatocytes without direct

contact (Tazreiter, 2010; Ali IK et al., 2012).

1.7 Transmission, diagnosis, and treatment

The foremost factors involved in the transmission of parasitic infections in developing countries

are poverty and low educational background (Karan et al., 2012). Defecation near water supplies

and indiscriminate squatting are important sources for transmission (Karan et al., 2012; El-Dib,

2017). In addition, unhygienic irrigation practices and fecal contaminated food result in high

infection rates mainly in endemic areas (Mohamed et al., 2016; Tharmaratnam et al., 2020). In

developed countries, where good sanitation, education and water supplies are available to most

members in the community, increased risk of infection is linked to infected asymptomatic food

handlers, immigrants and tourists returning from endemic areas (El-Dib, 2017; Shirley et al.,

2018). E. histolytica infections are diagnosed in the laboratory by detecting parasite in the

specimens or by immunologic or molecular technique (Ali IK et al., 2012). Metronidazole is the

7
first-choise for the treatment of amoebiasis and is recommended by WHO (Ali IK et al., 2012;

El-Dib, 2017).

1.8 Entamoeba cyst

It has been discussed earlier that amoebiasis is caused by ingesting food or water contaminated

with Entamoeba cysts. This form of parasite is responsible for disseminating from one host to

another thus spreading the disease. In the small intestine, excystation of cysts occurs releasing

trophozoites that cause for the symptoms of amoebiasis (Zulfiqar et al., 2019). Most studies on E.

histolytica infection have focused on the trophozoite stage which is relatively easy to grow in

axenic culture media. The information of the cyst stage is limited due to its incapability to replicate

and insufficiency of reproducible methods for endorsing trophozoite encystation and cyst

excystation in vitro (Naiyer et al., 2019). Currently, amoebiasis is a serious health problem globally

due to its epidemiological and clinical status. The encystation process, common within many

organisms, is defined as the process of differentiation from a reproductive form to a dormant,

resistant cyst form. Much of the knowledge gained regarding E. histolytica encystation has been

assembled using the model organism E. invadens, a reptile pathogen (Siegesmund et al., 2011).

Three main methods for inducing encystation in E. invadens are commonly used: glucose

deprivation, osmotic shock, or a combination of the two (Mi-ichi et al., 2016; Siegesmund et al.,

2011). These processes produce cysts which are similar to those of E. histolytica, having a

chemically similar composition of the cyst wall, containing four nuclei and chromatoidal bodies

(aggregations of ribosomes) which are resistant to osmotic lysis (Aguilar-Díaz et al., 2010).

However, there are noted differences between E. invadens and E. histolytica. The commonly used

E. invadens IP1 strain is “super encysting”, withstanding up to 100-fold dilutions of growth

medium and complete removal of glucose, both of which would kill E. histolytica (Herman et al.,

8
2017; Turner et al., 2007). E. invadens is also more distantly related to E. histolytica than other

Entamoeba species when 18S phylogeny is examined (Wilson IW et al., 2019; Das & Ganguly,

2014). However, little work has been conducted on these species and no model for the induction

of encystation has been discovered. Despite these differences a lot has been learnt regarding

encystation of Entamoeba parasites from the E. invadens system (Singh M. S. et al., 2015).

Developing strategies to regulate encystation and excystation will lead to novel measures to block

amoebiasis transmission by disrupting the life cycle of Entamoeba (Aguilar-Díaz et al., 2010; Mi-

ichi et al., 2016). Various chemicals that can target molecules contributing to encystation can be

effective. Ideal targets are those molecules that are exclusively present in parasite but not in host

(Mi-ichi et al., 2016). Luna-Nacar and coworkers reported the in vitro production of cyst-like

structures (CLS) having similar features like cysts such as reduction in size, round shape having

multiple nucleus and chitin wall formation together with overexpression of glucose 6-phosphate

isomerase (Luna-Nacar et al., 2016). They extracted the total proteins from trophozoites, CLS, and

partly purified cysts obtained from amoebic human patients’ feces. In trophozoites, 1029 proteins

were identified out of which 539 proteins were trophozoites specific. Similarly, 550 proteins in

CLS and 411 in cyst were identified with 299 and 84 proteins unique to each sample, respectively.

Only 74 proteins were shared by all three stages. They observed that almost 70% of proteins of

CLS were shared with trophozoites although the relative abundance is different. Proteins linked to

metabolically active cell were found abundant in trophozoites while proteins related to proteolysis,

stress response and redox homeostasis were highly expressed in CLS (Luna-Nacar et al., 2016).

Unlike trophozoites and CLS, proteomic analysis of cysts presented 40% of hypothetical proteins.

Luna-Nacar et al concluded that the nature of CLS is more like trophozoites as compared to cyst

and they could be produced as a rapid survival response of trophozoites to a stressful condition.

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These CLS allow the parasite to endure and live temporary in chitin-like resistant covering having

Jacob proteins (cyst wall-specific glycoprotein) (Frisardi et al., 2000; Luna-Nacar et al., 2016).

These findings suggested that CLS and cyst formation could be different stress responses. Also, it

has been found that cysts showed higher expression of proteins with unknown function (Luna-

Nacar et al., 2016).

Various eukaryotic parasites undergo cyst formation that transmits infection. This process is found

in several organisms such as Giardia, Entamoeba, Cryptosporidium, Toxoplasma, Naegleria, and

several nematodes and tapeworms. For enteric protozoa, the transmissible stage is the cyst which

allows survival outside the host cell. Understanding the molecular processes controlling stage

conversion is crucial for the development of transmission-blocking therapies as well as novel

diagnostics (Ali IK et al., 2012; Ehrenkaufer G. M. et al., 2007). Thus, disease transmission can

be controlled by blocking the stage conversion from trophozoite to cyst. The knowledge about cyst

form of Entamoeba histolytica is limited due to inability to produce cyst in vitro. This also hindered

the development of cyst specific diagnostic tools. To identify proteins in cyst samples and to

identify candidate proteins to develop cyst specific diagnostic tools, Ali et al. (2012) purified cysts

from stool of infected persons and proteins were identified using LC-MS/MS mass spectrometer.

A total of 417 proteins were identified out of which 195 proteins were never detected in

trophozoite-derived proteomes or EST datasets, suggesting these proteins are cyst specific. Jacob,

Jessie and chitinase, which are cyst wall specific glycoproteins (Chatterjee et al., 2009), were

positively identified. Antibodies against Jacob identified cysts in fecal specimens and have

potential value as a diagnostic reagent (Ali IK et al., 2012). Some protein kinases such as small

GTPase signaling molecules, epigenetic regulators, DNA repair proteins and surface associated

proteins were also identified. Proteins identified in this study are probably the most abundant in

10
excreted cysts and consequently show potential as diagnostic targets (Ali IK et al., 2012). This

study provided the first data for naturally occurring E. histolytica cysts and provided the vital

insights into the infectious cyst form. Moreover, several unique proteins were identified which will

assist the development of novel diagnostic markers for E. histolytica (Ali IK et al., 2012).

1.9 Change in gene expression during encystation and excystation

During early encystation, various gene families which are involved in signal transduction were

found up-regulated (Ehrenkaufer G. M. et al., 2013). These proteins include signaling molecules

like protein kinases, GTPase activating proteins and lipid signaling. These proteins may be

involved in transducing and affecting the signals that initiate encystation (Ehrenkaufer G. M. et

al., 2007; Ehrenkaufer G. M. et al., 2013). A cyst wall is formed from the secretion of lectins and

chitin although the underlying processes of membrane trafficking which support encystation is still

unclear. Evaluation of expression of membrane trafficking genes during encystation in E. invadens

identified the up-regulation of genes involved in secretion during encystation (Ehrenkaufer G. M.

et al., 2013). Moreover, in mature cyst, endocytic and phagocytic genes showed higher expression

suggesting that mRNA and other proteins are preserved in cysts in preparation for excystation. It

was found that two distinct dynamin related proteins in E. histolytica which are highly expressed

during cyst formation (Herman et al., 2017). Phylogenetic analysis of these dynamin proteins

revealed that they are paralogous but quite different from human dynamins making it attractive

target for drugs to block cyst formation (Herman et al., 2017).

Phospholipase D (PLD) is strongly up-regulated early in encystation and belongs to the class of

hydrolases (Dennis, 2015; Ehrenkaufer G. M. et al., 2013). Hydrolases initiate numerous digestive

and physiological processes. These enzymes consume a water molecule to cut down all four

biological molecules i.e. carbohydrates, proteins, fats or lipids and nucleic acid (Dennis, 2015).

11
Phospholipases are involved in the catalysis of phospholipids into fatty acids and lipophilic

substances. These enzymes cleave ester bonds within phospholipids (Dennis, 2015; Bandana et

al., 2018).

Phospholipases are primary digestive enzymes playing important role in various physiological

processes like signal transduction, lipid mediator production, metabolite digestion and aggregation

of snake venom (Aloulou et al., 2018). Phospholipids are classified into four major classes which

are distinguished based on type of reaction they catalyze.

❖ Phospholipase A (PLA) is further divided into PLA1 and PLA2 according to the specific

hydrolysis site within the phospholipid molecule (Bandana et al., 2018). The phospholipase

A hydrolyzes the carboxylic ester at the sn-1 site of the glycerol backbone is termed

phospholipase A1 while phospholipase A2 hydrolyze the sn-2 position. PLA most notably

is found in mammalian cells such as plasma of rat livers and bovine brains, and present in

metazoan parasites, protozoan parasites, and snake venom (Selvy et al., 2011).

❖ Phospholipase B (PLB) is also termed as lysophospholipase, is an enzyme having

activities of both PLA1 and PLA2, that is, it can hydrolyze the acyl-chain from both sn-1

and sn-2 positions of a phospholipid. PLB is widely distributed in microorganisms,

mammalian tissues and venom (Selvy et al., 2011).

