Biochemical Studies on Entamoeba histolytica with Special

Reference to its Host Invasive Functions

A DISSERTATION SUBMITTED TO THE

ALIGARH MUSLIM UNIVERSITY, ALIGARH FOR THE DEGREE OF

Master of PhHosophi/ IN

Biochemistri/

BY ShaUendra Kumar

M. Sc. (Biochem.)

DIVISION OF BIOCHEMISTRY CENTRAL DRUG RESEARCH INSTITUTE

LUCKNOW-226001 December, 1988

DS1315

This Work Is

Ded icated

IN

MEMORY OF

MY LOVING FATHER

T«l«x :053S-28S Teleflram : CENDRUG Phone : 32411-18 PABX STTT Jtf^, Tt?? 5nf?r flf» 173

*rePTS 226 001 ( m r w )

CENTRAL DRUG RESEARCH INSTITUTE Chattar Manzil, Post Box No. 173

LUCKNOW-226001 (INDIA)

No. Date Dec . 30 , 191

Dr.PREM SAGAR, ASSISTANT DIRECTOR, DIVISION OF BIOCHEMISTRY

CERTIFICATE

This is to certify that the work in this thesis entitled " B iochemical Studies on E_nt_a_nioe_b_a_ hi_s^to_l_y_t_i_c_a_ with Special Reference to its Host Invas ive Functions" has been carried out by Mr.Shailendra Kumar, M.Sc. (B iochem.) under my supervision.

He has f ulf illed the regu irements of the Aligarh Muslim U nivers ity regarding the prescribed period of invest igational work for the award of M.Phil degree.

The work inc luded in this thesis is original unless stated otherw ise, and has not been subm itted for any other degree.

(PREM SAGAR)

-ACKNOWLEDGEMENTS

I am very thankf ul to Prof.A.H.S idd ig i , Chairman,

Department of Biochemistry, Aligarh Muslim University,

Aligarh, for his inspir ing gu idance, constructive

cr it ic ism and encouragement during the progress of

this work.

I express my deep sense of grat itude and indebted­

ness to Dr.Prem Sagar, Assistant Director, Div is ion

of B iochemistry, CDRI, for suggest ing the problem

and taking keen and helpful interest during the entire

course of this study.

My grateful thanks are due to Dr.S.R.Das, Assist­

ant Director, D iv is ion of Microbiology, CDRI, for

his invaluable suggestions, timely advice and for

prov id ing all laboratory f ac i l i t ies includ ing amoebic

cultures without which this work could not have been

undertaken.

Words will not suffice to express rry feel ings

and gr atef ulness to Dr.L.M,T r ipathi, Sclent i s t , Division

of Biochemistry, CDRI for his suggestions, unf1 inching

co-operations and fruitful criticism.

I am highly grateful to Prof.B.N.Dhawan,Director,

C.D.R.I., Dr.M.M.Dhar, Ex-Director and K.C.Saxena,

Head, Division of B iochemistry for prov id ing laboratory

f a c i l i t i e s .

Technical ass istance of Messrs. L.M.P.S ing b,

R.K.V aish, S.K.Base, R.B.Lai and A.K.Gupta in various

aspects of ex per imental work is grateful'ly acknowledged

Last but not the least I express my f eel ings

of obiig at ion and gratitude towards ever helpful coll­

eagues who have ex tended all possible help in my work

and preparation of this d issertat ion.

AkaDu^J^^ (SHAILENDRA KUMAR)

CONTENTS

PREFACE

ABBREVIATIONS

1-11

iii-iv

CHAPTER I

CHAPTER II

INTRODUCTION AND REVIEW OF

LITERATURE

MATERIALS AND

METHODS

CHAPTER III RESULTS

CHAPTER IV DISCUSSION

1-33

34-49

50-79

80-86

CHAPTER V SUMMARY AND CONCLUSIONS 87-90

BIBLIOGRAPHY 91-102

.0. . .

-PREFACE-

The world of parasites presents a unique and

diverse panorama of biological species. The interact­

ions of variety of parasites with man is a major

human health and hygiene problem all over the world/

specially in tropical countries like India/ where

a wide variety of parasitic infection like amoebiasiS/

filariasis/ malaria/ leishmaniasis and leprosy etc.

pose major health hazards. Amoebiasis caused by

protozoal anaerobic amoeba Entamoeba histolytica is

a ubiquitous and serious disease in India.

The parasite (E .histolytica) has an indistingui­

shable dual character pertaining to its virulent

and avirulent forms. As a virulent form its importance

lies in its intestinal tissue necrotic activity resul­

ting into high incidence of dysentery and liver abscess,

As an avirulent form/ though its trophozoites dwell

in the intestinal lumen yet they do not invade the

clonic mucosa and hence present no apparent clinical

symptoms of the disease. The problem of relapse

and recurrence of infection of E .histolytica despite

best of available chemotherapeutic treatments is

still a serious one.

11

Very limited information concerning factors aff­

ecting host-parasite relation is available. One does

not know the chemical weapons employed by the parasite

in invading the host. Equally interesting is the quest­

ion how this parasite protects itself from the immuno­

logical mechanisms and relatively aerobic conditions

while in the host. The research embodied in the present

dissertation represents a humble effort to obtain some

insight into biochemical aspects of E.histolytica virul­

ence or its tissue invasive property.

Ill

ABBREVIATIONS

BHK

BSA

°C

CHO

Con . A

DMSO

UNA

DNAse

DTT

EDTA

EGTA

9

GPI

HK

hr

KD

lb

M

MAI

mg

mm

MW

NAD

NADH

NADP

NADPH

NBT

OD

PAGE

PBS

pCMB

Baby hamster kidney

Bovine serum albumin

Degree Celsius

Chinese hamster ovary

Coneanavalin A

D imethy1 sulphox ide

Deoxy r ibonucleic acid

Deoxyribonuclease

Dithiothreitol

Ethylened iamine tetr a-acetate

Ethylenegly col tetra-acetate

G r am/Acceler at ion due to gravity (cent­rifugal force)

Glucose phosphate isomerase

Hexokinase

Hour

K ilod alton

Pound

Molar

Microscopic aggregation induce

Milligr am

M illimetre

Molecular weight

N icot inamide adenined inucleotide

Reduced NAD

N icotinam ide adenine d inucleotide phosphate

Reduced NADP

N itroblue tetrazolium

Optical density

Poly aery lam id e gel electrophoresis

Phosphate buffered saline

p-chloro metcur ibenzoate

IV ,

PGM

Pl

RBC

RNA

RNAse

r pm

SDS

TPS

TYI

Phospho glucomutase

Isoelectric point

Red blood cell

R ibonucleic acid

R ibonuc1 ease

Revolution per minute

Sodium dodecyl sulphate

T ry pt ic ase panmade serum

T ry pt icase yeast ex tr act ironserum

CHAPTER - I

INTRODUCTION AND REVIEW OF LITERATURE

-1-

Amoebiasis is the infection of man and other

mammals caused by a protozoan parasite, Entamoeba

histolytica (Schaudinn, 1903)/ which belongs to the

class Rhizopoda. At present time this disease has

a cosmopolitan distribution with greater prevalance

in tropical and subtropical countries and still remains

one of the major unconquered human affliction despite

the development of a variety of antiamoabic drugs.

A more generalised definition of amoebiasis has been

advanced by an expert group of World Health Organization

(WHO) according to which 'It is a condition of harbour­

ing E.histolytica with or without clinical manifest­

ations ' .

The first evidenc " of amoebic aetiology of dysent­

ery came in the year 1875 from the observation of Russian

physician Losch (1875)/ who found a positive correlation

between the number of active amoebae in the stool of

his patient and the severity of disease symptoms during

the coarse of his illness. Losch also observed that

the disease could be induced in dogs by administering

the infected stools of the patients.

Robert (1975) reported the presence of amoebae

in histological sections of dysentery-associated liver

abscess in human patients-

Quincke and Roos (19r75) reported that life cycle

of dysentery amoeba comprises of the 'cystic' and

-2-

' trophic' forms.

VIRULENCE OF E_. hls_tol_^ti^c_a

The term pathogenicity is used to denote the

general ability of an organism to produce disease,

whereas the virulence also refers to that ability,

but has a more specified connotation that implies degree

(Gladstone, 1970).

Although pathogenicity of E.histolytica was dis­

covered more than a century ago, the factors responsi­

ble for its virulence are yet to be properly elucidated.

Clinicians have observed that human infections with

E.histolytica are associated with diarrhoea or dysentery

or may be without symptoms. The work on the virulence

of E.histolytica showed that strains isolated from

asymptomatic cases vary in their virulence from comp­

letely non-invasive to highly invasive ones (Singh,

et al. , 1963; Schensnovich and Saloview, 1963;Sarkisyan

and Vaskanyan, 1966; Kasprzak e_t al . , 1973). According

to the work of Beaver e_t_ aj^ (1956) the virulence of

E .histolytica has been determined in animals by the

degree of ulceration.

Neal (1954,1965) found that encystation does

not increase virulence. He also observed that when

amoebae were maintained in j ^ vitro cultures for pro­

longed periods their virulence was lost inspite of

periodic encystment. ^

-3-

The study of amoebic virulence has necessitated

the development of suitable animal models for studying

the disease. Many model systems have been used includ­

ing intestinal infection of kittens (Hoare, 1925)/

dogs (Faust, 1932), guinea pigs (Jervis and Takeuchi,

1979; Phillips and Gorstein, 1966),mice and rats (Gold

and Kagan, 1978; Neal, 1960) and hepatic infection

in hamsters (Jarumilinta, 1966 and Mattern and Keister,

1977) and monkeys (Martinez-Reyes £t_ a_l-> 1980). It

may be emphasized that there is no universal correlation

of virulence observed in animals with the actual extent

of the human disease. In particular, a strain of amoeba,

H 303:NIH totally avirulent in animals was isolated

from the contents of a liver abscess of a human patient

(Diamond et al . / 1976). The animal models do, however,

provide a semi-quantitative measure of disease potential

of specific strains of amoebae, and in studies where

different animal models were compared, similar rankings

of virulence were generally obtained (Mattern e_t_ al. ,

1978) .

Zlgop^Qg.QJ'T.g_g^gglMg ..J RE^^Ti_q_ti_TjD__i_T_s^ INVASIVE FUN^CT^ION

(a) I_nt_ac_t__t^£0£hoz^oJ^te^s_

The capacity of E.histolytica to move through

pseudopodia and ingest host erythrocytes by phagocytosis

was recognised by Losch (1875) himself who suggested

the presence of a flexible binding membrane in this

amoeba. Significant portion of membrane at the tropho­

zoite surface is involved in endocytosis, pinocytosis

transitional mobility (Jarumilinta and Kradolfer, 1964;

Weisman and Korn, 1967 and Bov/ers & Olszewski, 1972),

contact dependent invasion and eventual engulfment

or lysis of host cells, and host cell adhesion (Takeuchi

and Phillips, 1975; Ravdin et ^ - ' 1980; Ravdin and

Guerrant, 1981 and Ravdin et ^2^-' 1985). Transitional

mobility of the trophozoites, on account of amoeboid

movements and process of pinocytosis and phagocytosis

imparts distinct characters to the plasma membrane

of amoebae, such as presence of contractile proteins

and actin filaments. Serrano and Reeves (1974,1975)

reported that whole cell surface of the trophozoites

of Entamoeba sp. as well as their isolated plasma memb­

rane has the ability to actively transport glucose.

