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2014-01-01

Spingolipid Metabolism In A Reptillian Protozoan,Entamoeba InvadensDuran Dogan DebonsUniversity of Texas at El Paso, Valinor626@hotmail.com

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SPINGOLIPID METABOLISM IN A REPTILLIAN PROTOZOAN, ENTAMOEBA INVADENS

DURAN DEBONS

Department of Biological Sciences

APPROVED:

Siddhartha Das, Ph.D., Chair

Carl Lieb, Ph.D.

David Carmichael, Ph.D.

Charles H. Ambler, Ph.D.

Dean of the Graduate School

Copyright ©

by

Duran DeBons

2014

Dedication

I dedicate this thesis to my mother and father, both of whom encouraged me to always pick

myself up and keep moving forward.

This dedication also goes to my closest friends; Eddie L, Joseph R, Raul Z, and Hector Z. I never

had any siblings, but to me, these friends are my brothers.

A final dedication goes to my bosses Sarah C and Javier R, both of whom worked with my

schedule so that education always came first.

SPINGOLIPID METABOLISM IN A REPTILLIAN PROTOZOAN, ENTAMOEBA INVADENS

by

DURAN DEBONS, B.S

THESIS

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Biological Sciences

THE UNIVERSITY OF TEXAS AT EL PASO

August 2014

v

Acknowledgements

I would love to gratefully thank my committee chair and mentor Dr. Siddhartha Das, along with

Dr. Atasi Chatterjee and Dr. Trevor Duarte. Thank you all for your patience and support.

Without your guidance, knowledge, and passion, this journey would have ended before it had

even begun.

I wish to acknowledge and also thank my committee members Dr. Carl Lieb and Dr. David

Carmichael; two professors who to me were more than just educators, but my confidantes.

A special thanks to Dr. Lillian Mayberry, for without you I would have taken a different path and

not have discovered my true passion.

Finally, a most solemn and gracious bow goes to Dr. Larry Jones; a great professor and an even

greater friend who unfortunately passed away before his time.

vi

Abstract

Entamoeba invadens (hereinafter E. invadens) is an anaerobic parasitic protozoan that can be found

in water and soil. Although the host organisms for this parasite are snakes, lizards, and other reptile

species, the parasite is morphologically identical to Entamoeba histolytica, a causative agent of human

amebiasis. E. inavdens exists in two morphological forms—(1) a replicative trophozoite, and (2) a

relatively dormant cyst. While the transformation of cyst to trophozoites (called “excystation”) takes place

in the stomach, the differentiation of trophozoites to cyst (known as “encystation”), occurs in the small

intestine. Unlike the cyst, a trophozoite is unable to survive outside the host and therefore must encyst to

survive. Various factors in the milieu of the small intestine trigger encystation. Newly encysted cysts are

then excreted from the host and infect a new a host to continue the life cycle. In recent years,

sphingolipids (SLs) have emerged as important molecules in biological systems, and, in particular, these

molecules are involved in regulating the encystation of a related intestinal protozoan, Giardia lamblia.

Therefore, the major goal of my thesis is to examine whether SLs also play an important role in

determining the growth and encystation of E. inavdens. My first hypothesis is that serine-

palmitoyltransferase (SPT), a rate-limiting enzyme of SL biosynthesis, regulates the growth and

encystation of E. invadens. I also hypothesize that blocking of SPT activity with a suitable small molecule

inhibitor should interrupt the life cycle of this intestinal pathogen. In Specific Aim-1, I asked whether

myriocin, an inhibitor of SPT, inhibits the growth of E. invadens trophozoites in culture. The goal of Aim-

2 was to investigate whether the inactivation of SPT can interfere with encystation and cyst production.

Briefly, I found that SPT is an important Entamoeba enzyme and that manipulating its activity by

myriocin has a profound effect on its life cycle. The knowledge generated from the current study will shed

light on the possible role of SLs in regulating the growth and viability of Entamoeba histolytica, a parasite

that infects over 50 million people worldwide every year.

vii

Table of Contents

Acknowledgement………………………………………………………………….………vi

Table of Contents……………………………..………………………………………..….vii

List of Figures……………………………………………………………………………..viii

Chapter 1: Introduction……………………………………………………………………...1

1.1 Pathogenesis…………………………………………..………..……………......2

1.2 Molecular mechanism of encystation and exystation…....……..………..….......3

1.3 Sphingolipids………………………………………………..……..……...……..5

1.4 Goal and hypothesis…….……………………………………...….……..……...8

Chapter 2: Methods………..………………………………………………………………...11

2.1 Materials……………………………………..…………………………………..11

2.2 Parasites……………………………………………………………………….....11

2.3 Parasite, culture, and encystation………………………………………………..11

2.4 Treatment with glucosylceramide inhibitors…………………………………… 11

2.5 Myriocin treatment....………………………………………………………...….12

2.6 Myriocin and cyst production……………………………………………….......12

2.7 Staining with propidium iodide……………………………………………….…13

2.8 Staining with calcofluor white…………………………………………………...13

Chapter 3: Results……………………………………………………..……………………..14

3.1 Specific Aim-1: Does Serine palmitoyltransferase regulate the growth of E.

invadens?..................……………………………………………………..……....14

3.2 Experimental strategy and results…..……………………………………….......16

3.3 Specific Aim-2: Does myriocin influence the cyst production?...........................20

3.4 Approach, experiments, and results……...…………………………………........21

Chapter 4: Discussion……………………………………………………………………......25

References…….......................................................................................................................28