❖ Phospholipase C (PLC) is a phosphodiesterase responsible for hydrolysis of the

glycerophosphate bond. This bond is responsible for linking the polar head group to a

glycerol backbone. PLC is found in mammal and bacteria (Kadamur & Ross, 2013; Selvy

et al., 2011).

❖ Phospholipase D (PLD) is responsible for the cleavage of the phosphate releasing

phosphatidic acid and alcohol. PLD is also considered phosphodiesterase. PLD widely

12
 present in bacteria, yeast, animals, plants, and viruses (Haas & Stanley, 2007; Selvy et al.,

2011); Two main isoforms of phospholipase D have been found in mammalian cells which

include PLD1 and PLD2, each encoded by distinct genes. PLD1 is a 120 kDa protein

mainly located on the inner membranes of cells. It is mostly present at the Golgi

complex, endosomes, lysosomes, and secretory granules. On the other hand, PLD2 is a

106 kDa protein that primarily localizes to the plasma membrane, residing in light

membrane rafts (Jenkins & Frohman, 2005)

It has been observed that during encystation and in mature cysts, basic metabolic processes such

as glycolysis were significantly decreased, and glucose metabolism is redirected to cell wall

synthesis (Ehrenkaufer G. M. et al., 2013; Naiyer et al., 2019). Similarly, numerous virulence

factors were down-regulated in mature cyst which is expected as this stage does not cause disease

symptoms in the host. Moreover, during excystation, the down-regulation of carbohydrates

metabolism observed in the mature cyst is reversed with increased transcription levels of glycoside

hydrolases as well as hexokinases (Ehrenkaufer G. M. et al., 2013).

1.10 Structure of PLD

PLD is a widely distributed enzyme involved in the fine regulation of metabolism of cells with

different organization (Leiros et al., 2000). This enzyme is necessary for growth and development

of animals and plants, especially under conditions of stress and illumination. Apart from

phospholipid metabolism, PLD enzymes are involved in vesicle formation, protein transport,

signal transduction and mitosis. One of the products of the hydrolytic action of PLD, phosphatidic

acid (PA) is a putative second messenger (Li et al., 2020). Under certain circumstances, the

formation of phosphatidic acid is also suggested to cause changes in lipid bilayer properties that

would facilitate fusion events and vesicle budding in the course of intracellular membrane traffic

13
(Kooijman et al., 2005; Leiros et al., 2000). Lipids can modify PLD in plant and animal cells. In

particular, some phospholipids usually act not as substrates but as cofactors of the enzyme

(Kolesnikov et al., 2012). The and three-dimensional structures of PLD from Streptomyces sp.

strain PMF was determined by the X-ray crystallography and structure was solved by the

multiwavelength anomalous dispersion (MAD) phasing method as shown in Figure 2 (Leiros et

al., 2000).

Figure 2. Stereographic illustration of the crystal

structure of phospholipase D (PLD) from
Streptomyces sp. Overall view of 3D-structure.

The structure is colored from dark blue to red according to the

amino acid sequence. Two flexible loops (dashed and labeled L1
and L2, accordingly) are the part of the enzyme that docks onto the
membrane when hydrolysis takes place, and two phosphate ions
(red) are found on the surface of the protein (Leiros et al., 2000).

PLD from Streptomyces sp. strain PMF consists of a single polypeptide chain that is folded into

two domains. An active site is located at the interface between these domains (Leiros et al., 2000).

The structure includes 35 secondary structure elements forming two β sheets, with nine and eight

β strands, respectively. There are 18 α helices that flank the two β sheets; two of these have a

14
somewhat irregular orientation, running diagonally on one side of each of the β sheets. An close-

up view from of PLD structure at the outer membrane view is shown in Figure 3.

Figure 3. The stereographic illustration of the

PLD in Streptomyces sp PMF. The view from the
outer membrane.

The two β sheets are sandwiched between surrounding α helices, with the more usual orientation

of the helices on the water-exposed side of each β sheet. The α helices α8 and α16–α17 lie between

the β sheets and are rotated with respect to the general strand direction. The overall structure of

PLD from Streptomyces sp. (strain PMF) is a α-β-α-β-α-sandwich (Leiros et al., 2000; Kolesnikov

et al., 2012). A 1.8 Å resolution crystal structure of the human PLD1 catalytic domain was

presented by Bowling et al (2020). It is characterized by a globular fold with hydrophobic cavity

of funnel shaped leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the

membrane interface that is crucial for activity. The C-terminus folds into and adds part of the

catalytic pocket, which form a phosphohistidine that imitate an intermediate stage of the catalytic

cycle. Mapping of PLD1 mutations that interrupt RhoA activation identifies the RhoA-PLD1

15
binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors,

allowing inhibitor design for this well-studied therapeutic target (Bowling et al., 2020).

1.11 Role of PLD in encystation

Genes encoding PLD were amongst the ones up-regulated during encystation in E. invadens

(Naiyer et al., 2019; Ehrenkaufer G. M. et al., 2013). Its activities have been observed in simple to

complex organisms and has been linked to several important biological processes such as vesicle

transport and transduction of signals which are essential for cell shape changes and proliferation.

In E. histolytica, PLD might be important for efficient cyst formation. Its regulatory role in

encystation was examined in E. invadens by observing changes in PLD activity during

development (Ehrenkaufer G. M. et al., 2013). Its level was increased during early stage of cyst

formation and falling back later in encystation supporting its active role in encystation. However,

RNA sequencing did not show peak RNA levels of PLD genes indicating that its activity is being

regulated at protein level (Ehrenkaufer G. M. et al., 2013). Furthermore, inhibition of PLD activity

was tested by suppressing the formation of phosphatidic acid by adding 0.6% n-butanol which

serves as a non-productive substrate for PLD. Marked reduction of encystation efficiency was

observed (Ehrenkaufer G. M. et al., 2013).

Most of the genes expressed in cysts encode hypothetical proteins i.e. without any known function

(Ehrenkaufer G. M. et al., 2013; Ali IK et al., 2012). These hypothetical proteins were also found

up-regulated during encystation (Ehrenkaufer G. M. et al., 2013). I screened proteins from these

studies that are up-regulated during encystation and which received very little or no work (Table

1). PLD is one of the proteins which is found to be up-regulated during encystation and very little

work has been done on it.

16
 Table 1. Proteins up regulated during encystation in E. invadens with little (one or two papers) or no published
literature
Gene ID Common Name Identity percentage with Homo sapiens Identity percentage with E. histolytica
EIN_059870 chitotriosidase-1 precursor, putative 30.06% 40.27
EIN_186850 Embryonic protein DC-8, putative 33.04% 83.61%
EIN_313910 DTDP-glucose 4,6-dehydratase, putative 47.58% 84.37%
EIN_147990 vacuolar amino acid transporter, putative 23.94% 71.76%
EIN_186080 transmembrane protein R144.6, putative 26.89% 27.80%
EIN_059470 M-phase inducer phosphatase, putative no match 40.00%
EIN_136750 glucosamine--fructose-6-phosphate aminotransferase [isomerizing], putative 32.33% 80.00%
EIN_058910 pyruvate-flavodoxin oxidoreductase, putative no match 35.42%
EIN_053310 chitotriosidase-1 precursor, putative 36.57% 58.56%
EIN_289620 carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase, putative no match 40.00%
EIN_160630 D-alanine aminotransferase, putative no match 51.27%
EIN_135440 protein TRANSPARENT TESTA GLABRA, putative 34.13% 62.38%
EIN_107320 A/G-specific adenine DNA glycosylase, putative 33.47% 60.97%
EIN_168100 ER lumen protein retaining receptor, putative no match 30.81%
EIN_104460 G2/mitotic-specific cyclin-B, putative 30.52% 41.82%
EIN_020180 ankyrin repeat-containing protein, putative 28.32% 42.79%
EIN_024440 actophorin, putative 27.78% 68.12%
EIN_034650 N-acetyltransferase ECO1, putative 21.79% 36.82%
EIN_107750 ubiquitination factor E4, putative 26.01% 51.06%
EIN_037710 protein NUF1, putative 36.54% 42.11%
EIN_243030 inhibitor of apoptosis 1, diap1, putative 47.06% 42.80%
EIN_111270 zinc finger protein DHHC domain containing protein, putative 29.71% 49.51%
EIN_196190 transcription initiation factor IIB, putative no match 52.84%
EIN_080170 metal resistance protein ycf1, putative 34.93% 36.51%
EIN_161520 proline synthetase associated protein, putative 40.09% 57.96%
EIN_311760 chaperone protein DNAJ, putative no match 33.99%
EIN_058900 branched-chain-amino-acid aminotransferase, mitochondrial precursor, putative 47.27% no match
EIN_181450 O-acyltransferase (membrane bound) domain containing protein, putative 27.50% 72.54%
EIN_160910 pyruvate-flavodoxin oxidoreductase, putative no match 35.45%
EIN_134880 long-chain-fatty-acid--CoA ligase, putative 32.23% 66.25%
EIN_289150 serine palmitoyltransferase, putative 42.39% 69.86%
EIN_111460 protein phosphatase 1E, putative 33.33% 45.50%
EIN_218620 NAD-dependent deacetylase, putative 31.66% 71.64%
EIN_147760 phosphoglycerate mutase, putative no match 59.95%
EIN_249340 meiotic recombination protein dmc1, putative 60.49% 88.47%
EIN_133760 rab7, putative 47.92% 79.70%
EIN_026170 Phospholipase D, putative 28.95% 59.06%

1.12 Aims of this study

Knowledge about the Entamoeba histolytica cyst form is limited due to the inability to produce

cysts in vitro, this also hinders the development of cyst specific diagnostic tools. Various proteins

have been found to be up-regulated during encystation, targeting these proteins can help in

developing targeted interventions to disrupt transmission.