Equilibrative transport of glucose across the membrane

was several order higher than that by pinocytosis (Serrano

and Reeves, 1975).

Ronadelli e_t aj^. (1977) observed a external fuzzy

coat, glucocalyx, peripheral to the plasma membrane

of the trophozoites. Siddique and Rudzinska (1965)

in the electron micrographs of E.invadens noted the

presence of a fuzzy layer on the outer surface of its

plasmalerama. El-hashimi and Pittman (1970) too, in

their electron microscopic studies, observed a similar

-5-

fuzzy layec in E .histolytica trophozoites obtained

from Colon aspirates of acute amoebic patients and

also from monoaxenic cultures However, they claimed

absence of naked cell membrane in case of axenic

E.histolytica, although the presence of a surface layer

(referred to as glycocalyx) was observed by Feria-velasco

et al . (1977) by cytochemical staining methods in axenic

E.histolytica. The glycoproteins of the outer layer

of glycocalyx may play an important role in the biology

of amoeba, particularly in its cellular recognition,

adhesiveness and antigenic properties.

Lushbaugh and Miller (1974) using histochemical

stains viz., alcian blue, periodic acid^shiff's stain

(PAS), phosphotungstic acid, colloidal iron and Con-

canavalin A (Con A) along with horse raddish peroxidase,

confirmed the acid mucopolysaccharide nature of glyco­

calyx. They also observed that surface coats of mono-

axenically cultured amoebae taken from intestinal lesions

are about twice as thick as those of axenic cells.

These workers further noticed a thicker deposition

of mucopolysaccharide layeC on the amoeba from colon

aspirates of acute amoebic patients when the same was

compared with the one obtained from axenic cultures.

Whether this fuzzy coat of E .histolytica cells is a

separate layer unrelated to plasmalemma or jUst an

extension of cell membrane components is still an open

question. However, some^ observations which support

-6-

the second possibility are given below:

Ito (1965) while studying surface coat of cat

intestinal microvilli by cytochemical staining and

electron microscopy established its 'glycan' nature.

He further reported that this coat presented itself

as a continuous layer throughout the inner surface

of microvilli and was composed of filamentous protrusion

from its epithelial cells/ suggesting that this did

not exist as a extraneous coat but as an integral part

of their plasma membranes. Winzuler (1970) has reviewed

in detail the evidence which supports the view that

glycocalyx on eukaryotic cell is nothing but an exten­

sion of saccharide moieties from globular plasma membrane

proteins.

hore convincing evidence that 'surface coat'

of these cells is an integral part of plasmalemma came

from the work of Forstner (1968), who incubated in

vitro the microvilli epithelial cells with[ C]-glucos-

amine and recovered the radiolabel in their plasma

membrane fraction after their subcellular fractionation.

( b) Propert ies of isolated §_, hi^s_to_l^^ti£a plasma membrane

Plasma membrane, the limiting boundry of the

amoeba, is the primary site of interaction with host

cell during infection. Several methods based on density

gradient centrifugation have been reported for isolation

-7-

of plasma membrane (Schultz and Thompson/ 1969; Korn

and Wright, 1973; Ulsamer et_ al . / 1971; Mclaughlin

and Meerovitch, 1975; Van Vliet e_t_ £l_. / 1976; Serrano

et al . , 1977 and Aley et al. , 1980).

On SDS gel-electrophoresis the proteins of the

plasma membrane of E .histolytica prepared by Serrano

and Reeves (1975) and Serrano e^ a_l_. (1977) resolved

into eight bands ranging in molecular weight from 15-

20 KD. Carbohydrates were also present in the membrane.

The membrane was composed of 0.57 mg phospholipid and

0.11 mg cholesterol per mg of protein and cholesterol:

phospholipid molar ratio^ was 0.39, while the plasma

membrane of E .histolytica prepared by Aley et_ aj^. (1980)

resolved in SDS-PAGE into twelve iodinated peptides

of molecular weight, rangini ' from 12-200 KD, all of

them giving positive reaction with glycoprotein staining.

Cholesterol:phospholipid molar ratio in the membrane

was 0.87. Constituents of phospholipids were: Phospha­

tidyl choline, 12, Ceramide aminoethylphosphonate ,38,

phosphatidyl ethanolamine, 35, phosphatidyl serine,

10 (% of total phospholipids), phosphatidyl inositol

and sphingomyelin were present in traces.

Most of the plasma membrane carbohydrates exist

either in the form of glycoproteins or glycolipids.

The most common monosaccharides found in the plasma

membrane are glucose, galactose, N-acetyl glucosamine, r

-8-

N-acetyl galactosamine, mannose, fucose, neuraminic

acid and sialic acid. These are thought to form branched

oligosaccharides and are linked to the proteins or

lipids. The available evidence suggests that carbohydrate

of plasma membrane glycoproteins are located on the

outer surface of the membrane. Both the glycoproteins

and glycolipids are considered to have important role

in immunological response, cell-cell adhesion and cell

surface transformation in animal cells (Hall and Baker/

1977).

^2.ki.„9.^-,9.^9.ki.§l.i.E.9.k-^L!i-.ZLi.Zk^^^§.

Polyaxenic as well as axenic cultures of amoeba

generally lose their virulence, or invasive property

on prolonged cultivation ii vitro (Phillips/ 1973),

when tested in terms of their lesion-producing ability

in the liver of hamsters or in the caeca of rats and

guinea pigs. However such attenuated cultures may regain

this function on rn_ vitro passage in host livers.

Sharma (1959), Singh (1959) and Singh et_ al.

(1971) for the first time reported that avirulent strains

of E .histolytica became invasive when their bacteria

associated cultures were supplemented with cholesterol

or when animals were fed with cholesterol along with

their normal diet before being intracaecally inoculated

with non-invasive amoeba. This finding has since been

supported by numerous other--invest igators which suggest

-9-

that virulence-enhancing action of cholesterol occurs

also in axenic cultures (Das & Ghoshal/ 1976; Vinayak

et al . 1978; Meerovitch and Ghadirian, 1978a). Thus

Bos and Van de Griend (1977) working with two axenic

strains HK-9 and HB-301 were able to restore the virul­

ence with cholesterol i_n vitro and establish a higher

degree of virulence also by two successive passages

through hamster liver. They further observed that epi-

cholesterol/ the 3 a-hydroxy isomer of cholesterol/

which unlike cholesterol, does not induce changes in

the permeability of the cell/ had the same effect.

Meerovitch and Ghadirian (1978a and 1978b) demonst­

rated revival of the lost pathogenicity of three strains

of E.histolytica/ HM-1/ IMSS and DKB (growing axenically

for the last five and six years respectively) through

a number of successive transfers in culture medium

supplemented with extra-fine cholesterol. The number

of passages in cholesterol-enriched medium necessary

to restore a certain degree of pathogenicity of these

strains in hamsters/ was proportional to the time of

in vitro cultivation of the strain and not just the

time of cultivation/ under axenic conditions. Pathogeni­

city, once restored persisted for a long time after

cholesterol treatment was stopped.

-10-

Parallel to enhancement of E .histolytica virulence

in terms of its lesion forming ability/ potencies or

levels of a number of biochemical factors (which are

quantitatively higher in virulent strains) have been

found to increase on cultivation of the amoebae in

cholesterol enriched environment. Some of these para­

meters are listed below:

1. Concanavalin-A agglutinability (Prasad et^ al. /

1981, Katiyar £t al. , 1987).

2. Haemolytic activity (Prasad et^ ^ - ' 1982; Katiyar

et_ aj^. , 1988a and 1988b).

3. Activity of 'lysosomal' and certain other enzymes,

viz. Acid DNase, Acid RNase, Acid phosphatase.

Acid proteinase, Phosphoglucomutase, Hexokinase,

Phospholipase A, Sphingomylenase (Rai et_ al. ,

1980; Lai and Garg , 1979; Lai et al. , 1980; Dutta

et al. , 1982; Katiyar £t al_. , 1987; Katiyar e^

al. , 1988a; Mishra et_ aj^. , 1988).

The precise mechanism of virulence enhancement

of E.histolytica by cholesterol, however, is still

unclear, although a number of hypotheses have been

putforward in this context. Rothman and Engelman (1972)

demonstrated that cholesterol exerts a dual effect

on the fluidity changes at higher and lower temperatures

in synthetic membranes constituted of lipids/phospholipids,

-li­

lt has been proposed that the levels of cholesterol

in amoebae may thus affect the structure and function

of the plasma membrane of the trophozoites and thus

facilitate the release of the lysosomal enzymes from

the 'surface active lysosome' situated at the tip of

filopodia of E .histolytica (Eaton et_ al . / 1970; Proctor

and Grigory, 1972; Lushbaugh e_t_ aj^. , 1978; Katiyar

et al., 1988b).

Several early workers (Synder and Meleney, 1943;

Rees e_t_ aJ -/ 1944; Hansen and Anderson/ 1948) demonst­

rated that cholesterol is a growth promoting factor

in E .histolytica and this property of the sterol has

also been considered a possible factor in its virulenece

enhancing action.

Chen et aj^. (1975) suggested that a period of

enhanced cholesterol synthesis is prerequisite for

the subsequent phase of DNA synthesis and for the comp­

letion of mitotic cycle in lymphocytes after phyto-

haemagglutination activation. Kandutsch and Chen (1977)

showed that DNA synthesis showed a linear decline soon

after the addition of inhibitors of sterol biosynthesis.

Hradec and Dusek (1968) and Hradec et a_l_. (1971) reported

that cholesterol may play an important role in some

of the reactions involved in cholesterol's ability

to stimulate such anabolic functions of cells. This

has been regarded as one,- of the possible mechaninms

-12-

where by a general increase in acid hydrolases may

occur in E .histolytica (Lai and Garg/ 1979). However

recent observations of Katiyar e^ al. (1988b) showed

that the sterol does not bring about any significant

change in E .histolytica hydrolases activities at alkaline

pH. They also noted that although Hexokinase (HK) and

Phosphoglucomutase (PGM) were enhanced by high medium

cholesterol; glucose phosphate isomerase (GPI) maintained

itself at its basal level. This seems to exclude the

possibility that observed enzyme changes are due to

general modulation of protein synthesis. Such effects

moreover should be uniform for all proteins and should

not cause much change in enzyme specific activity values.

Martinez-Palomo (1982) proposed that the mechanism

of virulence escalating action of cholesterol may com­

prise of selection of pre-existing virulent clones

in the cultures. The hetrogeneity of E.histolytica

cultures was indicated from their observation that

poisonous bacteria induce significant reduction in

culture virulence. This hypothesis has found support

from recent observations of Katiyar et_ £l_. (1988a),

who observed that cholesterol enriched medium resulted

in marked enhancement of haemolytic potency and acti­

vities of acid hydrolase in a parent avirulent culture

( D K B ) , however, no such changes occurred in the clonal

cultures derived from the same strain by similar chol­

esterol treatment. The same investigators inferred

-13-

that all these data are apparently in agreement with

the concept that high cholesterol may be instrumental

in selecting out certain subpopulations in a hetero­

genous mixture of genetic variants (Martinez-Palomo/

1982; Das e_t_ aj^-/ 1982)/ this being particularly true

for strains showing low virulence.

VIRULENCE RELATED BIOCHEMICAL FUNCTIONS OF E_NT_AMOE_BA

1. Enzymes related to its virulence

Co uncilman and Lafleur (1891) for the first

time reported destruction of intestinal submucosa by

the cytolytic action of E.histolytica and Dobell (1919)

showed that the amoebae in sub-mucosal ulcers in pat­

ients were surrounded by clear lytic areas. He proposed

that such areas were possibly formed through the action

of some cytolytic enzymes released by amoebae.