Vita…………………………………………………………………………………………...35

viii

List of Figures

Figure 1: Life Cycle of E. invadens……………..……………….………………………………2

Figure 2: Staining E invadens Cyst Walls with Calcofluor White………….…………………...5

Figure 3: The Structure of a Sphingolipid……………………………………………………….7

Figure 4: A General View of Sphingolipid Metabolic Pathway in Mammalian Cells..…………8

Figure 5: Structure of Sphingolipid……………………………………………………………...9

Figure 6: Myriocin blocks Serine-palmitoyltransferase Enzyme and Inhibits Sphingolipid

Biosynthesis…………………………………………………………………………..10

Figure 7: Effects of Glucosylceramide Inhibitors (PPMP and PDMP) on E.invadens

Trophozoites………………………………………………………………………….17

Figure 8: Effects of Myriocin on the Trophozoites of E. invadens……………………………..18

Figure 9: Myriocin Inhibits the Growth of Trophozoites in Culture……………………..…….19

Figure 10: Myriocin Alters the Morphology and Viability of Trophozoites……………..…....20

Figure 11: Effects of myriocin on the encystation of E. invadens………………………..…….22

Figure 12: Encystation Process of Non-treated Trophozoites from Days 1-5…………..……...23

Figure 13: Myriocin Disintegrates Cyst Wall of Entamoeba invadens…………………..………..24

1

Chapter 1:

Introduction

Entamoeba invadens (E. invadens) is an anaerobic parasitic protozoan that infects snakes, lizards, and

reptiles. Like all Entamoeba, E. invadens exists in two forms; trophozoites and cysts. While the disease is

caused by trophozoites, the infection is transmitted by cysts. Once ingested, via contaminated water, the

cyst travels through the stomach. Acid in the stomach then induces excystation (i.e., transformation of

cyst to trophozoites). The excystation releases trophozoites from cysts, and newly emerged trophozoites

move downstream into the digestive tract to colonize the small intestine (Lopez-Romero, E et al. 1993).

During colonization in the small intestine of reptiles, immune and non-immune components are activated

and interact with the parasite to neutralize its growth and replication. Alternatively E. inavdens follows

several strategies to avoid this fate and ultimately cause infection (amebiasis). The parasite undergoes

encystation in the small intestine (as shown in Fig. 1) to continue the life cycle and infect a new host.

Newly encysted cysts then travel to the large intestine and are finally excreted from the host. The factors

responsible for encystation are not known.

2

Fig. 1. The life cycle of Entamoeba invadens. (A) Cysts are ingested by reptiles through contaminated water. During the passage

through the stomach, the cysts are broken down and release trophozoites.

(B) Trophozoites cultured in the laboratory and when subjected to encystation, they are

transformed to cyst. (a) trophozoite (b) encysting cells and (c) water-resistant cysts.

1.1 Pathogenesis.

Once E. invadens reaches the lower section of the intestine, it burrows into the intestinal lining,

causing ulcerations and necrosis of the infected area (Donaldson M et al. 1975). At this point, the parasite

has the potential to invade the blood stream and make its way into the lungs, kidneys, liver, and, at the

farthest extent, the brain. Amebiasis disseminates from the primary intestinal lesion via the blood stream

as trophozoites entering capillaries in the large intestine are carried to other organs. The most commonly

affected organ is the liver, as direct invasion by trophozoites from the large intestine through the

mesenteric blood vessels feed into the hepatic portal vein (Chia M.-Y et al. 2009). Hematogenous spread

of trophozoites to other sites, such as the lungs or the brain, has also been reported, and the lung is the

3

second most common extra-intestinal site after the liver. Pulmonary infections generally result from a

direct extension of the hepatic lesion across the diaphragm and into the pleura and lungs (Houpt E et al.

2013).

1.2 Molecular mechanism of encystation and excystation

As mentioned above, the life cycle of E. invadens is much like E. histolytica and Giardia lamblia—all

of which exhibit a fecal-oral life cycle (Fig. 1). Infection begins at the point of cyst ingestion. Entamoeba.

invadens cysts, as with E. histolytica cysts, are primarily found in contaminated food, soil, and water

sources—sources that are not properly sanitized and consumed by reptilian species. Usually, Entamoeba

cysts shown in Fig 2 are 10–20 M wide and excyst in the terminal ileum (Diamond and Clarke, 1993;

Schmidt and Roberts, 2000). The presence of chitin (a polymer of -N-acetylglucosamine, GlcNAc) has

been reported in the cyst wall of E. invadens by X-ray diffraction studies (Arroyo-Begovich et al. 1980),

and the enzymes for the synthesis and degradation of this polysaccharide (i.e., chitin) have also been

identified in this organism (Das and Gillin, 1991; Villagomez-Castro et al. 1992; Ghosh et al. 1999). Said-

Fernandez et al. (2001) demonstrated that various bivalent metal ions (i.e., Mn2+

, Mg2+

, and Co2+

) were

effective in stimulating the synthesis of a chitin-like substance in E. histolytica. In addition to chitin, the

Entamoeba cyst walls were also shown to contain a lectin; carbohydrate-binding proteins that are utilized

in binding of bacteria and viruses, which is thought to be involved in cyst wall synthesis (Fisaardi et al.