The aim of the study was to investigate PLD1 and PLD2 genes which are up-regulated during

encystation, with a focus on unique factors that are absent in humans which therefore may provide

targeted approaches to interrupt cyst formation and transmission. The role of PLD in cyst

17
formation was investigated, as it is found to be up-regulated during cyst formation and to analyzed

whether perturbation of PLD pathway inhibits stage conversion in E. histolytica. To achieve this

aim, few objectives were made, which includes amplification of PLD1 and PLD2 from genomic

DNA of E. invadens, construction of E. invadens specific vector with amplified PLD1 and PLD2

genes, then transfection of these cloned vectors into E. invadens trophozoite cells. Furthermore,

examination of the cyst’s formation activity by addition of n-butanol and t-butanol.

18
 2 MATERIAL AND METHODS – BIOINFORMATICS ANALYSIS
In silico approaches were used for analysis of PLD protein in different ways.

2.1 Sequence retrieval of PLD

The FASTA format sequence of PLD protein were retrieved from the National Centre for
Biotechnology Information (NCBI) database (NCBI, n.d.). These sequences were selected from
different organisms such as plants, animals, fungi, Entamoeba, and protists as shown in Table 2
with accession number, organism name and group. These sequences were chosen for further
analysis.

Table 2. List of sequence retrieval of PLD

Organisms Accession
Animals
Diachasma alloeum XP_015117785.1
Nematostella vectensis XP_032241257.1
Anolis carolinensis XP_008123686.1
Erpetoichthys calabaricus XP_028656955.1
Plants
Zea mays BAA11135.1
Oryza sativa Japonica BAA19467.1
Arabidopsis thaliana AAC49656.2
Entamoeba’s
Entamoeba nuttalli P19 EKE42518.1
Entamoeba histolytica HM-1: IMSS XP_651826.1
Entamoeba dispar SAW760 XP_001735606.1
Entamoeba invadens IP1 XP_004257539.1
Fungi
Fusarium oxysporum NRRL 32931 EWY91099.1
Moesziomyces antarcticus XP_014659599.1

19
2.2 Identification of conserved sequence
Conserved sequences are those regions in a sequence which are the backbone across different
species. The FASTA format sequences were subjected one by one to Conserved Domain Database
(NCBI, Conserved Domain Database, n.d.) for the identification highly conserved region across
the organisms in PLD protein.

2.3 Multiple sequence alignment and phylogenetic tree construction

The sequences were imported into BioEdit software for alignment of PLD protein. The
revolutionary relationship among organisms is represented by a diagram which is called
phylogenetic tree. It depicts the evolution of a set of taxa from their most recent common ancestor
The phylogenetic analysis was carried out by constructing a tree using the WAG Substitution
model that includes maximum likelihood approach with phylogeny.fr online tool (Methods of
Algorithms in bio-informatique, n.d.).

2.4 Proteomics study of PLD

2.4.1 Homology modelling
Protein modelling or homology modelling is a method to compare structurally unknown protein
structure with a structurally known similar protein with above 30% identity. The structurally
similar proteins were searched on protein database such as PDB (Protein Database, n.d.). This
method is also known as comparative modelling as the protein sequences were compared with
known template structures (Krieger et al., 2003).

2.4.2 Superimposition
Super imposition of protein structure is important factor for studying its characteristics.
Superimposition is to overlay the image (template) over another image (model) to check whether
it stays or differ from the template. In PLD1 case 6ohr.1 chain A (human Phospholipase D1
catalytic domain) was been selected as template and for PLD2 6ohm.1 (human Phospholipase D2
catalytic domain) chain A were taken from protein data bank, furthermore, the constructed model
was superimposed over the template model by using Chimera tool for analysis (Chimera molecular
modeling system, n.d.). On the other hand, E. histolytica PLD sequence was taken from NCBI and
protein structure was created by SWISS model (SWISS-model, n.d.). This structure was then
superimposed on the E. invadens model and structure similarities differences were analysed.

20
2.4.3 3D model of PLD
Every sequence is determining the structure of protein and the structure determine the function of
every protein. The 3D structure of our targeted protein PLD was not present in protein data bank
(PDB) that is why we apply the homology modelling approach to predict the 3D structure of PLD.
We used online server Swiss model (https://swissmodel.expasy.org/) for homology model.
6ohr.1.A was used as a template with 54% identity and maximum coverage.

2.4.4 Validation of model (Ramachandran plot)

Ramachandran plot was proposed in 1963 by G.N. Ramachandran a biophysicist. To validate the
constructed PLD model is legitimate, Ramachandran plot was produced, that determines which
torsional angles are acceptable and can achieve perceptiveness into the protein peptide structures.

21
 3 MATERIAL AND METHODS – MOLECULAR WORK
Molecular biology work overview demonstrated as follows: Initially E. invadens trophozoite cells
were grown in LYI-S media for 14 days at room temperature (23 °C). Then the culture was ready
for genomic DNA (gDNA) isolation, which was performed by CTAB protocol specific to
Entamoeba. After gDNA isolation, PCR amplification was done with PLD 1 and PLD2 specific
primers attached with restriction sites and HA-tag. Amplified products were ligated with pGEM-
T-Easy (standard vector) and transformed into E. coli JM109. After white and blue screening mini
cultures were performed on cloned colonies. Cloned plasmids were analyzed by restriction
digestion and sanger sequencing. These steps are illustrated in Figure 4.

Figure 4. gDNA isolation, PCR amplification & cloning into pGEM-T-Easy standard vector.

22
After confirmation of PLD1 and PLD2 sequences, these genes were cloned to E. invadens specific
vector pEiNEO which was firstly digested by BamHI and KpnI restriction enzymes for removal of
LUC gene from the vector to ligate PLD1 and PLD2 into it. After ligation and transformation to
E. coli JM109, different colonies were chosen, and mini cultures were prepared. Mini cultures led
to isolation of cloned plasmids which were PLD1+pEiNEO and PLD2+pEiNEO. Before
transfection, to ensure the presence of gene of interests (PLD1 and PLD2), these cloned plasmids
were confirmed by restriction double digestion. E. invadens trophozoite cells were then transfected
by successful cloned plasmids by use of electroporation technique. These steps layout can be seen
in Figure 5. For screening the transfected E. invadens cells, G418 was added, respectively.

Figure 5. Cloning into pEiNEO (E. invadens vector)

23
Second part of the molecular work was to check the activity of n-butanol and t-butanol in cyst
formation. E. invadens trophozoite cells were transferred to 50% LYG media wild type, 50% LYG
media containing 0.6% n-butanol and 50% LYG media containing 0.6% t-butanol. After 24 hrs,
48 hrs, and 72 hrs, cell samples were taken out from the respectively medium and were stained
with Calcofluor white stain with 4% paraformaldehyde fixation at the end, just before 72 hrs 0.05%
sarkosyl was added to remove remaining trophozoite cells. These stain cells were then observed
under 63x objective lens of Leica TCS SP8 FALCON confocal microscope for chitin formation in
cyst. These steps are shown in Figure 6.

Figure 6. Cysts formation activity in three different 50% LYG media such as wild type, n-butanol & t-butanol

24
3.1 LYI-S media for culturing Entamoeba invadens
Table 3. LYI-S medium composition

Component's Quantity
K2HPO4 1g
KH2PO4 0.6 g
NaCl 2g
Yeast extract 25 g
Liver digest neutralized 5g
Glucose 10 g
Cysteine 1g
Ascorbic acid 0.2 g
Ferric ammonium citrate 0.045 g/ 2 ml
Sodium hydroxide 1 M to adjust pH 6.8
Distilled water 880 ml (final volume)

All the components in Table 3 were dissolved and aliquoted 88 ml in 10 borosilicate glass of 250
ml followed by autoclaving at 121 °C for 15 mins. Afterwards, media was stored at -20 °C.

Frozen adult bovin serum (biovit) was heat inactivated by letting the tube in 56 °C water bath for
30 mins. Before using the media, in one of the 88 ml media flask, 15 ml heat inactivated adult
bovine serum was added along with 2 ml vitamin Mixture#18 as described by Clark and Diamond
(DIAMOND & CLARK, 1993). Further media was distributed in small glass tubes.

3.2 Culturing of E. invadens

Entamoeba invadens IP1 (kindly provided by Prof. Avelina Espinosa, Roger Williams University,
USA) trophozoites cells were grown axenically in LYI-S media containing heat inactivated adult
bovine serum and vitamin mixture #18 (recipe shown in Appendix A). 0.5 ml of cultured cells
were inoculated in 15 ml of media in glass tube. They were incubated at 23 °C at upright position
for two weeks.

25
3.3 Trophozoite cell culture storage
1 ml of E. invadens cell culture was poured in 2 ml Eppendorf tube and centrifuged for 1 min at
500 x g. Supernatant was discarded and pellet was resuspended in 1 ml sterile water. Resuspension
was performed for 15 mins and later the cells were kept at -20 °C for further use.

3.4 Recombinant DNA technology steps

Recombinant DNA technology is a technique of joining of DNA molecules from two different
species that are inserted into a host organism to produce new genetic combinations that are of value
to science, medicine, agriculture, and industry. Recombinant DNA technology comprises the
following steps.

• Isolation of genetic material

• Amplification using PCR
• Ligation of DNA molecules
• Insertion of recombinant DNA into host
• Enzyme restriction digestion
• Isolation of recombinant cells

3.4.1 Growing E. coli JM109

JM109 is a K strain that is recA– and endA– to minimize recombination and improve the quality
of plasmid DNA. In addition, the cells carry the F´ episome, which allows blue/white screening.
E. coli JM109 frozen stock was kindly provided by Prof. Cathrine Lilo’s lab and inoculated on
fresh LB agar plates. Plates were left at 37 °C incubator for overnight.