Neal (1960) and Jarumilinta and Maegraith (1969)

reviewed available information on the role of E .histo­

lytica enzymes in its host tissue invasive process.

However interpretation of most of the earlier work

in this area was difficult due to presence of a complex

microflora in amoebic cultures.

Neal (1960) demonstrated the proteolytic activity

in various strains of E .histolytica (both intact amoebae

and their extracts) on Gelatin and Casein. However/

-14-

the caseinase activity found in E .histolytica (Neal/

1956) could not be correlated with its virulence since

this was found in avirulent strains also.

Further/ Jarumilinta and Maegraith (1969) showed

both pathogenic and non-pathogenic E .histolytica possess

tryptic and peptic activity although they were both

devoid of chymotryptic activity.

Recently Munoz et al. (1982), and Gadasi and

Kobiler (1983) reported presence of collagenase in

E.histolytica which existed at a markedly higher level

in its virulent strains. A close correlation between

the ability to produce liver lesions in hamster and

collagenolytic activity of various strains of E.histo­

lytica has been found by Munoz et_ aJ . (198'').

The mechanism by which E.histolytica gains a

successful entry into the deeper tissues was suspected

by Jarumilinta and Maegraith (1960) to possibly depend

on the enzymes of the spreading type such as hyaluro-

nidase. However/ experimental results of different

investigators (Bradin, 1953; Neal/ 1960; Jarumilinta

and Maegraith, 1962) on this enzyme activity of E.histo­

lytica are conflicting and do not provide conclusive

evidence of involvement of this enzyme in the invasive

mechanism of the amoeba.

-15-

Another enzyme which possibly may be involved

in the invasive function of E .histolytica is acid phos­

phatase which was reported for the first time in the

year of 1948 by Carrera and Changus (1948). Eaton et_

al. (1970) and Trevino et_ al . (1971), showed high acid

phosphatase activity in both 'surface active' and cyto­

plasmic 'lysosomes'/ food vacuoles and also in the

intracellular bodies. The former investigators (Eaton

et al./ 1969, 1970) and also Feria-Velasco and Trevino

(1972) proposed that this enzyme associated with cup-

shaped surface lysosomes plays an important role in

the tissue invasion function of E.histolytica. More

recent results suggest that acid phosphatase may be

associated both with plasma and phagosomal membrane

of E .histolytica (Aley e^ J -' 1980; and Warren et_

al. , 1982). Pandey et_ al_. (1977) studied both alkaline

and acid phosphatases in the crude homogenate of axenic

E .histolytica and reported markedly higher activity

of acid phosphatase as compared to alkaline phosphatase.

They also studied the acid phosphatase activity in

subcellular fractions of the homogenate which revealed

maximum activity in the 'lysosomal' and 'nuclear fract­

ions', the former showing nearly two fold higher acti­

vity .

The release of hydrolytic enzymes on the surface

of host cells when attacked by amoeba was postulated

-16-

by Trevino and Castaned (1974), Miller et_ aj^. (1972),

Feria-velasco and Trevino (1972). They proposed that

this release may result from mechanical contact or

more likely, on contact with another living organism

which carries a charge on its surface.

A number of acid* hydrolases (found generally

in lysosomes) other than acid phosphatase have been

found to be associated with sedimentable structures

in E.histolytica. These include phosphatases (Mclaughlin

and Meerovitch, 1975; Serrano e_t_ a_l. , 1977; Van Vliet

et al., 1976), esterase (Mclaughlin and Meerovitch,

1975), Proteinases (Mclaughlin and Feubert, 1977) and

RNase (Weller e_t aj^. / 1981). Mclaughlin and Meerovitch

(1975) demonstrated that these hydrolases were contained

in sedimentable structures, displayed latency, and

phosphatase and N-acetylglucosaminidase at least, were

not released by repeated cycles of freezing and thawing.

Katiyar et aj^. (1988b) recently reported higher basal

levels of acid hydrolases viz. phosphatases, ribonu-

cleases (RNase), deoxyribonucleases (DNase) and protein­

ases in virulent (IP:106) strain as compared to NIH:200

strain of E.histolytica.

Possible involvement of phospholipases in the

virulence of E.histolytica is indicated from the obser­

vations suggesting its role in the haemolytic activity

of its homogenates and the cytotoxic effect of tro-

-17-

phozoites on cellular monolayers (Mclaughlin and Aley,

1985). Long-Krug £t a_l_. (1985) studied the sub-cellular

localization of phospholipase A in the trophozoites

of E.histolytica strains IMSS. They reported two phos­

pholipase A enzymes of E .histolytica/ one calcium indep­

endent and optimally active at acidic pH, and the other

calcium dependent and most active at alkaline pH. Calcium-

independent phospholipase A activity was predominantly

localized in soluble sub-cellular fractipn. Calcium

dependent phospholipase A activity was localized in

fractions derived from the plasma membrane. Calcium

ions can serve as a direct cofactor for the binding

of the enzyme to a phospholipid substrate or can be

indirectly required for cellular release of enzymes

located in cytoplasmic compartments. Intracellular

calcium ions serve as the source of calcium for plasma

membrane-bound phospholipase A enzymes.

Ravdin et al_. (1985) postulated the role of calcium

as a second messenger leading to the release of cyto­

toxic lyso-compounJs produced by amoebic phospholipase

A. A putatine antagonist of intracellular calcium flux,

8-(N/N-diethyl amino)actyl 3,4/5-trimethoxy benzoate

(TMB-8/250 [i M) and calcium chelators [ethylane diamine

tetraacetate (EDTA) and ethylene glycol bis ( P-amino

ethyl ether)-N,N'-tetraacetate (EGTA), 10 mM] inhibited

cytolysis of the target cells. Hence, these workers

-18-

concluded that extracellular calcium ions/ amoebic

intracellular calcium flux and amoebic phospholipase

A activity are required in this invasive function of

E.histolytica. Calcium could serve as an enzyme cofactor

of phospholipase A or be required to initiate vesicle

degranulation, resulting in immediate lysis of the

target cell membrane- Alternatively/ amoebic phospho­

lipase A may alter the integrity of target cell membrane

and render the target cell permeable to attack by other

amoebic enzymes (Ravdin e_t aj^. / 1985).

since glycosidic linkages of membrane proteins

project out of the bilayer and lectins possess specific

affinity for different sugar, they have been widely

used as probes to elucidate surface features of diverse

cells (Sharon and Lis , 1972). A great interest has

been developed by the discovery that some lectins can

differentially agglutinate viral or carcinogen transf­

ormed malignant cells as compared to their normal count­

erparts (Martinez-Palomo et_ ajL. , 1972; Burger and Gold­

berg, 1967). Thus concanavalin A (Con A) a lectin,

isolated by Sumner and Howell (1935) from Jack bean

(Concanavalia ensiformis), which is specific for glucose

and mannose moieties, has been used to identify the

surface differences of normal and tumor cells (Martinez-

Palomo et al./ 1972). They also reported that tropho-

-19-

zoites isolated from acute cases of amoebiasis patients

were selectively agglutinated by Con A while strains

from asymptomatic cases failed to agglutinate.

Das (1977) also observed that Con A gave strong

agglutination reaction with pathogenic strains of

E .histolytica while there was little or no reaction

with non-pathogenic strains. Pathogenic and non-patho­

genic strains of free-living amoebae, however, could

not be differentiated on the basis of Con A agglutinat­

ion since both gave little or no reaction. Prasad

et al. (1981) similarly showed that Con A agglutinabili­

ty was markedly higher in virulent strain IP;105 as

compared to avirulent, DKB strain of E .histolytica.

Trissl £t_ al . (1977) observed that treatment

of motile forms of Entamoeba with low concentration

of Con A and subsequently by peroxidase resulted in

the formation of clear cut caps of Con A receptors

at the posterior pole of the cell. These caps could

be separated from the amoebic cells by simple shaking

and low speed centrifugation. Treatment with a-methyl

mannoside dissociate Con A from the caps and the surface

coat sheats. These observations demonstrate the mobili­

ty of Con A receptor in amoebic membrane.

Mirelman and Bracha (1982) recently demonstrated

that a number of Escherichia coli and Serralia marcescens

-20-

strains which contain mannose binding component on

their cell surface can bind with mannose receptor on

the amoeba membrane. Further/ other bacterial strains

such as Shigella flexneri and Staphylococus aureus/

which do not posses mannose binding capacity can attach

themselves with amoeba with the aid of Con A. These

results suggest possible role of E.histolytica Con

A receptor in attachment and ingestion of bacterial

cells.

Prasad et al. (1985) examined agglutinability

of three E.histolytica strains (DKB, NIH:200 and IP:106)

with six other lectins viz. Ricinus communis agglutinin

and peanut lectin (both galactose specific)/ glycin

max lectin (N-acetyl galactosamine specific), wheat

germ agglv::tinin (N-acetyl D-glucosamine specific) ,

Limulus polyphemus lectin (sialic acid specific) and

Lens culmaris agglutinin (glucose and mannose specific)

+ 2 . .

was investigated. A Ca dependent positive response

was indicated with the last two which also produced

precipitin lines in agar diffusion test against sodium

dodecyl sulphate solubilized sedimentable homogenate

fraction. The sugar specificity of Lens culinaris lectin

is known to be similar to Con A and relative agglutinat­

ion of the three strains by both of them ran parallel

to each other. The sialic acid specific lectin was

however more effective towards the DKB (avirulent strain)/

-21-

whose treatment with trypsin and neuraminidase signi-

ficantiy increase its Con A agglutinability. These

results indicate that lower Con A agglutinability of

avirulent E.histolytica may possibly be due to greater

prepondance of sialic acid moieties on their surface.

Sialic acid is known to be the main contributor of

the surface charges in living cells which can possibly

retard the agglutination phenomenon. Further, enhance­

ment of Con A agglutination of the avirulent strain

by trypsin and neuraminidase treatment indicates that

sialoproteins may possibly mask the Con A receptors

in the avirulent E.histolytica. Prasad (1984) following

the binding of [ C] -Con A with amoebae noted that

the number of Con A receptors on IP:106 was 2.5 times

greater as compared to DKB strains. This again may

reflect 'masking' of Con A receptors in the latter,

although their lower density in this case is also possi­

ble.

Distinctions between isoenzyme patterns as indica­

ted from their electrophoretic mobilities have been

exploited as a method of characterizing strains of

a variety of parasites eg Plasmodium, Trypanosoma and

Leishmania etc. Isoerizymic patterns have been claimed

to be specific and stable properties and strains showing

-22-

the same isoenzyme profile have been considered to

represent a specific 'zymodeme'. Reeves and Bischoff

(1968) for the first time examined electrophoretic

behaviour of five amoebal enzymes/ viz./ glucokinase/

phosphoglucomutase, phosphoglucoisomerase, Oxidored -

uctase and NADP diaphorase from 21 cultures of amoeba

on cellulose acetate gel.

Sargeaunt and his colleagues under took extensive

investigations on isoenzymic profile of hexokinase,

phosphoglucomutase/ glucosephosphate isomerase and

malic cnzynie or\ clinical specimens of E .histolytica

(in non-axenic cultures derived from faecal samples)

collected from all over the world (Sargeaunt and Cuilian

1978; Sargeaunt et_ al . , 1978,1980). They thus claimed

identification of as many as 20 zymodemes of E .histo­

lytica (Sargeaunt £t aj^. , 1982a,b,c). All E .histolytica

strains isolated from clinical amoebiasis patients

were found to belong only to any one of the following

seven zymodames: II, VI, VII, XI, XII, XIII, XIV.