2000). All the polysaccharides identified in both species have a β-configuration and present a positive

fluorescence with the dye calcofluor, which specifically reacts with polymers in this conformation (Maeda

and Ishida 1967; Arroyo-Begovich et al. 1978; Chávez-Munguía et al. 2003, 2005).

During the encystation process of E. invadens, the molecules that constitute the cyst walls are

encapsulated in transport vesicles, which are then deposited on the membrane of the parasite to form the

4

cyst walls. These transport vesicles carry specific proteins and fibrillar contents similar to those observed

in cases of Giardia lamblia (Chávez-Munguía et al.2003; Mendez et al. 2013). The motile trophozoite of

E. invadens reestablishes itself after the excystation process. The in vitro excystation of this parasite takes

place when cysts are cultured in complete TYI-S-33 culture medium (García-Zapién et al. 2000).

Microscopic and biochemical studies indicate that increased proteolytic activity and the synthesis of dense

granules are associated with exystation (Chávez-Munguía et al. 2007). Electron-dense granules are present

in the parasite’s cytoplasm and likely attach to the inner leaflet of the plasma membrane by fingerlike

projections to the fibrillar cyst wall (Chávez-Munguía et al. 2007). Once the trophozozoite emerges from

the cysts, the motile amebic trophozoite undergoes one round of nuclear divisions along with several

rounds of cytokinesis. Trophozoites, 15–30 m in length with an amorphous shape, form colonies within

the large intestine; masses of intestinal bacteria there in will that serve as E. invadens food sources (Das et

al. 2002). The parasites continue to multiply in the large intestines through binary fission, and

trophozoites begin the process of encystation. It has been suggested this trophozoite aggregation process

in the mucin layer may be the cause of encystation. During this process, the trophozoites become

spherical in shape and chromatoid bodies begin to appear in the cytoplasm. The chromatoid bodies are

stained through use of basic dyes, and are elongated structures with round ends and represent the

aggregation of ribosomes (Jeelani G et al. 2012). The fully formed cysts are 12–15 m in diameter and

single- to multi-nucleic. As the cyst matures, the chromatoid bodies disappear. Once the trophozoites have

become fully formed cysts they are egested from the intestines through fecal ejection and become

potentially infective, thus renewing the life cycle. The ejected cysts are viable for infection for weeks and

up to months depending upon the environmental conditions in which they reside such as heat and pH.

5

Cysts

Calcofluor DIC Overlay

A B C

Fig. 2. Staining E. invadens cyst walls with calcofluor white. (A) Image showing

the labeling of cyst-wall by calcofluor white (B) DIC image of the same field and (C)

Merged image of A and B.

1.3 Sphingolipids

Sphingolipids (SLs) are sphingosine-based phospholipids that are found in plasma membranes and

raft-like microdomains. Sphingolipid synthesis begins in the endoplasmic reticulum (the area where most

proteins and lipids are synthesized) and is completed in the Golgi apparatus (responsible for packaging

and sending out proteins and lipids to their destinations throughout the cells). Sphingolipids are

characterized as having long-chain bases (sphingoids or sphingoid bases) that include long-chain aliphatic

amines containing two or three hydroxyl groups, and, in most cases a trans-double bond in position 4

(Fig. 3). Figure 4 depicts the SL synthesis pathway in mammalian cells. The de-novo synthesis of SL is

initiated by a rate-limiting condensation between serine and palmitoyl-CoA, catalyzed by the serine

palmitoyltransferase (SPT) enzyme to synthesize 3-ketosphinganine (3KS), and then converted to

sphinganine (also known as dihydrosphingosine or DHS) by the action of 3KS reductase (3KSR).

6

Sphinganine acts as a precursor of dihydroceramide (DHC), catalyzed by dihydroceramide synthase

(DHCS). Ceramide is synthesized from DHC with the help of dihydroceramide dehydrogenase (DHCD).

The conversion of DHS to DHC is catalyzed by ceramide synthase (CS), the product of “longevity

assurance genes” (LAG) in mammals and DHC to ceramide by dihydroceramide desaturase (DHCD).

Ceramide acts as a precursor of bioactive SLs that regulate a wide variety of cellular and biological

processes (Pandey et al. 2007; Bartke N and Hannun, 2009). Bioactive lipids are generated by the

phosphorylation of ceramide to ceramide-1-phosphate, or hydrolysis of ceramide to sphingosine (by

ceramidase enzyme). Sphingosine which is subsequently phosphorylated to the bioactive molecule

sphingosine-1-phosphate, etc., as shown in Fig 3. Sphingomyelin (SM), glucosylceramide (GlcCer),

galactosylceramide (GalCer), lactosylceramide (LacCer), and other complex ceramide-containing

glycolipids are also synthesized from ceramide. In ceramide salvage, or in its recycling pathway, the

enzyme sphingomyelinase (SMase) hydrolyzes sphingomyelin (SM) to generate ceramide (Kitatani et al.