3.4.2 Preparation of competent cells

Competency is defined as the ability of the cell to take up DNA. Competency can be induced by
chemical method by using divalent or multivalent cations, for example, calcium, magnesium,
manganese etc. These cations cause alternation in the permeability of the membranes (outer, inner
membrane and cell wall), allowing the DNA to cross the cell envelop. Treatment of E. coli cells
with CaCl2 at low temperature is the most used method to allow the cells to uptake naked foreign
DNA. The selected colony from LB agar plates plated with E. coli JM109 was incubated on 5 ml
LB broth media at 37 ℃ overnight on 240 rpm shaker. Later, 500 µl from the overnight culture
was added into the 50 ml LB media in Erlenmeyer flask. The culture was incubated at 37 ℃ shaker

26
for about 2-3 hours until the OD600 reached between 0.4 and 0.5. Afterwards, the sample distributed
equally into two centrifuge tubes with labels. Cooled on ice and centrifuged for 10 min at 2509 x
g (4 ℃). After discarding the supernatant, the pellet was treated with 5 ml ice-cold 0.1 M MgCl2
followed by spinning and the supernatant was discarded. Before mixing the sample in a new tube,
5 ml ice-cold 0.1 M CaCl2 was added into each centrifuge tube and again spined down. The cells,
without the supernatant, were resuspended in 500 µl MOPS glycerol and 100 μl competent cells
were aliquoted in Eppendorf tubes and stored at -70 ℃ until use.

3.4.3 Transformation
Bacterial transformation is the direct uptake of exogenous DNA resulting in the acquisition of new
genetic traits. Transformation of bacterial cell is mostly carried out by CaCl2 heat shocked method.
The CaCl2 neutralizes the charges on DNA and bacterial outer layer and cold-heat shock helps to
modify the fluidity of membrane and allow the DNA molecule to enter the cell. 40 ml LB agar
containing 40 µl of ampicillin antibiotic was poured in two petri plates. On other hand, 5 µl of
pEiNEO-LUC vector (grateful to Prof. Sudha Bhattacharya, Jawaharlal Nehru University, India.
for providing E. invadens specific vector) was drawn into 100 µl of E. coli JM109 competent cell.
Incubated on ice for 1.5 hrs followed by heat shock at 42 °C for 1 min and again on ice for 5 mins.
Afterwards 750 µl of LB media was poured over same Eppendorf tube. The tunes were incubated
for 1 hour at 37 °C on shaker. Media was then spread on the LB agar plates with 100µg/ml
ampicillin, which was then incubated for overnight at 37 °C.

3.4.4 Plasmid isolation

Plasmids are very beneficial for bacterial cells because they contain certain genes which allow
bacteria to survive in stressful environment. As plasmid is present inside the cell along with other
cell components such as genomic DNA, RNA, and proteins, therefore these molecules must be
removed in order to isolate pure plasmid. The purification or extraction of plasmid has three basic
steps; lysis of the cell followed by neutralization of the lysed cell for the renaturation of plasmid
DNA and precipitation of plasmid. Alkaline lysis method (Sambrook & Russell, 2006) is the most
widely employed method for plasmid isolation and many commercially available kits are also
based on this method. Transformed colony of E. coli JM109 containing pEiNEO-LUC plasmid
was grown in LB media with 100 µg/ml ampicillin. At their stationary phase i.e after 16 hrs of
cultivation, the cells were harvested by centrifugation at 14000 x g for 1 min. Plasmid DNA

27
purification was followed by manufacturer’s instruction in Nucleospin® Plasmid kit (Macherey-
Nagel company). Plasmid were stored at -20 °C for further use.

3.4.5 Vectors for cloning

Two separate vectors were used in this research project. One was standard vector pGEM-T-Easy
vector and second Entamoeba specific vector pEiNEO-LUC.

A: pGEM-T-Easy vector

This is a linearized 3 kbp size, commercially available vector (Figure 7) that contains 3´-T
overhangs at the insertion site for compatible overhang towards PCR products. Universal T7 and
SP6 RNA polymerase promoters are present within the α-peptide coding region for β-
galactosidase, which helps in identification of Blue/White screening on plates by insertional
inactivation of these peptides.

Figure 7. Commercially available Promega pGEM-T-Easy vector map Taken from SnapGene.

Showing different restriction sites, multiple cloning sites for PCR products, having
ampicillin resistance gene and universal T7, SP6 promoter sites.

28
B: pEiNEO-LUC vector

This is a non-commercial E. invadens vector 8.1 kbp size, modified from pBlueScript and kindly
provided by Prof. Sudha Bhattacharya (Jawaharlal Nehru University, India) (Singh N. O. et al.,
2012). This vector contains S10 promotor for E. invadens ribosomal protein represented as
neomycin phosphotransferase gene (NEO) of approximately 0.8 kbp, L3 promotor which controls
luciferase reporter gene (LUC) of 1.6 kbp and having two restriction sites of KpnI and BamHI
(Figure 8). The gene of interest (PLD1 and PLD2 HA-tagged) to be inserted were added to the
vector at LUC gene site, which was removed by using restriction digestion with KpnI and BamHI.

Figure 8. E. invadens specific vector pEiNEO-LUC

Figure taken from Dr Maulood Turfah University of Exeter Phd thesis. This illustration based
on (Singh N. O. et al., 2012).

3.4.6 pEiNEO-LUC restriction digestion

As mentioned in previous section (3.4.5), the pEiNEO-LUC vector contains luciferase gene which
is around 1.6 kbps and was needed to be removed for insertion of desired gene (PLD1 and PLD2).
For this purpose, pEiNEO-LUC vector was digested by two different restriction enzymes known
as BamHI and KpnI. Reaction mixture for the double digestion was prepared according to the
following Table 4, which was then incubated at 37 °C for 2 hrs. Afterwards, the reaction mixture
was ran on 1% agarose gel using TAE buffer for 40 mins at 90 V. The gel was observed under UV

29
radiation and 6.1 kbp band (plasmid pEINEO without LUC gene) cut out the gel. The same Qiagen
Gel cleanup Kit and its instructions were used for removal of other impurities.

Table 4. Reaction mixture for the double digestion of pEiNEO-LUC plasmid

Reagents Volume (µl)

1X fast digest buffer 2

Fast digest BamHI 1

Fast digest KpnI 1

pEiNEO-LUC vector Up to 1 µg

Sterilized Water Up to 20 µl

3.5 DNA extraction

Isolation of DNA from E. invadens trophozoite cells

The fast CTAB (cetyl trimethylammonium bromide) method was used for genomic DNA isolation
from E. invadens trophozoite cells as described by Ali (Ali et al., 2005). This method was used for
removal of carbohydrates and polysaccharides from cells which inhibits the PCR reaction. Initially
cultured cell tube was placed on ice for 10 mins and centrifuged at 2000 x g for 5 mins. The pellet
was washed with sterile water and transferred to sterile 1.5 ml Eppendorf tube. Afterwards, 250 µl
lysis buffer (0.25% SDS in 0.1M EDTA pH 8.0) and 100 µg/ml Proteinase K (1.4 µl from stock
of 21.5 mg/ml from Sigma manufacture) was added to the culture pellet. The tube was vortexed
and incubated at 55 °C for 20 mins. After incubation, 75 µl of 3.5 M NaCl was added and mixed.
Then, preheated 10% CTAB in 0.7 M NaCl at 55 °C was added to the pellet tube for about 42 µl,
mixed and incubated at 65 °C for 10 mins. Later, 400 µl chloroform was added and mixed by
inversion. The tube was centrifuged at maximum speed for 5 mins. The supernatant was transferred
to new sterile 1.5 ml microcentrifuge tube. 400 µl of phenol:chloroform:isoamyl alcohol (25:24:1)
was added, mixed by inversion and centrifuged as described above. This results in supernatant and
pellet in liquid phases. The supernatant whitish liquid phase was transferred to new sterile
centrifuged tube and 2 volumes of 100% ethanol was added to it, mixed properly and stored at
room temperature for 5 mins. The tube was again centrifuged for 10 mins as mentioned above and

30
this time supernatant was discarded carefully from the pellet. Pellet was washed with 200 µl of
70% ethanol and centrifuged for 5 mins at maximum speed. Later the pellet was air dried,
resuspended in 50 µl sterile water and left for overnight at 4 °C. By use of GC-Health care
MicroSpin S-200 HR Columns, the resuspended DNA was passed over the column and the pellet
then used for further PCR and recombinant DNA steps.

3.6 Polymerase chain reaction amplification

Polymerase chain reaction (PCR) is a fast and economical method used to copy small segments of
DNA as significant amounts of DNA are essential for molecular and genetic analyses, studies of
isolated pieces of DNA are approximately impossible without PCR amplification. Reaction
mixture was prepared having volume of 25 µl by adding 14.75 µl nuclease free water, 2.5 µl of
dNTP’s, 2.5 µl of DreamTaq buffer, 2 µl for each forward and reverse primers (Table 6), 1 µl of
DNA template and 0.25 µl of DreamTaq polymerase. Negative control was also used having 15.75
µl of nuclease free water. The PCR cycling steps used in this research are summarized in Table 5
as given below.

Table 5. PCR conditions using DreamTaq Polymerase for DNA template amplification

Cycle Steps Time Temperature

1 3 min 95 °C

2 0:30 min 95 °C

3 0:30 min 60 °C

4 2 min 72 °C

5 34x step 2 95 °C

6 10 min 72 °C

7 Forever 4 °C

31
Primers used for PCR amplification given in Table 6.

Table 6. Specific primers used for gene amplification (uppercase indicates perfect match with target gene; lowercase
indicates newly introduced sequence with introduced restriction sites indicates by italics).