Further, they claimed zymodeme XIV was the sole patho­

genic zymodeme in the Indian sub'^ont inent (Sargeaunt

et a_l_. , 1984) .

No evidence of alteration in isoenzyme patterns

i.e. shifts from nonpathogenic to pathogenic profile

or vice versa, was ever demonstrated in longitudinal

-23-

culture studies (Sargeaunt, 1985; Sargeaunt e_t al. /

1982d) till recently. However, Mirelman et_ aj^. (1987)

demonstrated that the isoenzyme electrophoretic pattern

of an axenic nonpathogenic CDC strain 0784:4 of E .histo­

lytica changes from a nonpathogenic to pathogenic pattern

when the nutritional supplement consisting of irradiated

bacterial cells were provided in the culture medium.

This culture retains the newly acquired pathogenic

zymodeme after continuous treatment with a variety

of antibiotics which finally yielded an axenic culture-

IN VITRO E.HISTOLYTICA EFFECTS ON ISOLATED MAMMALIAN

CELLS, CELLULAR MONOLAYERS OR INTACT TISSUES AS MARKERS

OF ITS VIRULENCE

L°.- -.^Il—B.Il—L—^2.£!IlAD.3.^P.L9.^^i.Il ( Amoebapore)

Lynch £t_ aj^. (1982) identified an ion-channel

forming polypeptide (M.W. 13000 daltons) in virulent

S.histolytica cultures which they designated as 'amoe­

bapore'. This was associated with high speed sediment­

able fraction of E .histolytica homogenates and also

occurred in the used culture medium/ indicating that

it may be secreted by the cells during their growth.

This amoebapore has the distinctive property of getting

spontaneously incorporated in the lipid bilayer, lipo­

somes and cells leading to progressive and irreversible

-24-

changes in ion conductance of target membranes. The

ion channel conductance is cation selective and capable

of rapidly collapsing valinomycin induced potential

in K in multilamellar liposomes.

The amoebapore differs from the cytotoxin of

E.histolytica because it is insensitive to serum and

stable at high temperatures (100 C for 2 mins). This

protein is insoluble in triton X-100 which suggests

its existence in a highly aggregated form. However,

it is soluble in sodium dodecyl sulphate.

Lynch et^ aJ . (1982) proposed that contact dependent

cell cytolysis may be initiated by the insertion of

parasite-derived channels into host cell plasma membrane.

Young e_t_ al . (1982) detected pore forming activity

in both freeze-thawed lysates and membrane pellets

of E.histolytica. Since similar pore forming activity

occurs also in some bacteria and yeastS/ it may repre­

sent a widespread mechanism of cell killing through

colloid-osmotic lysis, comparable to that induced by

other effector cells and complement. Young e^ al . (1982)

also demonstrated that this channel forming protein

is released from amoebaeafter cell surface stimulation

with Con A, a calcium ionophore or bacterial lipopoly-

saccharide. This may also be involved in the lysis

of red blood cells, since fractions enriched in this

protein lysed rat and rabbit erythrocytes. Prasad et

-25-

al. (1982) noted that ionic permeability of these cells

are markedly increased through the action of E .histo­

lytica homogenates much before actual haemolysis.

fi3_e_m_o_l_y_ti_c__a£t_i_v_i_t^^

Lopez-Revilla and Said Fernandez (1980) devised

an i_n vitro system to correlate the haemolytic activity

of cell free extracts of E.histolytica trophozoites

or certain other amoebae with their tissue necrotic

activity by the help of an end point titre method.

The four species tested included E .histolytica, E. inva-

dens / E .moshkovskii and Laredo type of E .histolytica

and the activity was found to be highest in E.histolytica,

Further/ the potency of activity varied in different

strains of E .histolytica and was highest in the most

virulent strain (HM-3:IMSS) followed by HM-2:IMSS (mod­

erately virulent) and least in HK-9 (avirulent). Prasad

et al. (1982) similarly showed that haemolytic potency

was highest for the virulent strain IP:105, followed

by moderately virulent NIH:200 and was least in the

avirulent strain (DKB).

Said Fernandez and Lopiz-Revilla (1988) have

recently reported that free fatty acids released from

phospholipids are major heat stable haemolytic factors

of E .histolytica trophozoits.

-26-

CytotoKins/En terotoxi^iis

Disruption of monolayers of mammalian cells in

tissue cultures through the action of E.histolytica

cells was reported by a number of research groups betw­

een 1975 and 1980 (Knight et_ aj^. , 1975; Bos and Van

De Griend, 1977; Mattern et_ aj -r 1978; Lushbaugh e^

al. y 1978; Bos; 1979). Most of these investigations

emphasized the necessity of contact between trophozoite

and target cells for this cytolytic effect. Ravdin

et al. (1980) systematically studied the lysis by E.histo­

lytica of Chinese hamster ovary (CHO) cells and noted

the importance of contact in the lytic process. Stat­

istical comparison of target cell killing with a one-

hit hypothesis demonstrated that only a single contact

or 'hit' was necessary for target cell destruction.

Both phagocytosis and cytotoxicity by E .histolytica

were inhibited at temperature of 4 C/ however/ binding

of trophozoites to target cells proceeded normally.

Using low temperature to inhibit the other processes

(Ravdin and Guerrant/ 1981), the same group demonstrated

that divalent cations had no effect on amoebae-target

cells binding and that trypsin treatment of trophozoites

did not affect binding affinity. Binding was inhibited

by cytochalasins B and D and by a high concentration

of N-acetyl-galactosamine. The former observation suggests

-27-

involvement of microfilaments in the binding process.

Lopez-|tevilla and Cono-Manera (1982) found by contrast

that divalent cations were required for binding of

erythrocytes to E.histolytica, but in agreement with

Ravdin and Guerrant (1981), they also produced evidence

for the involvement of microfilaments in binding.

A possible role for cations in target cell lysis induced

by E.histolytica was indicated by the protection of

target cells from lysis by slow Na -Ca channel blockers

added after amoeba/target cell interaction (Ravdin

et al. r 1982). This is compatible with the isolation

from E .histolytica trophozoites of a amoebapore capable

of causing pronounced alternations in the ion conduct­

ance of target membranes (Lynch e_t_ a_l. / 1982; Young

et al. , 1982) as already discussed under the heading

ion channel forming proteins-

The inhibition of trophozoite/target cell binding

by the addition of a simple sugar (Mirelman et_ al. ,

1983) suggests that a lectin-like activity may be invol­

ved in the proces of cytolysis. At least two lectin

activities have been reported for E.histolytica. One

is identical to or associated with the amoebal 'toxin'

described below and the other is soluble in sonicates

of E .histolytica but is associated with sedimentable

material when amoebae are lysed by repeated freezing

and thawing (Kobiler and Mirelman/ 1980). It is inhibited

-28-

by chitin and other N-acetylglucosamine containing

glyco-con jugates and is active between pH 5.7 and 6.0

but inactive at physiological pH (7.2). While this

lectin is probably not directly responsible for the

cytolytic activity of E.histolytica / it could play

a role in target recognition and in amoeba-cell binding

that can lead ultimately to lysis. Bracha and Mirelman

(1983) have also described the lectin-mediated binding

of bacteria to E.histolytica/ which involved either

mannose residues or N-acetyl glucosamine or galactose

residues.

In addition to contact dependent or contact regu­

latory cytotoxins discussed above, there are reports

indicating presence of amoebal lytic and toxic factors

in the parasite extracts. Mclaughlin and Aley (1985)

have classified such factors as 'contact independent

cytotoxins'. Amongst these, the haemolytic factors

found in E.histolytica homogenates were discussed under

the heading 'Haemolytic' activity'.

Lushbaugh e_t aj^. (1979) demonstrated that a solu­

ble factor obtained from sonicates of amoebal tropho­

zoites exhibited a cytopathogenic effect on target

cell monolayers. This also had 'toxin like' effect

when tested on guinea pig ileum. This factor was appar-

antly a protein having molecular weight between 35

KD and 45 KD and a pi of 4.5 to 5.0 (Bos, 1979; Bos

-29-

et al. / 1980; Lushbaugh et al. , 1979). It was heat

sensitive but could tolerate pH variation in the range

4 to 10 (Bos et al,, 1980). lodoacetamide and N-acetyl

D-galactosamine inhibited this cytopathogenic effect

while cysteine enhanced the activity (Kobiler e^ al. ,

1981; Mattern £t_ aj^. / 1980). Serum proteins including

feutin (Mattern e_t_ aj^w 1982) and alpha 1-antiprotease

and alpha 1-macroglobulin (Lushbaugh and Pittman, 1979)

also inhibit this activity. McGowan et a^. (1982) claimed

that virulent strains of amoebae contained more activity

than less virulent strains. A powerful toxin, secreted

by amoebae and quickly inactivated in the presence

of serum, could explain many of the observations of

the cytotoxication of E.histolytica. However, the toxin

like substance described does not kill target cells,

either in the presence or absence of serum proteins.

It will disrupt a target cell monolayer in a manner

analogous to the action of either proteases or lectins,

however even after five days of continuous culture

with this 'toxin' Baby Hamster Kidney (BHK) cells remain

viable and resume normal morphology after the addition

of an inhibitor protein such as fetunin (Mattern et_

al. , 1980). Lushbaugh et al. (1981) suggested that

the toxin they isolated was possibly a proteinase,

based on the inhibitory effect of serum antiproteases

and the similarity of chemical properties of the toxin

-30-

to a thiol proteinase (Mclaughlin and Feubert/ 1977).

Subsequently Munoz et_ ajL_. (1982) demonstrated a specific

collagenase-like activity in E.histolytica which they

proposed was involved in invasiveness. Gadasi and Kobiler

(1983) correlate virulence of a given strain with colla-

genolytic activity. However, in all these investigations

the evidences presented are coincidental, there being

no direct evidence for this or any other enzyme being

involved in the invasive process.

Another surface property of Entamoeba is the

ability to phagocytose a variety of particulate mater­

ials including starch grains, bacteria, various protozoa,

inert polystysene beads and erythrocytes (Lushbaugh

et al. , 1975; Neal, 1966; Shaffer and Balsom, 1954).

More than hundred years ago Lesh (Losch) demonstrated

that amoebae in stool samples from human dysentary

contained erythrocytes. Since then it has been claimed

repeatedly that only invasive strains of £. histolytica

inhabiting human intestine ingest erythrocytes and

erythrophagocytosis has been traditionally considered

as an important criteria in identifying pathogenic

E.histolytica trophozoites. On the other hand Entamoebae

other than E .histolytica have occasionally been shown

to engulf erythrocytes both in_ vivo and i_n vitro (Zaman,

1970; Lynch, 1924; Tyzzer and Quentin, 1938).

-31-

Trissl et aJ - (1978) showed significant differen­

ces in the erythrophagocytotic ability of different

B.histolytica strains cultivated axenically. Thus patho­

genic strains were found to phagocytose relatively

higher number of erythrocytes as compared to non-patho­

genic ones. They also demonstrated that this proces

is time dependent upto 30 min.