2008). Unlike mammalian cells, however, protozoa, fungi, and plant cells synthesize inositol

phosphorylceramide (IPC) by transferring phosphorylinositol from phosphatidylinositol to ceramide

(phytoceramide in the case of plants), a process catalyzed by IPC synthase. Furthermore, in fungi and

plants, DHS can carry an additional hydroxyl group to form 4-hydroxydihydrosphingosine

(phytosphingosine). Phytosphingosines are predominant sphingoid- base in fungi and plants, and their N-

acylation at C-2 position generates phytoceramides. Phytosphingosine 1-phosphate is synthesized by the

phosphorylation of phytosphingosine by sphingoid-based kinases (Cowart and Hannun, 2005).

Reports indicate that the enzymes and products of SL biosynthesis pathways act as regulators of

various cellular processes. Ceramide synthase activity is shown to be associated with tumor formation and

growth (Separocic et al. 2012). Sphingosine-1-phosphate has been shown to generate initial signals for

apoptotic signaling and ceramidase activity, which is important for retinal function (Gu et al. 2012;

7

Acharya et al. 2003). The mutation in the long-chain sphingoid base subunit-1 (LCB1) of serine-

palmitoyltransferase (SPT) causes autosomal, dominant, peripheral, and sensory neuropathy (Gable et al.

2010). SLs are also critical for regulating various biological processes in unicellular organisms such as

fungi and protozoa. In budding yeast, SLs contain C26 acyl moieties and are connected with the target of

rapamycin complex 2 (TORC2) and phosphoinositide signaling (Dickson 2008). In Leishmania and

Trypanosoma parasites, the synthesis of large amounts of non-glycosylated inositol-phosphorylceramides

(IPCs) and ethanolamine-phosphorylceramides (EPCs), along with other SLs, has been reported. The SLs

in trypanosomatids contribute greatly to the growth and survival of parasites within a host and provide

important functions during host-parasite interactions (Zhang et al. 2010). More recently, our laboratory

has shown that glucosylceramide synthase (GS), an enzyme involved in synthesizing glucosylceramide

(GlcCer), a glycosphingolipid, plays a critical role in encystation and cyst production by Giardia

(Mendez et al. 2013). For example, the overexpression of GS induces non-viable cysts and the

knockdown of this enzyme generates mostly dead cysts. Interestingly, when the overexpressed activity

was lowered by knocking down the GS gene (i.e., rescued), normal and viable cysts were again produced

(Mendez et al. 2013). This report by Mendez et al. (2013) strongly suggests that the modulation of the SL

pathway in Giardia is involved in encystation and cyst production.

Fig. 3. The structure of a sphingolipid

8

Fig. 4. A general view of sphingolipid metabolic pathway in mammalian cell.

1.4 Goal and hypothesis

It is possible that like Giardia, E. invadens also uses the SL pathway to induce encystation and cyst

production. I found that blocking SPT, but not GS, appears to be more important for regulating growth

and encystation of E. invadens. SPT catalyzes the first product (i.e., 3-keto-sphinganine) of the entire SL

9

pathway. Therefore, it can be hypothesized that blocking the SPT reaction in E. invadens will block the

entire pathway. It has been shown that myriocin (2-Amino-3, 4-dihydroxy-2-(hydroxymethyl)-14-oxo-6-

eicosenoic acid ISP-I thermozymocidin) (Fig. 5) is a potent SPT inhibitor and blocks mammalian and

fungal SPT. Myriocin is an atypical amino acid produced by thermophilic fungi and competes with serine

for the SPT catalytic site (Fig. 6). In my thesis research, I examined how SPT inhibitor (myriocin)

regulates the growth and encystation in E. invadens. Therefore, the specific aims of my thesis were:

Aim-1. To evaluate the effect of myriocin (a potent SPT inhibitor) on the growth of E. invadens

trophozoite.

Aim-2. To determine if myriocin has an effect upon the encystations process of this intestinal parasite.

Fig. 5. Structure of myriocin (2-Amino-3, 4-dihydroxy-2-(hydroxymethyl)-14-oxo-6-eicosenoic acid ISP-I thermozymocidin)

10

Fig. 6. Myriocin blocks serine-palmitoyltransferase enzyme and inhibits

sphingolipid biosynthesis.

11

Chapter 2:

Methods

2.1 Materials

Unless otherwise specified, all chemicals were purchased from the Sigma and were of the

highest purity available. Myriocin, PPMP and PDMP were purchased from Matreya LLC (Pleasant Gap,

PA).

2.2. Parasites

Entamoeba invadens IP-1 strain was provided by Professor Daniel Eichenger, Director,

Division of Parasitology (New York University).

2.3. Parasite, culture and encystation

The trophozoites of E. invadens were cultured at room temperature for 7 days in T25 (65 ml)

flasks containing Diamond’s TYI-S-33 media supplemented with bovine serum (Diamond et al. 1978).

Trophozoites were harvested by centrifugation (3000 rpm for 7 min at 4°C), re-suspended in sterile

phosphate buffered saline (PBS) and counted. For encystation, ~ 4 x 105

cells were inoculated in diluted

growth medium (2:1; water: medium) and subjected to encystation for 5 days. The cysts were harvested,

washed three to four times with sterile Milli-Q water, re-suspended in water and kept at 4oC for future

use.