Names Primers
Ei_DLP1-KpnI-F (PLD1 forward Primer) aga aga ggt acc ATG AAG AAG ATT TTG
TGG AGA G
Ei_DLP1-BamHI-R-HA (PLD1 Reverse tct tct gga tcc cta TGC ATA GTC TGG AAC
Primer) GTC ATA TGG ATA CTC AAC CAT AAT
GTT TCC G
Ei_DLP2-KpnI-F (PLD2 forward Primer) aga aga ggt acc ATG CGA AGC CTT TAC
TAT CGC C
Ei_DLP2-BglII-R-HA (PLD2 Reverse tct tct aga tct tta TGC ATA GTC TGG AAC
Primer) GTC ATA TGG ATA GGT GAC TAT AGC
CGT CAG TCC

3.6.1 Agarose gel electrophoresis

Electrophoresis is the technique which is used to separate macromolecules especially nucleic acids
based on size, conformation, and charge. Nucleic acids move towards the anode due to negative
charge present on it when placed in electric field. DNA has negative charge due to the presence of
phosphate backbone. Due to this negative charge and pH of the buffer (8 or above), the DNA
moves towards the anode.

The success of DNA extraction was confirmed by agarose gel electrophoresis. The PCR amplified
product was ran on 1% agarose gel using 1% TAE buffer. 1% agarose gel was prepared in 1x
TAE/TBE buffer by adding the mixture in an Erlenmeyer flask. The mixture was dissolved by
heating it in an oven for 1 min, then letting it cool down to about 50 °C. Gel stand was arranged
by placing the comb in the stand and the agarose gel solution was poured in it and left for
solidification. The samples were loaded in the wells with appropriate amount of loading buffer and
Gel Red (Sigma-Aldrich). The gel was ran with the voltage of about 90 V for 45 mins. Later, the
gel was analyzed under UV radiation by ChemiDoc Touch Imaging system.

32
3.6.2 Purification of PCR products
To ensure that only the main band gets sequenced, purification of PCR products was carried
out. PCR amplified product which were PLD1 and PLD2 of appropriate molecular size were cut
off from the agarose gel with the help of clean scalp and placed in sterilized 1.5 ml Eppendorf
tube. Gel bands in two tubes were weighed and processed according to Qiagen QIAquick Gel
Extraction kit. The kit protocol includes dissolving of agarose gel band in buffer QG at 50 °C for
10 mins, vortexed and followed by addition of isopropanol. Later passed through the kit provided
column and washed by addition of PE buffer, which was then eluted with sterilized milli-Q water
in new 1.5 ml centrifuged tube.

3.7 Cloning of purified PCR products into vectors

PCR cloning is a rapid method for cloning genes, and it allows for the cloning of DNA fragments
that are not available in large amounts. A PCR reaction is performed to amplify the sequence of
interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to
transformation. Early PCR cloning often used Taq DNA Polymerase to amplify the gene. This
results in a PCR product with a single template-independent base addition of an adenine (A)
residue to the 3' end of the PCR product, through the normal action of the polymerase. These "A-
tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase,
followed by transformation.

3.7.1 Standard vector (pGEM-T-Eeasy)

Ligation reactions were prepared as described in Table 7 (Promega kit) and incubated at 4 °C for
overnight. Later, the plasmid construct was centrifuged and mixed with 50 µl E. coli JM109
competent cells (which were thawed on ice for 5 mins), and that was followed by incubation on
ice for 20 mins. After that heat shock was given at 42 °C for 50 sec and immediately reactions
were returned to ice for 2 more minutes. 950 µl SOC media was added to each ligation reactions,
which were then incubated on shaker (150 rpm) at 37 °C for 1 hr. and 30 mins. 100 µl of each
transformant cultures were plated on LB Agar containing ampicillin, IPTG and X-Gal for white,
blue screening of the cells. Lately, the plates were incubated at 37 °C for overnight.

33
 Table 7. Setup of ligation reaction for pGEM-T-Easy vector and PLD1/PLD2

Reagent Negative control Positive control Reaction mixture

(µl) (µl) (µl)
2X ligation buffer 5 5 5

T4 DNA ligase 1 1 1

pGEM-T-Easy vector 1 1 1
(50 ng)

PCR product (3:1) - - 2

Control insert DNA - 2 -

Deionized water up to 10 10 10
final volume

3.7.2 E. invadens vector (pEiNEO)

The same process for cloning E. invadens vector with PLD1/PLD2 was performed as described in
previous section, and the setup for reaction mixture was followed as given in Table 8.

Table 8. Setup of ligation reaction mixture for pEiNEO vector and PLD1/PLD2

Reagent Negative control Positive control Reaction mixture

(µl) (µl) (µl)
2X ligation buffer 5 5 5
T4 DNA ligase 1 1 1
pEiNEO vector (50 1 1 1
ng)
PCR product (3:1) - - 2
Control insert DNA - 2 -
Deionized water up to 10 10 10
final volume

34
3.8 Mini cultures for mini prep
Transformed cells were inoculated in 5 ml LB broth containing 5 µl 100 mg/ml ampicillin, which
were allowed to grow overnight at 37 °C in shaking incubator (220-250 rpm). The next day,
isolation of plasmid DNA was performed by using Nucleospin Plasmid kit (Macherey-Nagel
company), which includes cell lysis, neutralization of lysed cells and removal of protein and cell
debris by centrifugation. The impurities were removed by using the provided column and later
plasmid DNA was eluted with distilled water which was stored at -20 °C until further use.

3.9 NanoDrop One

The DNA concentration was determined by using Thermo Scientific Nano Drop One machine.
After selecting the appropriate option for the sample (DNA), 2µl of DNA samples were pipetted
(blanking it with the correct elution buffer). The machine was cleaned every time with soft tissue
before the examination of a new sample.

3.10 Subsequential restriction digestion

For confirmation of plasmid containing gene of interest, restriction digestion was performed on
both pGEM-T-Easy vector containing PLD1/PLD2 and pEiNEO with PLD1/PLD2. The double
digestion was done by using Fast digest enzymes from Thermo Scientific and their protocol for
plasmid digestion as mentioned in Table 9. For PLD1 gene; BamHI and KpnI restriction enzymes
were used, while for PLD2; BglII and KpnI used.

Table 9. Reaction mixture for restriction digestion

Reagents Volume (µl)

1X fast digest buffer 2

Fast Digest BamHI (PLD1) / BglII (PLD2) 1

Fast digest KpnI 1

pEiNEO-LUC/pGEM-T-Easy vector containing gene of interests Up to 1 ug

Sterilized Water Up to 20

35
3.11 Long term storage of bacterial clones
Bacterial clones were prepared by adding 500 µl of 50% sterilized glycerol to 500 µl culture broth
and they were stored as -80 °C.

3.12 DNA sequencing

To verify the presence of desired inserts in the plasmid, the plasmids were sent for sequencing,
which was performed by Microsynth Seqlab GmbH. Universal SP6 forward and T7 reverse
primers were used for sequencing the pGEM-T-Easy based construct while designed Rpl3 forward
(GCA GAT GTC GCA CAG AAA GTT TG) and Rpl3 reverse (GAT AAC TTA GAG TTT CTT
TCC GG) primers were used for pEiNEO based construct containing desired inserts.

3.13 Transfection
Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods.
Using various chemical or physical methods, this gene transfer technology enables the study of
gene function and protein expression in a cellular environment. The transfection strategies can
generally be classified into two types based on whether the introduced nucleic acid exists in the
cell for a limited period of time (transient transfection) or whether it persists in the cells long-term
and is passed to the progeny of the transfected cell (stable transfection). E. invadens trophozoite
cells were grown for 7 to 8 days and then the tube containing these cells was placed on ice to
harvest these cells at 500 x g for 5 mins at 4 °C. After harvesting, the cells were washed with PBS
and, with an incomplete cytomix buffer to have better stability. Subsequently, the washed cells
were suspended in 600 µl of cytomix buffer (recipe in Appendix A), which was completed with 2
mM ATP and 5 mM reduced glutathione. This cell suspension was transferred to electroporation
cuvette (0.2 mm) which was followed by the addition of 60 µg plasmid DNA. Later, it was placed
in Bio-Rad Gene Pulser Xcell Electroporation systems shock pod and 1500 V intensity of two
consecutive pulses were applied with the capacitance of 25 µF. These transfected cells were
transferred to 13 ml glass tube containing LYI-S medium as well as heat inactivated adult bovine
serum (15%), vitamin mix #18 (2%), and penicillin-streptomycin solution (100 µg/ml). For
selection of transfected cells, G418 antibiotic (10 µg/ml) was added after 24 hrs to the culture
medium and this medium was refreshed in intervals of 72 hrs.

36
3.14 Encystation of E. invadens
50% LYG media (Table 10) was used for encystation of E. invadens trophozoite cells.

Table 10. 50% LYG media composition

Component's Quantity
K2HPO4 0.20 g
KH2PO4 0.12 g
NaCl 0.4 g
Yeast extract 5g
Liver digest neutralized 1g
Cysteine 0.2 g
Ascorbic acid 0.04 g
Ferric ammonium citrate 0.04 g/ ml
Sodium hydroxide 1 M to adjust pH 6.8
Distilled water 380 ml (final volume)

Media was aliquoted in 95 ml glass bottles and were autoclaved at 121 °C for 15 mins for
sterilization. Later, before using the media, it was completed by addition of 5 ml adult bovine
serum and 2 ml vitamin mix #18.

Trophozoites cells were allowed to grow at room temperature for 7 days, which were then left on
ice for 10 mins, followed by centrifugation at 400 x g for 5 mins. The cells pellet were then ones
washed with incomplete 50% LYG and then centrifuged same as above. After centrifugation, the
cells pellet was suspended in complete 50% LYG media. This media will allow the trophozoite
cells to grow and transform to their cysts stage.