Adhesion of E .histolytia trophoizoites to animal

cells is essential for ontact mediated phagocytosis

(Shaffer and Balsom, 1954). Lopez-Revilla and Cano

Manera (1982) demonstrated that adhesion rate depends

upon the concentration of amoebae and RBC but not on

the A/B and O human blood groups/ and was maximum at

37 C at pH 7.3. They concluded that E .histolytica adhesion

is an active process that depends on the amoebal cyto-

skeleton and metabolic energy and mobility of both

amoebal and RBC surface ligands. Cano-Manera e_t al.

(1985) also examined the effect of large number of

sugars on amoebic adherence with human erythrocytes

and inferred that this requires presence of atleast

three carbohydrate residues viz. galactose, mannose

and glucose on interacting cell surfaces. Ravdin and

Guerrant (1981) however, while examining the interaction

of amoebic trophozoites with Chinese hamster ovary

(CHO) cells, observed significant inhibition of both

-32-

adherence and cytolysin by N-acetyl D-galactosamine

with much tissues effect of the sugar on their phago­

cytosis. On this basis they suggested that different

surface receptors may be involved in adherence and

phagocytosis.

-33-

The main objectives of the research reported

in this dissertation were:

1. To elucidate the molecular factors related to

invasive action of the amoeba on host tissue.

2. To elucidate the role of cholesterol in the modu­

lation of amoebic virulence.

3. To identify specific biochemical features of

Entamoeba histolytica as potential targets for

selective chemotherapy.

The study employed axenic NIH:200 and IP:106

strains of E .histolytica maintained and cultivated

in modified Diamonds medium which show marked differen­

ces in their invasive properties. Further, the former

culture (NIH:200) was passaged more than 20 times through

cholesterol enriched medium and this also was inclu­

ded in the study. Investigations on erythrophagocytosis

employed in addition the DKB strain (which shows very

low virulence) and a few clonal cultures derived there­

from through cultivation of single cells picked up

by micromanipulative procedure. The parameters investi­

gated include erythrophagocytosis/ nitroblue tetra

Zolium reduction^alcohol dehydrogenase and also pyruvate

phosphate dikinate activities in this amoeba.

CHAPTER-II

MATERIALS AND METHODS

-34-

SOURCE OF ENTAMOEBA ILLiLI^LLLI^ CULTURES USED , IN

THE STUDY

Three strains of E .histolytica > viz. NIH:200,

IP: 106 and DKB were used in the study, which were

received from Dr.S.R.Das, Division of Microbiology,

Central Drug Research Institute, Lucknow. The strain

NIH:200, was obtained by him from Dr .L.S.Diamond,

National Institute of Health, Maryland, whereas other

two strains, DKB and IP:106 from Dr .E.Merrovitch,

Canada.

MAINTENANCE OF E_. H_I_S^T_0_L^YT_I_C_A CULTURES

The axenic NIH:200 and IP:106 strains were

maintained in RNA supplimented modified Diamonds TP-

S-II (Trypticase-Panmade Serum) medium (Diamond,

1968). The modification was based on research under­

taken at C.D.R.I. (Imam, 1986). The DKB strain of

E.histolytica was maintained in Diamonds TYI-S-33

(Trypticase-yeast extract-Ironserum) medium (Diamond,

1978). Both these media were constituted by mixing

a number of solutions whose composition and preparat­

ion procedure is described below.

- 3 5 -

C_o_m2_o_s_i^t_^o_n qf _t_/7£ ilii^£i.££jt ^££t.^ £.£.£ L—^-^LL EIS^iH.21

Trypticase (BBL) 10.0 g

Panmade (Paines and Byrne) 20.0 g

Glucose 5.0 g

L-cysteine (monohydrochloride) 2.0 g

Sodium chloride 5.0 g

Potassium phosphate monobasic 0.6 g

Potassium phosphate dibasic 1.0 g

Ribonucleic acid 2.5 g

Distilled water 895.0 ml

The nutrient broth was suction filtered in Buchner

funnel through Whatman No. 1 filter paper/ then auto-

claved at 15 lbs. pressure (121 C) for 15 min. and

allowed to cool at room temperature. pH was adjusted

to 7.0 with IN NaOH (before autoclave).

vitamin mixture No.107 of Evans et_ aj^. (1956)

was used which was prepared from five stock solutions

as follows:

1 . W ate r_ soluble B v itam ins^

Solution A

Niacin 62.5 mg

Para amino benzoic acid (PABA) 125.0 mg

-36-

These were dissolved In boiling water and final volume

brought to a total of 150 ml with distilled water.

Solution B

Niacinamide

Pyridoxine hydrochloride

Thiamine hydrochloride

Calcium pantothenate

i-inositol

Choline chloride

62.5 mg

62.5 mg

2 5.0 mg

125.0 mg

125.0 mg

12 50.0 mg

These were dissolved in distilled water and final

volume made to 150 ml.

Solution C

Twenty five mg Riboflavin was dissolved in

75.0 ml distilled water with the help of O.lN NaOH,

drop by drop, and final volume brought to 100 ml.

Solutions A,B and C were combined and final

volume made to 500 ml with distilled water.

2. B_io^t^n solution

Thirty mg D-Biotin was dissolved in 200 ml

distilled water with the help of O.lN NaOH, drop

by drop, and final volume brought to 300 ml with

distilled water.

Thirty mg folic acj.d was dissolved in 2^0 ml

-37-

distilled vater with the nelp o£ O.IN NaOH and final

volume brought to 300 ml with distilled water.

Solution A: Three hundred mg vitamin D (calciferol)

was dissolved in 63 ml of 95% (v/v) ethyl alcohol.

Then 300 mg vitamin A was added to this solution

and mixed.

Solution B: Sixty mg vitamin K (menadione sodium

bisulphite) was dissolved in 300 ml of 5% aqueous

solution of tween-80.

Twenty five mg vitamin E ( ci -tocopherol acetate)

was dissolved in 250 ml of ethyl alcohol.

To prepare final working solution/ these five

stock solutions were combined as follow -

Water soluble vitamins

Biotin solution

Folic acid solution

Lipid-soluble vitamins

Vitamin E solution

500 ml

?50 ml

250 ml

2500 ml

250 ml

This complete mixture was sterlized by passaging

through seitz filter and then stored at -20 C until use

-38-

a) Streptomycin 1.0 gtn dissolved in 5 ml sterile

distilled water.

b) Penicillin (10 lakh units) was dissolved in

5 ml sterile distilled water.

c) Terramycin (injectable) 0.1 ml was dissolved

in 20 ml sterile distilled water.

Eighty ml of inactivated (56°C for 30 mints.)

adult buffalo serum was mixed with 20 ml of vitamin

mixture prepared as described above. This serum +

vitamin mixture was sterilized by passing through

Seitz filter.

The constituents of complete TP-S-II medium

for 100 ml are:

Autoclaved TP-S-II nutrient broth 85 ml

Sterilized serum + vitamin 15 ml mixture

Penicillin 0.25 ml

Streptomycin 0.25 ml

Terramycin 0.50 ml

Aseptic conditions were maintained throughout the

preparation of medium.

-39-

Q.2.!!lE2.El^i2.B. 2.i. —!i^£l^Il——^k£°^h ^2.£ L^Lz§.zll HS.^ia.11

i£Aa_mond__^_1^9_7_8_l

Trypticase (BBL) 20.0 g

Yeast extract (BBL) 10.0 g

Glucose 10.0 g

Sodium chloride 2.0 g

Potassium phosphate 1.0 g

(monobasic)

Potassium phosphate (dibasic) 0.5 g

L-cysteine (monohydrochloride) 1.0 g

Ferric ammonium citrate 22.0 mg

purified

Ascorbic acid (Sigma Chem.Co.) 200 mg

Distilled water 875 ml

pH was adjusted to 6.8 by IN NaOH

The broth was suction filtered in Buchner funnel

though Whatman No.l filter paper, autoclaved at 15

lbs. pressure (121 C) for 15 minutes and allowed

to cool at room temperature.

Solution A - vitamin mixture No.107 (prepared as

described)

Solution B - vitamin B, (Sigma Chem. Co.) 40 mg

dissolved in distilled water and

final volume made to 100 ml.

-40-

Solution C - DL-6/8-thioctic acid (Sigma Chem. Co)

100 mg dissolved in 100 ml absolute

ethyl alcohol.

Solution D - Tween 80 (Sigma Chem. Co.) —50 g. Tween

80 was dissolved in 100 ml of absolute

ethyl alcohol.

The final mixture was constituted as follows:

Solution A

Solution B

Solution C

Solution D

100 ml

12 ml

4 ml

4 ml

Final volume was made to 180.0 ml with distilled

water. This working solution was sterilized by passing

through 0.2 l-l M polycarbonate filter (millipore) and

stored at -20 C in dark in brown bottles.

The constituents of complete TYI-S-33 medium

for 100 ml are:

Autoclaved TYI-S-33 nutrient broth 87 ml

Sterilized serum + working 13 ml solution

Penicillin

Streptomycin

Terramycin

0.25 ml

0.25 ml

0.50 ml

Aseptic conditions were maintained throughout the

preparation of medium.

-41-

The procedure of Bos and Van de Griend (1977)

was used to enrich the medium with cholesterol. Thirty

five milligrams of extra fine cholesterol (E.Merck)

was dissolved in 10 ml of chloroform and 100 M- 1 of

this solution (350 p. g cholesterol) was pipetted into

125 X 15 mm screw capped tubes and made to coat as

much of the inner surface as possible. With screw

caps loosened/ the chloroform was allowed to evaporate

at room temperature. The tubes, after being autoclaved

at 15 lbs pressure (121 C) for 15 minutes/ were allowed

to cool and dry at room temperature and were finally

filled up with 12 ml of appropriate axenic medium.

The complete medium was dispensed in screw

capped tubes (125 x 15 mm)/ to check the sterility

of this medium. The tubes were incubated at 37 C for

24-48 hr . before use to test their sterility. These

tubes were inoculated with 10/000 to 20/000 amoebae

from a 72 hrs old parent culture and incubated at

37 C in upright position. Such subculturing was carried

out every 72 hr.

CHEMICALS

N-acetyl D-galactosamine, N-acetyl D-glucosamine/

galactose/ Rhamnose and Acetaldehyde were obtained

-42-

from Sigma Chemical Co./ St.Louis, USA. L-cysteine

HCl, RNA; Glucose/ Lactose and Nitroblue tetra zolium

were obtained from Sisco Research Laboratory, Bombay.

AN_I_M_ALS_

Animals used in the present studies were obtained

from animal colony of Central Drug Research Institute,

Lucknow, housed in air conditioned rooms, and main­

tained on standard balanced pellet (Hindustan Lever,

Bombay, India).

The clonal cultures were developed by the pro­

cedure of Das and Meerovitch (1981) by growing single

cells of the amoeba isolated from the parent culture

(DKB strain) and growing them in TYI-S-33 medium

in a specially designed sealed 'Perspex chamber'.

When a fairly good number of trophozoites had developed

from the isolated single amoeba, the contents of

the cavity were transferred into screw capped culture

tubes containing fresh medium and the culture thus

developed were maintained and grown as usual.

Cultures, during their log phase of growth

(72 hr, at 37 C), were chilled in ice cold water

for 5-10 min. to dislodge the amoebae from glass

surfaces. The culture tubes were then centrifuged

-43-

at 2/000 rpm for 5 mins. followed by at least three

washings (for complete removal of medium constituents)

with 0.85% sodium chloride (normal saline). The super­

natant was decanted off and the amoebae in the pellet

were resuspended in phosphate buffer saline/ pH 7.2

(20 mM phosphate buffer containing 155 mM sodium

chloride/ PBS). The cell number in final amoebic

suspension was counted in a haemocytometer.