2.4. Treatment with glucosylceramide inhibitors

Cultured E. invadens trophozoites were harvested by centrifugation (3000 rpm for 7 min at

4°C). The experiments were conducted to monitor the effects of two glucosylceramide synthase inhibitors

12

(PDMP and PPMP) on the growth of E. invadens trophozoites. Approximately 4 x 105

trophozoites were

cultured in 4 ml culture tubes (borosilicate, screw cap) containing TYI-S-33 Diamond’s media

supplemented with bovine serum (Diamond et al., 1978) in the presence of PDMP (10, 25, and 50 m) or

PPMP (10, 25, and 50 m). Both were cultured and incubated at room temperature for 24 h and the

viability was checked by counting the cells under a microscope with the help of a hemocytometer. The

IC50 of both PDMP and PPMP trophozoites were calculated from the dose-response curves.

2.5. Myriocin treatment

Approximately, 4x105

E. invadens trophozoites were cultured as described above in the

presence and absence of myriocin (, and 50m) at room temperature for 24 h. The trophozoite

viability was checked by counting cells under a microscope with the help of a hemocytometer. To

examine the effect of myriocin on actively growing trophozoites, both control and myriocin (10 M)-

treated trophozoites were cultivated for nine days. Each day the trophozoites from both the control and

experimental sets were counted as described above.

2.6. Myriocin and cyst production

To examine the effects of myriocin on encystation and cyst production, trophozoites were

cultured and encysted following the protocol previously described by Das and Gillin (1991). Ten

micromolar of myriocin was added to the diluted medium (see above) and the encystation was carried out

for 0–5 days. Cyst formation was observed between 3–5 days and cells from control and treatment were

isolated, counted, and examined by confocal microscopy.

13

2.7. Staining with propidium iodide

E. invadens trophozoites were stained with propidium iodide (PI, a nucleic acid staining

reagent) to differentiate between live and dead cells. PI is a red florescent nuclear-binding molecule that

stains the nucleus of dead cells. PI penetrates the cell membranes of cells that have been damaged and are

no longer impermeable. Once it enters the cell, PI binds to the nucleus and emits red fluorescence color.

Trophozoites to be tested with PI were harvested by centrifugation, washed with PBS. Cells were re-

suspended in PBS (500 l), mixed with PI (10 l) and incubated at room temperature 10 min. PI-treated

cells were washed with PBS, fixed in 4% paraformaldehyde and examined with the help of a laser-

scanning Zeiss LSM 700 confocal microscope (Texas Red channel, excitation 589 nm; emission 615 nm).

2.8. Staining with calcofluor white

Calcofluor white is a fluorescent stain that binds with cellulose and chitin. Because E.

invadens cyst wall is made of chitin (Das and Gillin, 1991), I used this reagent to visualize cyst wall

formation by E. invadens. Twenty microliter calcofluor white solution (1%) was added to the samples,

followed by 20µl of KOH (10%). Cells were fixed in 4% paraformaldehyde before analyzing by confocal

microscopy (calcofluor white channel, excitation 300, emission 450).

14

Chapter 3

Results

3.1. Specific Aim-1: Does Serine palmitoyltransferase regulate the growth of E. invadens?

Background information:

The de novo synthesis of SL is initiated by a rate-limiting condensation between serine and

palmitoyl-CoA, catalyzed by the serine palmitoyltransferase (SPT) enzyme to synthesize 3-

ketosphinganine (3KS), which is then converted to sphinganine (also known as dihydrosphingosine or

DHS) by the action of 3KS reductase (3KSR). Sphinganine acts as a precursor of dihydroceramide

(DHC), catalyzed by dihydroceramide synthase (DHCS). Ceramide is synthesized from DHC with the

help of dihydroceramide dehydrogenase (DHCD). The conversion of DHS to DHC is catalyzed by

ceramide synthase (CS) a.k.a longevity assurance genes (LAG) in mammals, and DHC to ceramide by

dihydroceramide desaturase (DHCD). Ceramide acts as a precursor of bioactive SLs that regulate a wide

variety of cellular and biological processes (Pandey et al. 2007; Bartke, N., and Hannun, 2009). Bioactive

lipids are generated by the phosphorylation of ceramide to ceramide-1-phosphate, or hydrolysis of

ceramide to sphingosine (by ceramidase enzyme), which is subsequently phosphorylated to the bioactive

molecule sphingosine-1-phosphate, etc., as shown in Fig 3. Sphingomyelin (SM), glucosylceramide

(GlcCer), galactosylceramide (GalCer), lactosylceramide (LacCer), and other complex ceramide-

containing glycolipids are also synthesized from ceramide. In ceramide salvage or the recycling pathway,

the enzyme sphingomyelinase (SMase) hydrolyzes sphingomyelin (SM) to generate ceramide (Kitatani et

al. 2008). Unlike mammalian cells, however, protozoa, fungi, and plant cells synthesize inositol

phosphorylceramide (IPC) by transferring phosphorylinositol from phosphatidylinositol to ceramide

(phytoceramide in the case of plants), a process catalyzed by IPC synthase. Furthermore, in fungi and

15

plants, DHS can carry an additional hydroxyl group to form 4-hydroxydihydrosphingosine

(phytosphingosine). Phytosphingosines are predominant sphingoid- base in fungi and plants, and their N-

acylation at C-2 position generates phytoceramides. Phytosphingosine 1-phosphate is synthesized by the

phosphorylation of phytosphingosine by sphingoid-based kinases (Cowart and Hannun, 2005).