3.15 Calcofluor staining and fluorescence microscopy

Calcofluor stain is a fluorescent stain for rapid detection of yeasts, fungi, and parasitic organisms.
Calcofluor White is a non-specific fluorochrome that binds to cellulose and chitin in cell walls.
Immunofluorescence (IF) microscopy is a form of immunohistochemistry based on the use of
fluorophores to visualize the location of bound antibodies. When transferring the E. invadens
trophozoite cells to the 50% LYG media, cells samples were taken at different time intervals such

37
as 24 hrs, 48 hrs and 72 hrs. Cells were centrifuged at 500 x g for 5 mins and then pellet was
washed with 1X PBS. Afterwards, cells were suspended in 1% calcofluor white (Sigma-Aldrich)
stain for 30 mins, washed again with PBS. Furthermore, fixed by 4% paraformaldehyde and lastly
200 ul PBS was added to pellet. The cells were kept in fridge for later fluorescence microscopy.

3.16 n-Butanol and t-Butanol activity on PLD

Two 15 ml 50% LYG media tubes were taken having 100 µg/ml penicillin-streptomycin
antibiotics. In addition to the media, one tube had 0.6 % n-butanol, and another contained 0.6 %
tert-butanol (as negative control). Afterwards, 1×105 trophozoite cells were added in each tube and
incubated at room temperature for 72 hrs. Cell samples were taken at different time intervals 24
hrs, 48 hrs and 72 hrs. Before sample collection for staining at 72 hrs, 0.05% sarkosyl was added
to each tube and incubated for 1 hr. Afterwards, they were stained with calcofluor white and fixed
with paraformaldehyde as described in previous section (3.15).

38
 4 RESULTS
4.1 Bioinformatics analysis
PLD evolutionary relationship with other organisms via phylogenetic tree analysis and its protein
structure were analyzed by use of different tools and software’s.

4.1.1 Conserved sequences in PLD

Using NCBI database, total 13 sequences in FASTA format were retrieved and then these
sequences were applied in CDD database to identify the conserved sequence from all selected
organisms from different groups. PLN02866 was identified as the most conserved domain in all
species.

4.1.2 Phylogenetic tree

The phylogeny of PLD across different organism were constructed by Phylogeny.fr. The tree
showed a clear separation of four clades further dividing into sub-clades, the alignments lead to
phylogenetic tree construction and showed the relationship between different species from
different groups as shown in Figure 9.

Figure 9. PLD phylogenetic tree generated from Phylogeny.fr and editied in Figtree

39
4.1.3 Proteomics work
4.1.4 3D model of E. invadens PLD
The model of PLN02866 super family domain of PLD was developed by using online source server
Swiss model. The model showed 54% of similarities with reference crystal structure and maximum
coverage. The theoretical 3D model of PLD PLN02866 super family domain is shown in Figure
10.

Figure 10. 3D model of E. invadens PLD, superfamily domain

PLN02866.

4.1.5 Superimposition of PLD1 protein models

The superimposition of PLD1 template (6ohr.1 human Phospholipase D1 catalytic domain) and E.
invadens model was done by chimera, where the green color represented the template and the red
colour represented the model of our targeted protein (Figure 11), both the template and model
superimposed, in which template had 4566 atoms and 577 residues where as inPLD1 model it was
4343 atoms and 542 residues. The structures are overlapping well in nearly all secondary
structurally elements.

Figure 11. Superimposition of PLD1 template (6ohr.1 chain A) in light blue

and E. invadens PLD1 model in golden.

40
The constructed models using Chimera for PLD1 of E. invadens and E. histolytica were
superimposed by using chimera. It showed that the residues in E. histolytica model were 544
residues and 4464 atoms. In Figure 12, orange color represents E. invadens PLD model while dark
blue color represents E. histolytica PLD model.

Figure 12. Superimposition of E. invadens PLD in orange and E. histolytica constructed protein model
in dark blue.
4.1.6 Superimposition of PLD2 protein models
For PLD2 the template was a crystal structure of 6ohm.1 human Phospholipase D2 catalytic
domain in brown and constructed E. invadens PLD2 model in light blue in Figure 13. Both
structures were superimposed, where 6ohm.1 structure had 5251 atoms and 1153 residues while
PLD2 model had 4326 atoms and 535 residues.

Figure 13. Superimposition of E. invadens PLD2 model in orange and 6ohm.1 template in purple.

On other hand the superimposition between E. histolytica and E. invadens showed in Figure 14.
4359 atoms and 532 residues were found for PLD2 E. histolytica model.

41
 Figure 14. Superimposition of E. invadens PLD2 model in forest green and E. histolytica in yellow.

4.1.7 Validation of model (Ramachandran plot)

In Ramachandran plot, the protein confirmation is displayed graphically where specific region
were assigned. Different portion in the plot were the most favorable, generously allowed and
disallowed regions which are indicated by different color scheme. For ideal structure it is needed
to have 90% of residues in core region (Ho & Brasseur, 2005).

Our result showed that most of PLD residues are located on core region (Figure 15). This
orientation is suitable for phi-psi values. About 87% residues were in a favored region and 11%
were in allowed region. Approximately 1.02% were present on outlier region.

Figure 15. Ramachandran Plot

Ramachandran plot of E. invaden PLD structure showed 87.8% residues in favorable region while 0.2%
residues in disallowed region. Plot statistics are presented in Table 11

42
 Table 11. Ramachandran plot statistics for constructed PLD model

Plot statistics

Residues in most favored regions (A, B, L) 430 87.8 %

Residues in additional allowed regions (a, b, l, p) 54 11.0 %

Residues in generously allowed region (~a, ~b, ~l, ~p) 5 1%

Residues in disallowed regions 1 0.2 %

Number of non-glycine and non-proline residues 490 100 %

Number of end residues (excl. Gly and Pro) 2

Number of glycine residues (shown as triangles) 40

Number of proline residues 14

Total number of residues 546

4.2 Molecular Work

E. invadens trophozoites cells were grown at room temp (22 °C) in 15ml LYI-S medium for 2
weeks. Following results were analyzed from these parental cells.

4.2.1 Isolation and PCR amplification of E. invadens genomic DNA

The genomic DNA isolated from E. invadens trophozoite cells having concentration 238.9 ng/µl,
was amplified directly by using specific primers. The PCR product resulted in two genes of interest
with their predicted sizes: PLD1 1.7 kbp and PLD2 1.6 kbp. They were analyzed on 1% agarose
gel via Gel electrophoresis as shown in Figure 16.

43
 Figure 16. PCR amplification from E. invadens genomic DNA

Lane 1: L= Bioline 1 kb Ladder, Lane 2: Isolated genomic DNA from E. invadens trophozoite cells, Lane 3:
Negative PCR control for PLD1, Lane 4: Amplified PCR product PLD1, Lane 5: Negative PCR control for PLD2,
Lane 6: Amplified PCR product PLD2.

4.2.2 Determination of DNA concentration using NanoDrop One

The DNA samples concentration were determined by using Thermo Scientific NanoDrop One as
shown in Table 12. The One’s selected for further experiments were pGEM-T-Easy+PLD1 vector
(Plasmid purification) from colony 1, whereas pGEM-T-Easy+PLD2 vector (Plasmid purification)
from colony 1.

44
 Table 12. Concentration of pGEM-T-Easy vector+PLD1/PLD2 insert using Nanodrop ONE

Sample Name DNA Sample Type

Concentration
1. colony 1 pGEM-T-easy+PLD1 225.7 ng/µl DNA
2. colony 2 pGEM-T-Easy+PLD1 200.0 ng/µl DNA
3. colony 1 pGEM-T-Easy+PLD2 189.2 ng/µl DNA
3. colony 2 pGEM-T-Easy+PLD2 223.3 ng/µl DNA
For E. invadens vector, pEiNEO+PLD1 vector (Plasmid purification) with concentration 345.2
ng/µl, whereas pEiNEO+PLD2 vector (Plasmid purification) having concentration 241.3 ng/µl
were been selected for transfection into E. invadens trophozoite cells. Table 13 shows the
concentrations of selected pEiNEO cloned plasmid for transfection.

Table 13. Concentration of pEiNEOvector+PLD1/PLD2 insert using Nanodrop ONE

Sample Name DNA Sample Type

Concentration
1. colony 1 pEiNEO-PLD1 345.2 ng/µl DNA

2. colony 1 pEiNEO-PLD2 241.3 ng/µl DNA

4.2.3 DNA fragments on gel after restriction digestion

For confirmation of cloned vectors that either they contain gene of interest or not, subsequential
restriction digestion was performed. The restriction digestion products were analyzed on 1%
agarose gel as shown in Figure 17. All cloned confirmed the presence of gene of interest. The
undigested cloned vectors are shown in lane 1 of Figure 17 (a, b, c, d). Lane 2 and 3, Figure 17(a),
after single digestion with KpnI or BamHI, the clear shift in migration of plasmid is shown, which
size is 4.7 kbp. Whereas Lane 4, the double digestion by KpnI and BamHI shows the appearance
of two band of 1.7 kbp and 3 kbp size, respectively. For Figure 17 (b) pEiNEO+PLD1 vector, Lane
2 and 3 after single digestion with KpnI or BamHI, the migration of cloned plasmid is shown,
which size is 8.2 kbp. While Lane 4, the double digestion by KpnI and BamHI shows the presence
of two band of 1.7 kbp and 6.5 kbp size, respectively. In Figure 17 (c), single digestions with KpnI
and BgIII in Lane 2 and 3 respectively, gave 4.6 kbp size bands of cloned pGEM-T-Easy with
PLD2, while the double digestion results in two bands of sizes 1.6 kbp and 3 kbp. Figure 17 (d),

45
Lane 2 and 3 represent the band of 8.1 kbp, which appeared after single digestion with KpnI and
BgIII. Lane 4 shows the double digestion of pEiNEO+PLD2 with band sizes 6.5 kpb (pEiNEO
vector) and 1.6 kbp (PLD2 gene).