Con A agglutination was done according to the

procedure of Prasad et_ a_l. (1981). Lectin agglutina-

bility was assayed in terms of microscopic aggregation

index (MAI) determined through haemocytometer counting

of cells and their aggregates. Equal volumes (0.05

ml) of lectin solutions and amoebic suspension were

mixed together directly on the haemocytometer and

were allowed to interact at room temperature for

5 min. A control sample of the same amoebic suspen­

sion mixed with an equal volume of PBS was similarly

counted in all the experiments. The MAI were deter­

mined according to the method used by Chien (1975)

(to estimate erythrocytic aggregation)/ which was

defined as below

No.of units in control MAI =

No.of units in presence of lectin

-44-

H_AE_M0_LY_T_I_C_AS5AY_

Haemolytic assay procedure was' essentially

same as described by Prasad e_t a^. (1982). The E .histo­

lytica cell suspension (10 cells/ml) in phosphate

buffered saline (PBS), pH 7.2 were homogenised by

25-30 up and down movements of piston in a motor

driven ice-chilled Potter-Elvehjen homogeniser. Larger

cell debris and residual intact cells were removed

by low speed centrifugation. The sediment was resus-

pended in a small volume of PBS, and was similarly

rehomogenised and centrifuged to finally pool the

two supernates. The homogenate thus prepared showed

almost complete absence of intact amoebae on micro­

scopic examination.

Erythrocyte haematocrit (20%) in PBS was incu­

bated overnight (16-18 hr) with an equal volume of

appropriately diluted homogenate so that the amoebal

protein content in the assay mixture was equal to

250 ng/ml. The degree of haemolysis was finally esti­

mated in terms of spectrophotometric (Baush and Lomb

710) measurement of haemoglobin released in the cent­

rifuged supernatant at 440 nm. This was expressed

as percent of the total haemoglobin releasable by

osmotic lysis with distilled water in an equivalent

quantity of the erythrocytes. A similarly incubated

'control' in which ?moebic homogenate was replaced

-45-

by an equal volume of PBS, was invariably set up

and respective haemolysis value was substracted to

compute the presented relative haemolysis data.

Erythrophagocytosis by different amoebic cultures

was assayed using a modification of the procedure

employed by Trissl e_t_ al. (1978). A suspension of

erythrocytes for these studies was prepared from

blood drawn from the eye of the rats through a sharp

capillary. The erythrocytes were separated and washed

three times with the physiological saline rejecting

the top buffy coat. The density of RBC was adjusted

R finally to 10 cells/ml in saline by haemocytometer

counting.

For erythrophagocytosis assay, 0.2 ml of the

amoebic cell suspensions (10 /ml) in the appropriate

growth medium were incubated with 0.2 ml of above

RBC preparation for the desired time at room temper­

ature (28-30 C) . Ten ml of distilled water was then

added rapidly for eliminating the external RBC atta­

ched to amoebic surface. After centrifugation (600

g X 1 min) the pellet was resuspended and fixed for

15 min in glutaraldehyde (0.2% in phosphate buffer

saline, pH 7.0). The cells thus fixed were washed

with PBS and phagocytosed erythrocytes in atleast

-46-

100 amoebae were counted in a haemocytometer. The

degree of phagocytosis was expressed in terms of

average number of RBC per amoebic cells.

To study the effect of different sugars, cell

suspension of amoebae and RBC were preincubated at

room temperature (28-30 C) separately with desired

amount of sugars for 30 min. The two were then mixed

together and the erythrophagocytosis was assayed

microscopically in terms of average number of engulfed

erythrocytes per amoeba.

NITROBLUE TETRAZOLIUM (tjBT) REDUCTION ASSAY

The modified procedure of Kettis et_ aJ_. (1982)

was used to follow NBT reduction by E .histolytica

cells/homogenates.

The 72 hr growth of amoebae was harvested by

cooling the culture tubes in ice for 5 min and washing

the cells with 10 ml Hank's buffer to a density of

3.3 X 10 cells/ml. The cell suspension (0.5 ml)

was mixed with an equal volume of 0.1% NBT in Hank's

solution. The tubes were incubated for desired time

at 37 C in a water bath with a rocking device. The

reaction was interrupted by addition of 1 ml of 0.5N

HCl. The tubes were centrifuged at 1000 x g for 15

min and the formazan produced was extracted with

1.5 ml dimethylsulphoxide. The optical density of

-47-

this extract was read at 572 nm against the respective «

zero time control and its value was taken as the

index of relative NBT reduction.

ALCOHOL DEHYDROGENASE ASSAY

The activity of alcohol dehydrogenase in the

direction of acetaldehyde to ethanol, was assayed

following the slightly modified method of Lo and

Reeves (1978) in the presence of either NADH or NADPH.

The reaction mixture in a total volume of 3 ml consis­

ted/ potassium phosphate buffer, pH 6.5/ 20 ^ moleS/

acetaldehyde, 23 limoles, NADH or NADPH, 0.2 M- moles

and the suitable amount of enzyme protein. The rate

of oxidation of NADH/NADPH was measured by recording

decrease in absorbance at 340 nm in a Simadzu double

beam UV-190 spectrophotometer for 5- min. One unit

of the enzyme was defined as the amount causing the

oxidation of 1 j^mole of NADH/NADPH min under our

experimental conditions and specific activity was

expressed as units (mg protein) . Enzyme activity

was also assayed in the forward direction according

to procedure reported by Lo and Reeves (1978) i.e.

ethanol to acetaldehyde in 50 mM glycine/NaCH buffer,

pH 9.5 containing 0.2 mM NADP/NAD and 0.16 M ethanol

in terms of increase in absorbance at 340 nm.

-48-

PYRUVATE PHOSPHATE DIKINASE

Assay was done by the method of Milner et al

(1975)

Packed E.histolytica cells were suspended in

three volumes of cold 0.05 M potassium phosphate

buffer pH 7.0 containing 0.25 M sucrose and 1 mM

dithiothreitol and ruptured by 20 passess in a

Potter-Elvijem homogenizer. The homogenate was centri-

fuged at 35,000 x g for 30 min. The supernate was

used for the enzyme assay. Properly diluted enzyme

in 0.05 ml was added to the test tubes and then 0.1

ml each of pyrophosphate solution (30 mM, pH 6.7)

and 0.05 ml of AMP (60 mM, pH 6.7), The reaction

was initiated by adding 0.3 ml of the substrate mixt­

ure (1 ml mixture containing imidazole HCl-buTfer/

pH 6.7, sop. moles; (NH.)_SO., 40nmoles, p-enolpyruvate,

4.8 limoles and MgCl», 24 |i moles). The tubes were

incubated 25°C for 30 min . The controls in which

AMP was replaced by an equivalent amount of water

being run similarly in each experiment. The reaction

was terminated by adding 0.3 ml of dinitrophenyl

hydrazine reagent (0.1% in 2 N HCl). After 5 min

at 5° degree celcius > 0.2 ml of 2 N HCl

solution was added and the absorbance was read directly

in a spectrophotometer at 415 nm, 10 min later. For

-49-

calculating the enzyme activity the value of molar

extention coefficient was taken as z 415 = 1.1 x

7 -1 -1 10 mole lit. for the hydrazone formed during

the reaction. The enzyme unit was expressed as amount

of pyruvate formed/min and specific activity was

expressed as units/mg protein.

ESTIMATION OF PROTEIN

Protein was determined according to Low^ry

et al. (1951) using bovine serum albumin as standard-

CHAPTER III

RESULTS

-50-

VIRULENCE ASSAY

The virulence of the cultures used was checked

occasionally in terms of lesion forming ability in

hamsters (25-30 g) by inoculating 5 x 10 amoebae using

the procedure of Dutta (1970). This method reproducibly

induced very clearcut pus-containing lesions with

IP:106 and cholesterol passaged NIH:200, which in

terms of a semiquantitative morphology based grading

procedure fell under the ++ or +++ category. On the

other hand normal unpassaged NIH:200 and DKB did

not induce any lesions under similar conditions,

or at best only occasional small 'pin head' lesions

in case of NIH:200.

Lectin (Con A) agglutination and haemolytic

activity were measured in the culture as ijn vitro

indices of their virulence. Both the activities were

found to be consistently maximum in IP:106 and minimum

in the DKB strain. Further these activities increased

significantly after cholesterol passage of NIH:200

strain (Table 1) .

Comparative results on progress of erythropha-

gocytosis with time in three different E.histolytica

strains are presented in Fig. 1. The data show very

marked differences in the erythrophagocytotic capacity

- 5 1 -

m 0) u 3

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-52-

of IP:105 and DKB cultures, whereas this activity

of NIH:200 strain fell in between two. Statistically

significant differences were, however, indicated

in the clonal cultures derived from the DKB strain

which showed lowest erythrophagocytotic capacity

amongst the above 3 strains (Fig. 2). The erythro-

phagocytosis in clone 3 (DKB-3) isolated from DKB

culture was highest amongst the three clones tested

Serial passage of NIH:200 culture through cholesterol

enriched modified diamonds TPS-II medium resulted

in considerable increase in the erythrophagocytotic

capacity of the culture (Fig. 3).

Effect of different sugars on the erythrophago­

cytotic capacity of NIH:200 and IP:106 cultures are

presented in Table 2. The data show considerable

reduction in the degree of erythrophagocytosis in

both these cultures by N-acetyl galactosamine, galact­

ose and lactose and slight but statistically signi­

ficant decrease by fructose at all the time points.

The other sugars tested showed only marginal influence

on this activity, if at all any, in either strain.

NBT REDUCTIOJI

Results of relative rates of NBT reduction

by NIH:200, IP:106 and cholesterol passaged NIH:200

are presented in Fig. 4. These data demonstrate a

markedly higher NBT reduction rate in IP:106 (which

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tu in 4-1 O (0 A rH tu CL V J - ^

to 4-1

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a c

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-54-

TIME (MIN)

Fig-1 Comparison of the progress of RBC phagocytosis with time in IP:106 (T-f ) , NIH:200 (•- ) and DKB (•—• ) strains of E .histolytica. The bars represent mean ± SD. Values.

LlJ

w

• H

(DD!UloiS!L|-3) v a 3 0 W V / D a a

-56-

a o

o

11 -

10-

9 -

8 -

20 /;o 60

TIME (MIN)

180

Fig.3 Influence of serial passage of E .histolytica (NIH:200) through cholesterol enriched medium on its erythrophagocytotic ability. Kinetics of erythrophagocytosis before ( O — O ) and after ( •-# ) cholesterol passage.

-57-

also shows higher degree of erythrophagocytosis).

Further this activity of NBT reduction increases

after cholesterol passage of NIH:200 strain of E .histo­

lytica. Kettis et_ aj^. (1982) followed NBT reduction

in diluted Eagle's medium which contains a large

number of amino acids etc. To determine whether the

organic substrates present in this medium were having

any role in the reaction similar experiments were

done in normal saline, phosphate buffer and Hank's

buffer completely lacking any organic nutrients (Table

3). Almost the same degree of NBT reduction was still

observed in all three conditions, showing thereby

that the substrates involved in this process are

mostly endogenous- All subsequent studies on NBT

reduction by E.histo]ytica were, therefore, carried

out in Hank's buffer. The data showing NBT reduction

by E.histolytica cells at different pH are presented

in Fig. 5 which showsoptimum activity at pH 6.5.