For the past few years, researches have begun to demonstrate the role of SL metabolism in

inducing the growth and differentiation of Giardia. Protein BLAST searches of the Giardia genome

database (Morrison et al. 2007) have revealed that the majority of SL metabolic genes are missing in

Giardia except for the putative genes for giardial serine palmitoyltransferase 1 and 2 enzymes (gSPT1

and gSPT2), glucosylceramide transferase or synthase (gGlcT1), and acid sphingomyelinase-like

phosphodiesterase 3b precursors (gASMase B and gASMase 3b) (Fig. 3). On the other hand, mining the

Entamoeba histolytica genome (Loftus et al. 2005) has revealed the presence of nearly all of the genes

required for SL syntheses except for the genes of DHCD, galactosylceramide synthase, and ceramidase

enzymes. However, SL genes in Entamoeba invadens because they can be encysted in axenic medium

under low glucose conditions (Das and Gillin, 1991).

It has been previously reported SL genes play an important role in giardial biology. Blocking

the activity of SPT enzyme by L-cycloserine interferes with the endocytic pathway and inhibiting the

activity of GS by PPMP and PDMP blocks encystation and cyst production by Giardia (Hernandez et al.

2008). These results have encouraged us to examine if these inhibitors also block the SL pathway in E.

invadens and inhibit the growth of the parasite as both Giardia and Entamoeba are cyst-forming parasites

and infect humans, mammals, and reptiles via the fecal-oral route.

16

3.2. Experimental strategy and results

In the current study, I have used three commercially available SL inhibitors i.e., PPMP,

PDMP and myriocin to examine their influence the growth of the parasite. Myriocin is an atypical amino

acid produced by thermophilic fungi and competes with serine for the SPT catalytic site (Fig. 5). For GS,

I selected PPMP and PDMP (Kovacs et al. 2000; Hernandez et al. 2008). Both of these drugs are

structural analogues of ceramide and they inhibit the transfer of glucose from UDP-glucose to a ceramide

acceptor. After treatment with these synthetic ceramide analogues, the expression of glycosphingolipid

decreases while ceramide and sphingomyelin levels increase in the cells of higher eukaryotes (Sonda et al.

2008; Hernandez et al. 2008). Briefly, ~4 x 105

trophozoites were used to conduct the experiment in

which the cells were cultured with the presence of PDMP, PPMP or myriocin (0, 10, 25, and 50M) for

24 h. Results show that all three inhibitors inhibit the growth of E. inavdens trophozoites. Interestingly,

while inhibitory concentrations of PPMP and PDMP are ~25 M (Fig.7), IC50 for myriocin is ~10 M

(Fig.8), suggesting that myriocin is more effective than PPMP and PDMP. Next, I asked if myriocin could

be more effective in growing cells. For this the trophozoites were grown over a nine days period in the

presence and absence of myriocin. Fig. 9 shows that myriocin inhibits the growth of Entamoeba and kills

the trophozoites within four days of treatment, suggesting that myriocin is more effective on growing

cells. As myriocin is more potent than PPMP and PDMP, I concluded that the function of SPT is more

important than GS. A propidium iodide (PI) staining of control and 24 h myriocin-treated trophozoites

was carried out to separate viable and non-viable cells. The PI staining shows the saturation of the nucleus

of the treated cells due to the increased membrane permeability in non-viable cells (Fig.10). This

experiment shows that inhibiting SPT activity induces cell death. With these results it becomes apparent

that the SPT enzyme may play a crucial role in the viability and function of E. invadens trophozoites.

17

Fig: 7. Effects of glucosylceramide inhibitors (PPMP and PDMP) on E. invadens

trophozoites

The experiments were conducted to monitor the effects of two glucosylceramide synthase

inhibitors (PDMP and PPMP) on the growth of E. invadens trophozoites. Approximately, 4 x 105

trophozoites were cultured in the presence of PDMP (10, 25, and 50 m) or PPMP (10, 25, and

50 m). The IC50 of both PDMP and PPMP trophozoites were calculated from the dose-response

curves.

18

Fig. 8. Effects of myriocin on the trophozoites of E. invadens Trophozoite growth of E. invadens were monitored in the presence of myriocin.

Approximately, 4x105 E. invadens trophozoites were cultured in the presence of

myriocin (, and 50m) for 24 h and counted, or determining the effect of

myriocin on cysts.

Myriocin Concentration ( M

0 10 25 50

Nu

mb

er

of

cells x

10

5

0

1

2

3

4

5

19

Fig. 9. Myriocin inhibits the growth of trophozoites in culture

Growth curve showing 10M myriocin inhibits the growth of E. invadens in culture.

Control and inhibitor-treated trophozoites (~4 x 105) were cultivated for nine days and

counted as indicated in the figure. Results indicate that myriocin kills trophozoites after

4 days of treatment.

Number of days

0 2 4 6 8 10

Nu

mb

er o

f ce

lls x

105

0

2

4

6

8

10

12

Control

Myriocin, 10

20

Fig. 10. Myriocin alters the morphology and viability of trophozoites

E. invadens trophozoites were stained with propidium iodide (PI, a nuclear staining reagent) to

differentiate between live and dead cells. Photograph A: PI-stained trophozoite. Photograph B:

Corresponding direct interface contrast (DIC) picture. Photograph C: the overlay. Results show

that while control trophozoites exhibit typical ameboid structure, myriocin induces morphological

changes yielding small round-shaped cells. Live trophozoites are not stained with PI. The arrow

indicates the trophozoite plasma membranes and the arrowhead denotes fragmented nucleus after

myriocin treatment. Bar: 10 m.