Figure 17. Sub-sequential restriction digestion

Restriction digestion was analyzed on 1% agarose gel where: (a) gel representing the restriction
digestion of pGEM-T Easy + PLD1, (b) gel representing the restriction digestion of pEiNEO +
PLD1, (c) gel representing the restriction digestion of pGEM-T Easy + PLD2, (d) gel representing
the restriction digestion of pEiNEO + PLD2
46
4.3 Sequencing results
The sequences results were compiled, and alignment was performed using Clustal Omega multiple
alignment tool online. The DNA sequence was first translated to their protein and then alignment
was performed with the reference protein sequence. pGEM-T-Easy vector cloned with PLD1 and
PLD2 were confirmed by sequencing result that the vector contains desired gene of interest.
(Figure 18 and Figure 19). Alignment can be seen under appendices Appendix B: Sequence
alignment of cloned pGEM-T-Easy and PLD1/PLD28.2 (Appendix B).

Figure 18. pGEM-T-Easy vector cloned with PLD1 gene constructed by using Snapgene.

Insert in red color: amplified PLD1 with forward and reverse primers.

pGEM-T-Easy vector in black color: standard linearized vector for

TA cloning

47
 Figure 19. pGEM-T-Easy vector cloned with PLD2 gene constructed by using Snapgene

Insert in red color: amplified PLD2 with forward and reverse primers.

pGEM-T-Easy vector in black color: standard linearized vector for TA

cloning

4.3.1 Encystation
The encystation study of E. invadens was carried out and the effect of 0.6% n-butanol on PLD
activity was also tested as it is considered to repress the activity of PLD. Same amount of t-butanol
was also used as control encystation media. The encystation was allowed to proceed for 24, 48 and
72 hrs. Chitin production was observed by fluorescence microscope at mentioned time intervals
(Table 14). No effect of n-butanol on encystation was found. Same number of cysts cells were
found in wild type, n-butanol cultures and in control (Table 15). In wild type cultures, about 32%
cyst was formed after 24 hrs, 66% after 48 hrs and 93% after 72 hrs in stained sample. In n-butanol
cultures, about 36% cyst was formed after 24 hrs, 57% after 48 hrs and 100% after 72 hrs in stained
sample. In n-butanol cultures, about 28% cyst was formed after 24 hrs, 52% after 48 hrs and 100%
after 72 hrs in stained sample as shown in Figure 20. The experiment was performed once.

48
 Table 14. Cysts chitin observation by using bright field and fluorescence microscope

Media 24 hrs 48 hrs 72 hrs

type Bright field CFW stain Bright CFW stain Bright CFW stain
field field
Wild type

n-Butanol

t-Butanol

Table 15. Encystation percentage (%) of E. invadens at different time intervals

Time Wild type N-Butanol T-Butanol

24 hrs 32 36 28
48 hrs 66 57 52
72 hrs 93 100 100

Figure 20. Cysts formation at different time interval.

W.T= wild type 50% LYG media, N.B= 0.6% n-butanol added to 50% LYG media, T.B= 0.6% t-butanol added to 50% LYG
media, whereas cells for each sample were analysed at different time intervals such as 24 hrs, 48hrs and 72 hrs.

49
 5 DISCUSSION
Being an important protozoan parasite, knowledge of different stages of its life cycle are of
immense importance in interrupting the life cycle of Entamoeba. One such conversion stage is
encystation, which was the main research topic of this thesis. Encystation in Entamoeba has the
potential to be targeted in stopping infection and interpreting the life cycle, hence turning down
the transmission of this parasite (Aguilar-Díaz et al., 2010). It has been a challenge to study
encystation and its role in infection directly in Entamoeba (E. histolytica) however a related reptile
parasite, E. invadens has proved to be a competent model organism for such studies. Development
of in vitro encystation system in E. histolytica still remains high priority goal for researchers. To
investigate the proteins and genes involved in encystation, frequent studies have been attempted.
PLD, an enzyme involved in lipid second messenger signaling has been reported to strongly up
regulate during early encystation. Genome sequencing study of E. histolytica and E. invadens has
provided novel information related to amoeba development yet the cellular events, signal
transduction, regulation and molecular changes during encystation are poorly understood.
Ehrenkaufer and her co-workers (2013) used whole transcriptome sequencing for studying changes
in gene expression during encystation and excystation, It was reported that the genomic assembly
of E. invadens is large (11549 genes) compared to E. histolytica (8306 genes) mainly due to
intergenic regions expansion but overall number of genes and machinery for regulation of genes
were conserved between both species.

5.1 Genomic DNA isolation

Getting the high molecular weight genomic DNA of E. invadens was challenging and it was
initially difficult to get a good yield following the standard protocols of using CTAB. Primarily
15 ml cultures were used for isolating genomic DNA but that resulted in low yield and improved
concentration was attained using 100 ml cultures under the same conditions. Another reason could
be because the initial culture was taking time to adopt in the media and there were less visible cells
under the microscope. Different temperatures were also checked, and the best growth was observed
in room temperature of 23 °C. The genes encoding PLD1 and PLD2 were PCR amplified directly
from genomic DNA of E. invadens using the samples which were passed through S-200 HR
column for removing PCR inhibitors such as salts and restriction enzymes. In the initial experiment
of isolating genomic DNA, the S-200 HR column was not used, and it resulted in no amplification.

50
MicroSpin S-200 HR although reduced the concentration, improved the purity of the genomic
DNA and hence it was used before PCR amplification.

5.2 Amplification of genes

In this study, amplification of both genes was challenging, different polymerases were used to get
the required bands. Promega mix containing TaqDNA polymerase, dNTPs, MgCl2 and optimal
reaction buffer resulted in no amplification of PLD1 and PLD2. However, Dream Taq polymerase,
dNTPs mix and dream taq buffer resulted in PCR products which contained PLD1 and PLD2 gene
bands when run on the gel. The reason behind DreamTaq yielding the desired genes could be
because of its higher sensitivity and high yield ability compared to taq DNA polymerase (Fisher,
n.d.). There were a set of two primers used for the amplification of PLD1 and PLD2. One was
gene specific primer set and the other one was gene specific primer containing restrictions site and
HA tag. The ones containing restriction site and HA tag resulted in successful amplification
however the ones without it resulted in no amplification.

5.3 Transformation in E. coli JM109

The gene encoding PLD1 and PLD2 was cloned, and recombinantly expressed in E. coli JM109
using Promega standard vector pGEM-T-Easy. On PLD2 plate, only two colonies were obtained,
one white and one blue, according to the protocol given by Promega, white colored colonies means
that it is a transformed colony and blue colonies are the ones which hasn’t transformed
successfully. To confirm the transformation, miniprep for both colonies was done. Surprisingly,
sequencing of inserts of transformants revealed the presence of PLD2 in both blue and white
colonies. It was unexpected as it is mentioned in most literatures that blue colonies do not contain
gene of interest. However, mentioned on the Promega website was also that sometimes even
transformed E. coli JM109 can contain blue color cause of a possible mutation (Grooms et al.,
2019). They might have a second mutation, laclq which upsurges the production of repressor lacl
thus stopping transcription from the lac operon. The substrate, IPTG, dismisses the repression of
the lac operon and lets transcription to occur. These strains will need to be grown on media
containing IPTG as well as X-gal. Sometimes, colonies will not appear white but pale blue. So,
there is strong chance that pale blue colonies might contain gene of interest (Promega, n.d.).

51
5.4 Transfection in Entamoeba invadens
Previously, DNA-mediated gene transfer has been extensively used as a powerful tool for the
genetic alteration of cells or organisms. Transfection and expression of DNA in protozoan
parasites was reported to be more difficult as compared to higher eukaryotic cells, as particular
growth conditions are required for protozoan parasites and own various peculiarities in gene
organization and control of gene expression (Huo et al., 2016).

For the successful transfection in protists, electroporation is a highly effective method that has
been extensively used regardless of the high voltages that might upset the viability of cells.
Successful transfection of E. invadens can be achieved using thousands of volts (1000-4000 V).
High voltage is known to kill most of the E. histolytica trophozoites at 3000 V/m and 50% of
Leishmania enriettii at 3000 V/m. E. invadens transfection was performed by electroporation
through applying two consecutive electrical pulses of 3000 V using a 0.4 mm cuvette or 1500 V
using a 0.2 mm cuvette (Turfah, 2020). The transfection efficiency is greatly affected by viability
and number of cells, osmolarity, DNA purity and concentrations (Zu et al., 2014). Transfection of
E. invadens in this study was also challenging because the culture of E. invadens trophozoites cells
was not dense, instead it was a thin layer. That could be because of bacterial contamination in the
LYI-S media. It is common to have more bacterial contamination during cell culturing, although
penicillin and streptomycin antibiotics were added into media prior to inoculum addition.
Successful transfection was obtained by adding G418 antibiotic in the media. The constructed
vector contained neomycin phosphotransferase (NEO) gene as a selectable marker for the
transfection of the parasite. We choose NEO gene because studies showed that amoebae are
remarkably sensitive to G418 as compared to other eukaryotes (Hamann et al., 1995).