With a view to identify the endogeneous subst-

rate(s)/coenzymes which may donate electrons to NBT

and to obtain some insight into the mechanism of

this process, action of metabolic inhibitors /acti­

vators and antiamoebic compounds (Tables 4,5 and

6) on formation of formazan from NBT was examined.

The results show that this reaction is highly sensitive

to all the -SH blocking reagents viz. icdoacetate.

-58-

N-ethyl maMmide (NEM) and pCMB • This was also strong­

ly inhibited by quinacrine (an inhibitor of electron

transfer from E.histolytica dehydrogenase to flavin

moieties, Weinbach e_t £l_. 1980) ''^ antiamoebic

drug phanquinone and the metal chelator 0-phenanthrolene^

EDTA and EGTA on the other hand were found to be

activators of NBT reduction. Other chemicals tested

viz-F luoride, Rotenone and Antimycin A were almost

ineffective.

DL-Serine/ Glucose and pyruvate known to induce

an appreciable increase in oxygen uptake by E.histo­

lytica (Weinbach and Diamond, 1974^^ significantly

enhance NBT reduction (Table 7). Further a very high

degree of stimulation of this reaction was induced

by NADH and NADPH, suggesting that generation of

these coenzymes may precede the final transfer of

electrons to NBT (Table? )•

NBT redact ion by £ • i£^ °Ay. i.££ homogenate and its

f r act ions

The homogenates of E.histolytica prepared by

disrupting the cells in a Potter -Elvejem homogeniser

also exhibited NBT reducing activity although some

loss (approximately 40%) in this was indicated when

the cellular activity was compared with that in the

homogenate prepared from the same number of cells.

-59-

Fractionation of the homogenate through differential

centrifugation showed that a major portion of the

activity of the homogenate was recovered in the low

speed sedimentable fraction (Table 8) suggesting

its assocation with plasma membranes or intracellular

vesicles. A significant part of the activity was

however/ found to be associated with the supernatant

obtained from homogenate at 24/000 g.

—i.£.^—^^2.£.^^£y.^IlL9.E—^3.9.£.y.L2.^i.—^2.E———'L.—LB.^]iS.^i.2.E

Phagocytosis of bacteria has been reported

to enhance NBT reduction in E .histolytica as occurs

also in mammalian phagocytes (Kettis £t_ a_l_. / 1982).

The effect of erythrophagocytosis on the nitroblue

tetrazolium reducing ability was studied by comparing

formazan production from this dye by amoebae alone

and amoebae-erythrocyte mixtures. The DMSO extracts

of equivalent quantity of erythrocytes alone did

not show any significant change in their absorbance

with time and gave an O.D. value of 0.117 + 0.008.

The results after substracting this from the values

obtained at all the time points are presented in

Fig. 6. The results suggest a significant increase

in the dye reductive ability of both IP:106 and NIH:200

cultures of E .histolytica which were allowed to phago-

cytose erythrocytes.

- 6 0 -

o u a en e

e c

in

(0

O

c o

•rH 4-1 U

-o o a c (0 N (0

e o

c e o

C •H

e o

o

c •H

e o

00

ro

CD ro

o

03 ro

ro

1X1

CTi

O

ro

00

CM

O

ro

CM I—I

CO

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-61-

Table 4; The effect of some inhibitors/activators on

NBT reduction

Inhibitor/acti- Concentration Relative activity vators (% NBT reduction of

control)

Control - 100.0

lodoacetate 1.0 mM 30.5 2.0 mM 10.6

pCMB 0,6 mM 2 2.6 1.2 mM 7.6

N-ethyl malcimide 3.0 mM 12.5 (NEM) 6.0 mM 8.4

Sod.fluoride 1.0 mM 92.4 2.0 mM 88-5

Imidazole 2.0 mM 78.4 4.0 mM 63.7

0-Phenanthroline 3.0 mM 22.6 6.0 mM 16.9

Sod.deoxycholate 0.06 % 64.7 0.12 % 32.6

EGTA 1.0 mM 213.2 2.0 mM 258.2

EDTA 1.0 mM 232.0 2.0 mM 296.4

-62-

Table 5: Effect of some antiamoebic compounds on NBT

reduction

Compounds Concentration Relative activity (% NBT reduction of control)

Control - 100.0

Emitine 0.1 mM 64.7 0.2 mM 32.6

Metronidazole 0.1 mM 81.6 0.2 mM 78.9

Quinacrine 0.1 mM 87.5 0.2 mM 35.9

lodochloro 0.1 mM 98.8 hydroxyquone 0.2 mM 89.9

Diloxanide furoate 0.1 mM 96.7 0.2 mM 90.3

Diiodoquone 0.1 mM 87.5 0.2 mM 78.3

Phanquinone 0.02mM 57.0 0.04mM 52.9

-63-

Table 6: Effect of superoxide dismutase (SOD) on NBT

reduction

Activity source S.O.D cono/ml Relative activity (% NBT reduction of control)

E.histolytica 0 Unit (control) 100.0 cell suspension ^^ ^^.^^ g^^^

100 Units 92.4

E.histolytica 0 Unit (control) 100.0 homogenate

25 Units 76.1

100 Units 65.8

-64-

Table 7: Effect of exogenous substrates on NBT reduction

by E.histolytica

Substrates Concentration Activity (% NBT Reduction)

Control - 100.0

Glucose 0.5 mM 117.7

Pyruvate 0.1 mM 138.2

DL-Serine 0.5 mM 164.2

NADH 0.2 mM 810.0

NADPH 0.2 mM 1991.0

NAD 0.2 mM 101.9

NADP 0.2 mM 98.4

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a

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Time (min) Fig.4 Relative formazan production in different strains

of E.histolytica/ NIH:200 ( o—0 ); cholesterol passaged NIH:200 (A—A) and IP:106 (•—D) strains. The bars represent S-D. values.

-67-

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-69-

^k9.9.^9.k^£.i.^L£E.o.9.K!iA§.i.

Alcohol is one of the major end product of

carbohydrate metabolism in E .histolytica which is

generated through the mediation of alcohol dehydro­

genase. This enzyme plays an important role in regen­

eration of NAD (from glycolytically reduced coenzyme)

and thus continued operation of the glycolytic cycle.

Our experiments showed that equilibrium of this enzyme

reaction in E .histolytica highly favoured production

of alcohol from acetaldehyde and the backward reaction

had a very poor rate (Table 9). Further, the enzyme

showed a much higher activity in presence of NADH

as compared to NADPH and its activity was optimal

at pH 6.5 (Fig. 7).

A comparison of the specific activity of this

enzyme in NIH:200 and IP:106 culture (Table 9) gave

more than two fold activity in the latter. Further,

this activity in NIH:200 culture showed marked inc­

rease when the culture was cultivated in cholesterol

enriched medium.

Results showing the sensitivity of alcohol

dehydrogenase to various inhibitors in normal and

cholesterol passaged NIH:200 and IP:106 strains of

E .histolytica are presented in Table 10. The data

show that this enzyme activity was also sensitive

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E . h i s t o l y t i c a

-73-

to -SH blocking agentS/ pCMB in this case being most

potent. A very strong inhibition of this enzyme acti­

vity was observed with Zn and Borate ions. However/

the pattern of effect of the compounds tested was

generally similar in different cultures.

Pyruvate phosphate dikinase converts phosphoenol

pyruvate into pyruvate which is an important step

in the glycolytic cycle of E.histolytica. This enzyme

reaction in contrast to the corresponding activity

of mammalian cells was found to require AMP and pyro­

phosphate instead of ATP and inorganic phosphate

as reported earlier by Reeves (1968). The activity

was optimal at pH 6.7 (Fig. 8). Comparative results

of this enzyme activity in IP:106 and NIH:200 (normal

and cholesterol passaged) strains are presented in

Table 11. Unlike NET reduction and alcohol dehydro-

genase; no significant differences were indicated

in this activity in different cultures. The results

on sensitivity of this enzyme to various inhibitors/

activators and antiamoebic drugs are presented in

Table 12 and 13. The enzyme was found to be very

sensitive to pCMB and NEM but not significantly infl­

uenced by iodoacetate. Both EGTA and EDTA significantly

+ 2 + stimulated the enzyme activity and Ca and Zn ions

-74-

Table 11: Pyruvate phosphate dikinase activity of

E.histolytica cultures

Name of culture Specific activity

(ng product formed min mg protein )

NIH:200 42.14

NIH:200 (Cholesterol passaged)

52.58

IP: 106 49.87

-75-

Table 12: Effect of some inhibitors/activators on

pyruvate phosphate dikinase activity

Compound Concentration Relative specific activity

Control - 100.0

pCMB 0-02 mM 17-0

0.04 mM 12.5

Imidazole 2.0 mM 100.0

4.0 mM 94.2

0-Phanthrolene 2.0 mM 100.0

4.0 mM 76.9

lodaacetate 2.0 mM 100.0

4.0 mM 100.0

NEM 2.0 mM 15.5

4.0 mM 14.9

EDTA 2.0 mM 125.2

4.0 mM 143.3

EGTA 2.0 mM 124.0

4.0 mM 123.3

CaCl2 2.0 mM 62.8

4.0 mM 48.6

P-Mercapto-ethanol 4.0 mM 110.2

8.0 mM 99.0

ZnSO. 2.0 mM 10.5

4.0 mM 31.1

Deoxycholate 2.0 mM 92.2

4.0 mM 76.7

-76-

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0)

fO o u 3

OJ

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o iH

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CJD

O

CO

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rO >

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IT)

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Q) AJ (d £1

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00

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No Inhib i to r

Fig.9 :Non-competitivG type plot of pyruvate phosphate dikinase inhibition by Phanquinone (antiamoebic compound)-

-79-

were inhibitory. This enzyme was also found to be

highly sensitive to phanquinone (Entobex) an anti-

amoebic compound, while other drugs listed were more

or less ineffective. The inhibition due to phanquinone

was found to be noncompetitive type and gave a KI

value of 2.97 |jM (Fig. 9).

CHAPTER IV

DISCUSSION

-80-

Several experimental models which do take

infection with complex microflora-associated polyaxenic

stool cultures, are not readily invaded by the same

when axenised. Special models have, therefore/ been

devised to examine the pathogenicity of axenised strains.

A stable marked difference has been reported in the

virulence of DKB and IP:106 strains as tested in new

born and adult golden hamsters (Mattern and Keister,

1977; Ghadarian and Meerovitch, 1978), although both

these strains were initially obtained from acute amoe-

biasis patients. The virulence tests performed by us

in the present study by inoculating 5 x 10 amoebae in

hamster (25-30 g), using the procedure of Dutta (1970)

confirmed very low invasiveness of both NIH:200 and

DKB strains as compared to IP:106.

CHOLESTEROL AS AN INDUCER OF E.HISTOLYTICA VIRULENCE

Marked enhancement of the virulence of E .histo­

lytica by cholesterol supplementation of its culture

media was observed for the first time in the laboratories

of Central Drug Research Institute, Lucknow by Sharma

(1959) and Singh (1959). While Neal and Stewart (1960)

reported their inability to reproduce these findings,

this has since been amply confirmed in a number of labor­

atories by different groups of investigators (Singh

-81-

et al./ 1971; Das and Ghoshal/ 1976; Bos and Vande Griend,

1977; Meerovitch and Ghadirian, 1978a; Rai et_ al . , 1980

and Dutta et_ al . , 1982). Das and Ghoshal (1976) reported

that capacity of the parasite to colonise and induce

ulcers in rat caeca was not inherently lost in axenic

NIH:200 strains and manifested itself when such cultures

were supplied with bacteria together with cholesterol.