3.3. Specific Aim-2: Does myriocin influence the cyst production?

Background information:

Because myriocin blocks the growth of trophozoites in culture (see above), I speculated whether

myriocin also inhibits the encystation and cyst production by E. invadens. Cyst formation by E. invadens

is an important step of the amebic life cycle. By forming a cyst, this parasite can survive in the

environment and transmit disease via contaminated water.

21

3.4. Approach, experiments, and results:

To examine the effect of myriocin on encystation and cyst production, trophozoites were

cultured and encysted following the protocol previously described by Das and Gillin (1991). Myriocin (10

M) was added to the media and encystation was carried out from 0–5 days. Cyst formation was observed

between 3–5 days and cells from control and treatment were isolated, counted, and examined by confocal

microscopy. Fig. 11 shows that myriocin treatment decreased cyst production in culture. About 50%

inhibition was observed at 10 M (IC50 ~10 M). I have also examined the changes of morphology that

occurs during encystation. Fig. 12 demonstrates the synthesis of chitin wall by Entamoeba during

encystation. Newly synthesized chitin can be labeled with calcofluor. The synthesis of cyst wall (blue)

initiates in day 2 of encystation and is completed by day 5. Fully formed cysts are visible in day 5 (panels

A-E, Fig.12). DIC images and K-O are merged pictures are shown in panels F-J and K-O, respectively.

Next, I tested the effect of myriocin on chitin wall synthesis and overall encystation. While the control

cysts exhibited thick, chitin-containing cyst walls (nicely stained with calcofluor), myriocin broke the cyst

walls dramatically (Fig.13). The earliest changes were seen on day 2 of the treatment in which fragmented

cyst walls were observed. On day 3, the cysts showed amorphous structures with ruptured cyst walls that

were poorly stained with calcofluor. On day 4 and day 5 profound effects on the cysts were observed and

the majority of cysts were fragmented. Thus, myriocin has an adverse effect upon the encystation process,

and suggests that SPT and SL biosynthesis play an important role in cyst formation by E. invadens.

22

Fig. 11. Effects of myriocin on the encystation of E. invadens

Encystation of E. invadens trophozoites were monitored in the presence of

myriocin. For determining the effect of myriocin on cysts, trophozoites were

cultured in encystation media (please see the experimental section) for 5

days, and counted. The IC50 of cysts that were encysted through myriocin

treatment were calculated from the dose-response curve.

0 10 25 50

Nu

mb

er

of

cells x

10

5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Myriocin Concentration ( M)

23

Encystation Process (Days 1-5)C

alc

ofl

uo

rD

ICO

verl

ay

A B C D E

F

K L

G H

M

I

N

J

O

1 2 3 4 5

Water resistant

cysts

Fig. 12. Encystation process of non-treated trophozoites from days 1-5.

E. invadens trophozoites (~4 x 105) were subjected to encystation by culturing them in

diluted (1:2; media: water) and allowed to encyst for five days. Photographs A-E consist of

chitin formation through calcofluor white staining from days 1-5 of the encystations

process, images F-J are the corresponding DIC images, and photographs K-O are the overlay

between the calcofluor white stained images and DIC. The first day of encystation shows no

visible chitin synthesis, therefore no cysts have been formed and all trophozoites had been

lysed due to washing of cells before placing on slide. On days 2-4 the trophozoite cells have

begun to become rounded and slight chitin is being seen, indicating chitin synthesis. On day 5

complete water-resistant cysts are formed with the white arrow shows indicating the fully

formed cyst walls. Bar: 10 m.

24

Fig. 13. Myriocin disintegrates cyst wall of Entamoeba invadens E. invadens trophozoites (~1 x 10

5) were subjected to encystation by culturing them in diluted

1:2; media: water) and allowed to encyst for five days in the presence of 10 m myriocin.

Photographs A, myriocin was added immediately (0 h) to the culture, photograph B after 24 h;

photograph C, the myriocin was added after 48 h, photograph D shows the effect after the

addition of myriocin at 72 h, and photograph E is the control where no myriocin was added. The

calcofluor-white staining shows virtually no visible chitin synthesis on the first and second day

of encystation and therefore cysts were not formed. The DIC (photographs F–J) and overlay

images (K–O) demonstrate ruptured, damaged, and incomplete cysts. In contrast, control cysts

show fully formed cyst walls that can be stained with calcafluor white. The arrow shows fully

formed cyst walls and the arrowhead indicates disrupted cysts. Bar: 10 m.

25

Chapter 4

Discussion

Entamoeba invadens is a protozoan parasite that infects reptiles. The infection can be

spread from one organism to another via commensal interactions and is usually not harmful.