5.5 PLD and its role in Encystation

Role of PLD in E. invadens encystation was also assessed. PLD was one of the proteins which is
highly expressed during encystation. PLD is involved in lipid second messenger signaling. It is
responsible for the conversion of phosphatidyl choline to phosphatidic acid and linked with various
crucial biological processes such as vesicle transport and signal transduction. E. invadens encode
two PLD proteins; PLD1 and PLD2 and both were found up-regulated during encystation. It was
also up-regulated in E. histolytica cysts (Ehrenkaufer G. M. et al., 2013). To assess the cysts
formation during encystation, 0.6% n-butanol was used, which is known to inhibit PLD activity

52
by serving as non-productive substrate for PLD thus suppressing its activity. T-butanol was used
as control as it has no effect on PLD activity. The encystation was allowed to procced for 24 hrs,
48 hrs and 72 hrs. Astonishingly, no decline in encystation efficiency was found in n-butanol
treated samples and was approximately similar to that of wild type and t-butanol treated samples.
Contrary to our results, marked reduction of encystation efficiency was observed in n-butanol
treated samples by Ehrenkaufer and co-workers. There was no major effect on encystation in t-
butanol treated samples. So, they concluded that n-butanol is responsible for repression of
encystation (Ehrenkaufer G. M. et al., 2013). But in the current study, no change in cyst formation
activity was observed in all the three samples.

5.6 Bioinformatics of PLD

The phylogenetic tree of PLD across different organisms was constructed with 100% bootstrap
which showed that PLD of Entamoeba is distantly related to PLD of human beings. Similarity of
sequences or structures usually elicits a discussion on the evolutionary relatedness of the respective
proteins and structure/structure alignments are often used in the statistical analysis of protein folds.
Structures of different proteins is usually compared by superimposing the coordinates of protein
backbones or selected parts of the proteins (Nassoury & Morse, 2005). Superimposition of PLD1
protein (model) with 6ohr.1 (template) which is a human phospholipase D1 catalytic domain was
carried out. The superimposition between template and model showed 29.89% similarity.
Superimposition between E. histolytica PLD1 protein and E. invadens PLD1 protein was also
carried out which showed similarity of 54.54%. On the other hand, superimposition of PLD2
protein (model) and template (6ohm.1) showed 31.09 % similarity. E. histolytica PLD2 protein
and E. invadens PLD2 protein superimposition showed similarity of 55.45%. The structural
dissimilarities between human PLD catalytic domain and Entamoeba PLD is desirable as it will
help to design targeted approaches to disrupt cyst formation and disease transmission.

PLD protein sequence alignment of E. nuttali, E. histolytica, E. dispar, E. invadens, Zea mays,
Oryza sativa, Arabidopsis thaliana, Fusarium oxysporum, Moesziomyces antarcticus, Diachasma
alloeum, Nematostella vectensis, Anolis carolinensis, Erpetoichthys calabaricus and Homo
sapiens was performed. The sequence alignment showed that HKD is the main active site motif
and is present in all the aforementioned organisms. HKD motif appears twice, and both are
conserved as shown in Figure 21 and Figure 22.

53
 Figure 21. Alignment representation of active domain motif site (HKD) #1

Arrow showing conserved HxKx4D active motive site appeared first time in alignment

Figure 22. Alignment representation of Active domain motif site (HKD) #2

Arrow showing conserved HxKx4D active motive site appeared second time in alignment

These two HKD motifs confer hydrolytic activity to PLD and are critical for its enzymatic activity
both in vitro and in vivo. Two HKD motifs from the two domains form a single active site. HKD
motif comprises of histidine, lysine, and aspartic acid. The two histidine residues from the two
HKD motifs play key roles in the catalysis. Upon substrate binding, a histidine residue from one
HKD motif could function as the nucleophile, attacking the phosphodiester bond to create a
covalent phosphohistidine intermediate, while the other histidine residue from the second HKD
motif could serve as a general acid, stabilizing the leaving group (NCBI, Conserved Protein
Domain Family, 2020). Based on structural details, mechanism of human PLD2 was proposed.

54
Histidine 442 exerts a nucleophilic attack on phosphate moiety of phosphatidyl choline (PC), with
the help of aspartate 363. Histidine 756, lysine’s 444 and 758 might be involved in stabilizing the
tetrahedral intermediate. In the final step, histidine 756 donates hydrogen to the tetrahedral
intermediate and activates water, which then exerts a second nucleophilic attack, ultimately
leading to the release of choline and phosphatidic acid (PA) respectively (Mahankali, 2015). The
conserved catalytic domain might demonstrate the same function of PLD in human and
Entamoeba. In future, we can introduce site-directed mutagenesis in the active site of PLD protein
of Entamoeba to study whether PLD performs same function in human and Entamoeba or not.
Whereas when PLD1 and PLD2 sequence was searched in Pfam database, it showed two same
protein family that were PLDc_2 (PLD-like domain) and PLDc (Phospholipase D Active site
motif).

55
 6 CONCLUSION

Entamoeba’s infection are of immense importance in developing countries and more knowledge

about its life cycle will help scientists to identify the novel therapies in treating infections caused

by it. In the current study, the aim was to study the role of PLD1 and PLD2 in encystation as it has

been suggested in the literature that those two genes are upregulated during cyst formation, and

Entamoeba cells are the hardest to treat in cyst form. Cloning of these two genes (PLD1 and PLD2)

was successful and so was transfection to Entamoeba invadens trophozoite cells. Furthermore,

imaging of Entamoeba invadens cysts under different conditions was also attained. However, more

research will be needed to determine the role of PLD1 and PLD2 during encystation and how the

whole process works. Important things to understand would be what triggers the upregulation

when Entamoeba is inside the host and possible therapies to target these changes. N-butanol

showed no significant effect on encystation activity, but the experiment needs to be performed

multiple times with proper controls to conclude that as a fact.

56
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 8 APPENDICES
8.1 Appendix A: Chemicals and media
Incomplete cytomix medium for electroporation:

Ingredients Quantity (g)

KCl 8.95

CaCl2 0.02

HEPES 5.96

EGTA 0.76

MgCl2 0.48

1000 ml of buffer (10mM K2PO4/KH2PO4) with pH 7.6 is used to make solution, where, pH of the

solution is adjusted to 7.8-7.9 with the help of KOH. This follows with the filter sterilisation of the

buffer. Prior to electroporation 20 mg ATP is used to complete the 8 ml cytomix to achieve the

concentration of 4 mM and similarly, 24 mg of reduced gluthathione is used for the 10 mM

concentration.

LB agar:

Ingredients Quantity

LB agar 40 g

Distilled water 1L

Sterilized by autoclave at 121 °C for 15 minutes.

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Luria Broth (LB broth):

Ingredients Quantity

LB broth 25 g

Distilled water 1L

Autoclaved at 121 °C for 15 minutes.

Ampicillin:

Ingredients Quantity

Ampicillin 100 µg/ml

milliQ sterilize water 1

Ampicillin was dissolved in milliQ sterilize water and filter‐sterilized via 0.2 μm filter. The final
concentration used was 100 ug/ml whereas used concentration was 100 mg/ml.

SOC media composition:

Ingredients Quantity

Tryptone 20 g

Yeast extract 5g

5 M NaCl 2 ml

1 M KCl 2.5 ml

1 M MgCl2 10 ml

1 M MgSO4 10 ml

1 M glucose 20 ml

H2O 1000 ml

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Phosphate buffered saline (PBS):

Ingredients Quantity (g/l)

Na2HPO4 0.454

KH2PO4 0.068

KCl 0.097

NaCl 7.89

H2O 1

pH 7.4

Vitamin mixture #18 (Diamond and & CLARK 1993)

Solution 1

Ingredients Quantity

Niacinamide 45 mg

Pyridoxal hydrochloride 4 mg

Pantothenic acid 23 mg

Thiamine hydrochloride 5 mg

Vitamin B12 1.2 mg

H2O 25 ml

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Solution 2

Ingredients Quantity

Riboflavin 7 mg

0.1 M NaOH Minimum amount

H2O 45 ml

Solution 3

Ingredients Quantity

Folic acid 5.5 mg

0.1 M NaOH Minimum amount

H2O 45 ml

Solution 4

Ingredients Quantity

D-biotin 2 mg

H2O 45 ml

Solutions 1‐4 were combined in glass tube.

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Solution 5

Ingredients Quantity

DL-6-8-thioctic acid (oxidized form) 1 mg

95 % ethanol 5 ml

Tween-80 500 mg

H2O 30 ml

Combined solutions of 1-4 was mixed with the solution 5 and final volume adjusted to 200 ml
distilled H2O. Later, autoclaved for 15 minutes at the temperature of 121 oC.

DNA Marker

DNA fragments with the size greater than 400 bp was dealt with the Bioline hyperladder I, as
Figure 23

Figure 23. Bioline hyperladder 1

A DNA ladder by the Bioline Hyperladder I as shown in Figure 23, was used throughout this study.

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Calcofluor white stain (1%):

Ingredients Quantity

Calcofluor white stain 1 ml

sterilized water 99 ml

4% paraformaldehyde solution:

Ingredients Quantity

paraformaldehyde powder 4g

sterilized water 100 ml

NaOH 1 M to adjust pH 7.4

8.2 Appendix B: Sequence alignment of cloned pGEM-T-Easy and

PLD1/PLD2

Figure 24 and Figure 25 confirms the alignment of cloned pGEM-T-Easy vector with PLD1/PLD2
respectively. SP6 as forward primer, T7 as reverse primers used by Microsynth lab for Sanger
cycle sequencing, whereas alignment also showed with gene specific forward and reverse primers
used for amplification of PLD1/PLD2. EIN-017100 represents reference sequence of PLD1
(Figure 24) and EIN-196230 represents reference sequence for PLD2 (Figure 25).

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Figure 24. Alignment representation of cloned pGEM-T-Easy+PLD1 along with reference gene and primers

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Figure 25. Alignment representation of cloned pGEM-T-Easy+PLD2 along with reference gene and primers

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