The present study reports marked increase in the lesion

forming ability of E.histolytica NIH:200 culture in

hamster liver through its subculturing in cholesterol

enriched environment even in the absence of any bacteria.

CORRELATION OF__ERY_T^H_RO_P_H_AG_OCYTOS_I_S AND VIJRULENCE^

A possible correlation between erythrophagocytotic

capacity of E.histolytica and its invasiveness was indi­

cated from the observation of Losch (1875) who for the

first time demonstrated this to be responsible for amoebic

dysentry. Trissl et aJ . (1978) recently reported a corr­

elation between virulence and its erythrophagocytotic

capacity in axenic amoebic cultures. The present finding

that IP:106 shows maximum erythrophagocytosis amongst

the three strains tested support this view. However/

the clonal cultures derived from the same parent culture

(DKB) were found to exhibit differences suggesting that

the amoebic population in any single culture may not

be homogenous in this respect. Thus a strain designated

-82-

'avirulent' on the basis of animal testing may retain

some virulent amoebic cells and the degree of invasive

capacity of any culture may depend on the proportion

of the heterogeneous subpopulations. It is also mention-

worthy that the same DKB clone (DKB-3) which showed

maximum erythrophagocytotic capacity was found to possess

highest haemolytic capacity, Con.A agglutinability and

greater activity of acid hydrolases (Katiyar e_t_ al. ,

1987). Gitler and Mirelman (1986) recently reviewed

some observations which suggest bunched stimulation

of several other factors (viz. protease and collagenase

activities/ contact mediated killing of target cells

and resistance to direct and indirect complement cascades)

along with virulence restoration by a variety of treat­

ments .

The serial passage of NIH:200 culture through

cholesterol resulted in considerable increase in the

erythrophagocytotic capacity of the culture. This suggests

that cholesterol may somehow enrich the proportion of

virulent population.

Ravdin and Guerrant (1982) examined the influence

of various sugar molecules on adherence of E.histoly­

tica with Chinese hamster ovary (CHO) cells and compared

the action of these saccharides on the cytolytic and

-83-

phagocytotic functions of E.histolytica. They observed

a significant inhibition of both adherence and cytolysis

by the N-acetyl D-galactosamine/ though phagocytosis

of these cells by the amoeba was influenced to a much

lesser degree by the same sugar. On this basis they

suggested that different surface receptors may be invol­

ved in adherence and phagocytosis. Cano-Mancera e^ al.

(1986) recently examined the effect of large number

of sugars on amoebic adherence with human erythrocytes

and inferred that this requires presence of atleast

three carbohydrate residues viz. galactose/ mannose

and glucose on interacting cell surfaces. However/ they

observed that different strains differ from each other

in their adhesion-inhibition pattern by Lhe carbohydrates.

The present results suggest that galactose-containing

moieties on amoebic or erythrocyte cell surfaces are

probably involved in the erythrophagocytotic mechanism

directly/ or indirectly through controlling adhesion.

OXIDO-REDUCTIVE FUNCTIONS OF E.HISTOLYTICA IN RELATION

TO VIRULENCE

Kettis et_ al_. (1982) observed that phagocytosis

of bacteria significantly enhanced the nitroblue tetra-

zolium reducing ability of E.histolytica and we found

that erythrophagocytosis also enhances this activity.

-84-

A similar phenomenon occurs in imammalian phago­

cytes and it has been proposed that this may represent

induction of 'oxygen-burst', whereby reactive oxygen

intermediates (ROI) such as superoxides, singlet oxygen,

hydroxyl redicals and H„0„ may be generated, which exer­

cise a lethal effect on phagocytosed cells. On the other

hand, Bracha and Mirelman (1984) recently observed that

association of E .histolytica cells with various types

of gram-negative bacteria and also microaerobic conditions

markedly increase their capacity to destroy monolayers

of baby hamster kidney (BHK) cells. They also noted

that this effect was abolished by metronidazole and

inferred on this basis that virulence of the amoeba

may, to a considerable extent, depend on the activity

of its electron transport system or its 'reducing power'.

Thus both anaerobic conditions and ingested bacteria

may favour lowering of the redox-pptential and thereby

the efficiency of amoebic electron transport system,

facilitating protection of amoebae from ROI. Increased

efficiency of NBT reduction in phagocytosing amoebae

may thus represent enhanced scavenging of ROI and partial

inhibition of NBT reduction by superoxide dismutase

in E.histolytica homogenates seems interesting in this

context.

-85-

In addition to above mentioned enhancement in

NBT reduction due to erythrophagocytosis, the present

investigations revealed per se greater efficiency of

NBT reduction and also alcohol dehydrogenase activity

in the virulent IP:106 culture as compared to NIH:200/

and marked stimulation of both occurred by cholesterol

passage in the latter. Thus like many other functions

which have been listed earlier/ greater efficiency of

oxido-reductive capacity may also form a characteristic

feature of the virulent forms of this amoeba. Further,

the present finding that NBT reduction can proceed to

a considerable extent even in the absence of any exogenous

organic nutrients suggests that E .histolytica cells

probably possess an endogenous pool of substrates which

can transfer their electrons to NBT. Such a pool of

substrates may itself have a role in scavenging the

toxic products generated through oxidative metabolism

of the amoeba as envisaged by Bracha and Mirelman (1984).

PYRUVATE PHOSPHATE DIKINASE

Pyruvate phosphate dikinase of E.histolytica

is characterised by its requirement of pyrophosphate

and adenosine monophosphater instead of adenosine

diphosphate and inorganic phosphate required by corres­

ponding host enzyme (ReeveS/ 1958). This specific feature

-86-

renders this enzyme particularly interesting from the

point of view of developing selective chemotherapy against

the parasite. Among the various antiamoebic drugs tested,

phanquinone was found to be highly sensitive for this

specific enzyme of E.histolytica. Further work is nece­

ssary to determine whether the observed inhibition of

formation of pyruvate from phosphoenol pyruvate by phan­

quinone in E.histolytica is truly specific to establish

this amoebic function as a selective target for chemoth­

erapy -

Another interesting feature of this enzyme was

its stimulation by metal ion chelator, EDTA/ and specific

+2 . . . +2

Ca ion binder viz. EGTA, and its inhibition by Ca and

Zn . The NBT reduction and also alcohol dehydrogenase

and pyruvate phosphate dikinase activities of E .histolytica

were found to be generally very sensitive to -SH group

blocking agents viz. pCMB/ N-ethyl malimide and iodo-

acetate. This seems particularly interesting considering

the requirement of cysteine in the growth medium of

this amoeba and the mentioned findings of Bracha and

Mirelman (1984) that invasive action of the amoeba is

higher under conditions which favour lower redox-potential.

Specific importance of -SH groups in vital biochemical

functions of the amoeba may render this parasite parti­

cularly sensitive to higher oxygen tensions.

CHAPTER V

SUMMARY AND CONCLUSION

-87-

Ent amoeba histolytica is known to be the cause

of intestinal as well as ex tr a-intest inal amoeb ias is

in man. H owever, many infected ind iv idu als, who show

the presence of cystic or trophic form of the amoeba

in their stools, do not suffer from any symptoms of

this disease. Variable inv as iv e potency of different

^ • hAS.i.°.Ly.^A£.B. isolates is seen also in ex per imental

animals; and quite often such cultures which were ini­

tially pathogenic, lose their virulence on prolonged

cultivation in axenic or poly ax enic cond it ions. Such

attenuated cultures, however, are inv as iv e in the animals

fed with cholesterol and their v irulence is enhanced

markedly by repeated cult iv at ion in cholesterol enr iched

culture media.

The main objective of the present study was

to identify biochemical differences in 'invasive' and

' non- invas ive ' £ • i i_£^o^£^ £££ strains and to invest igate

the mod if icat ions of such par ameter s by cuIt iv at ion

in cholesterol supplemented medium. The salient results

are summar ised below:

ST_RAI_N_ VIRULENCE

Amongst the E_. tii_s_t^ol_^t^£C_a cultures used viz. NIH : 200 ,

DKB and IP:106 which were all obtained originally from

acute amoebiasis patients showed very low degree of

-88-

V irulence while IP:106 was still inv as ive as tested

in hamsters for its les ion-£ orm ing ability in liver.

The same order of v irulence was ind ic ated from tests

of Con.A agglut inab ility of the cells and haemoly t ic

activity of the homogenates which are i_n v_i^t_r_o_ indices

of E_-]li_s_^o_l_i^t^ic_a_ V irulence .

ERYTHROPHAGOCYTOSIS

The virulent IP:106 culture showed significantly

higher erythrophagocy tot ic c apac ity as compar ed to DKB

and NIH:200 strains. Statistically s ignif ic ant d if f er ences

were, however, ind ic ated in the clonal cultures derived

from the DKB strain which exhib ited lowest erythrophago­

cy tot ic capac ity amongst the three strains. The same

DKB clone (DKB-3) which showed max imum ery throphagocy to-

tic capac ity was found to possess highest haemoly t ic

capac ity, Con.A agglut inability and acid hydrolase acti­

vities amongst the clones. Serial passage of NIH:200

culture through cholesterol-enr iched medium r esulted

in cons ider able increase in the erythophagocy tot ic capa­

city of the culture.

A number of sugars viz., galactose, N-acetyl gala-

ctosam ine, lactose and f ructose inhibited ery throphago-

cytosis in both NIH:200 and IP:106 strains^ of E. histo-

^li^±c_a_. Further both these strains showed considerable

-89-

increase in their ability to reduce nitroblue tetrazolium

(NBT) during ery throphagocy tos i s .

O XI DO - RE DUCT I_V E FU NCTIO N S_

Comparison of NBT reduction and alcohol dehydrogenase

in the virulent IP: 10 6, the normal and the cholesterol

passaged NIH:200 showed more than two fold higher acti­

vities of both these parameters in IP:106 and cholesterol

passaged NIH:200 as compared to normal ( unpassaged)

NIH:200. Both these activities were highly sens it ive

to a number of -SH group blocking agents v i z . , iodoacetate

pCMB and N-ethyl maleimide (NEM). The NBT reduction

was strongly inhibited by ant iamoeb ic drugs phanquinone

( Entobex ) and guinacr ine and also by the metal chelator

0-phenanthrolene. Other inhibitors fluori^ e, rotenone,

antimycin A, were however almost inef f ect ive. DL serine,

glucose, pyruvate, NADH and NADPH stimulated NBT reduct­

ion .

The alcohol dehydrogenase activity highly favoured

product ion of alcohol from acetaldehyde and the back­

ward reaction had a very poor rate. Further, the enzyme

showed a much higher activity in pr esence of NADH as

compared to NADPH.

-90-

PYRUVATE PHOSPHATE DIKINASE

The pyruvate phosphate dikinase, in accordance

with the earlier results of Milner e_t a 2_. (1975), was

found to show the r egu irerPents of py rophos phate and

adenos ine monophosphate (instead of adenos ine d iphos phate

and inorganic phosphate required by the cor respond ing

host enzyme). Unlike NBT reduction and alcohol dehydro­

genase, no s ignifleant differences were ind icated in

this activity in different cultures . This enzyme was

highly sensitive to pCMB and N-ethyl malimide but was

not signif icantly influenced by iodoacetate. Both EGTA

and EDTA significantly stimulated the enzyme activity

+ 2 + and Ca and Zn ions were inhibitory. This enzyme was

also found to be highly sensitive to phanqu inone.

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