However, in some cases, E. invadens can cause colitis, diarrhea, and death in tortoises (i.e.,

Gopherus polyphemus and Geochelone pardalis) and snakes (e.g., Boa constrictor imperator and

Pythonidae) (Donaldson et al. 1975). Therefore, it will be interesting to investigate how and by

what mechanism this parasite selectively infects reptiles but not humans. Entamoeba histolytica,

on the other hand, is a human parasite that kills approximately 100,000 people annually

worldwide (WHO 1997). Both organisms have a two-stage life cycle—i.e., an invasive

trophozoite and a transmissible cyst. Although the cyst wall of Entamoeba is made of protein,

chitin, chitosan, and chitin-binding lectins (Chatterjee et al. 2009), chitin is the major component.

Unlike E. histolytica, E. inavdens can be encysted in culture and thus serves as a model organism

for the study of E. histolytica.

Because E. invadens can be cultured and differentiated in the laboratory, I took the

opportunity to study this organism to see whether sphingolipid (SL) biosynthesis could be linked

to encystation and cyst production. The hypothesis that SLs could be involved in regulating the

growth and encystation of E. invadens came from our observations that lipid molecules are key

regulators of giardial encystation (Yichoy et al. 2011).

I initiated my experiments by culturing E. inavdens trophozoites in the presence of

myriocin, PPMP, and PDMP. While myriocin inhibits SPT activity, PPMP and PDMP are

specific for GlcT1 (please see Fig. 6 for review). My initial experiments suggested that myriocin

was more effective in inhibiting the growth of trophozoites than PPMP and PDMP (Figs. 7 and

26

8). For example, while the IC50 of myriocin is ~10 M, PPMP and PDMP are effective at much

higher concentrations (IC50 ~25 M). Figure 9 shows that when myriocin is added in the

growing culture of trophozoites, it appears to be more effective and inhibits the growth of the

parasite completely. I also found that myriocin treatment alters the morphological structures of

Entamoeba trophozoites, causing a dramatic change of amoeboid structures to a round-shaped,

cyst-like form (Fig.10). My next goal was to test whether the viability of trophozoites is affected

by myriocin treatment. The viable and non-viable trophzoites were identified by staining with

propidium iodide (PI), which was used earlier by our laboratory to determine the viability of

Giardia trophozoites and cysts (Mendez et al. 2013). I found that myriocin induces cell death, as

evidenced by staining with propidium iodide (PI). This observation suggests that (1) the

expression of SPT is important for maintaining the growth of this parasite, and (2) actively

growing cells are more susceptible to myriocin. A recent study shows that SPT is involved in

regulating cellular multiplication and cytokinesis in yeast (Epstein, S. 2012), In the vascular

plant Arabidopsis. Another study showed that SPT activity is essential for male gametophyte

development (Teng et al. 2008). Thus, it is possible that SPT in Entamoeba is also involved in

regulating, the cell division and nuclear ploidy, but more in-depth work will be required to

support this conclusion.

As mentioned above, Entamoeba undergoes encystation in the small intestine and forms

water-resistant cysts. Such cyst formation is important for the survival of the parasite outside the

host. The Entamoeba cyst wall is made of chitin, and it has been found that inhibiting chitin

synthesis blocks cyst production. Because myriocin inhibits the growth and replication of

trophozoites, I asked whether myriocin also blocks cyst production. Figure 11 shows that

myriocin inhibits cyst production in a culture in a dose-dependent manner, with the IC50 of cyst

27

viability being ~10 M (similar to trophozoites). Because E. invadens cyst wall contains

microfibrillar structures of chitin, and because chitin can be stained with calcofluor (Arroyo-

Begovich et al. 1980), I used this fluorescence molecule to monitor the cyst wall formation. Fig.

12 demonstrates that the blue-colored chitin appears to synthesize in day 3 of encystation, with

completion by day 5, when water-resistant cysts are formed. When encystation is carried out in

the presence of myriocin (10 M) chitin synthesis is interrupted, and cyst-wall formation is

blocked (Fig. 13). My results clearly indicate that SL pathway in Entamoeba is linked directly

with chitin biosynthesis and that inhibiting SPT activity influences chitin synthesis and blocks

the cyst-wall formation. How myriocin inhibits chitin synthesis is not yet clear, but it is possible

that in E. invadens chitin synthesis and SL pathways are inter-dependent and “cross-talk” to each

other. It will be interesting to see if chitin inhibitors (polyoxin-D, nikomycin etc.) can also affect

SL biosynthesis.

In conclusion, I have demonstrated that the synthesis of chitin is essential for cyst

formation and that it does so by interacting with the SL pathway. This is a novel finding and

should be investigated thoroughly. It would also be important to synthesize a new generation of

chitin or SPT inhibitors and examine whether these inhibitors block the growth and cyst

production by E. histolytica, which is responsible for causing often-deadly childhood diarrhea

throughout the world.

28

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Curriculum Vitae

Duran DeBons was born in Marine City, Michigan. The only child of Josef DeBons and

Gulsen DeBons, he graduated from L’Anse Creuse High School North in Chesterfield Twp,

Michigan with full honors in the spring of 2003. Duran entered UTEP in fall of 2003 pursuing a

bachelor’s degree in biomedical science. While acquiring his degree Duran worked full time

continuously. He graduated from UTEP with his BSc in biomedical science in the spring of 2008

and entered the UTEP Environmental Science Masters Program in fall of 2010. Duran DeBons is

of dual citizenship of both the United States and Turkey and is fluent in both English and

Turkish.

Permanent Address

6304 Franklin Ridge

El Paso TX, 79912