INTRODUCTION

1.0 Overview:

. Members of genus Leishmania arc a biologically diverse group of

trypanosomatid protozoa. Some species are nonpathogenic to man and

parasitize lower vertebrates e.g lizards. Human Leishmaniasis, a spectrum of

. diseases ranging froi11 simple self limiting or asymptomatic cutaneous forms to

·horribly disfiguring, debilitating fatal disease is caused by different species of

Leishmania. This clinical diversity may be the result of genetic diversity of the

parasite. All Leishmania spp are transmitted by sandtlies belonging to the genus

Phlebotomus in the Old World and Lutzomyia in the New World. Leishmaniasis

represents three major classes of diseases viz (a) Cutaneous Leishmaniasis or

Oriental Sore, (b) Mucocutaneous Leishmaniasis or Espundia and (c) Visceral

Leishmaniasis or Kala-azar.

Visceral Leishmaniasis (VL or Kala-azar) is a maJor public ·health

problem in parts of India. The drugs that are commonly used for the treatment

of VL are pentavalent antimonials. Lately there has been an increase in the

cases of Leishmaniasis refractory to antimonial treatment. Hence there is an

urgent need to look either for alternative chemotherapeutic strategies or vaccine

targets or to devise diagnostics for drug resistance.

Gene amplification is a hallmark for drug resistance. The amplified

genomic DNA sequences encode genes that have survival value for the parasite

such as drug resistance thus circular DNAs encode genes with functions that are

useful to the parasite as well as to the researcher.

The work in this thesis relates to one such amplified DNA i.e LDI. It is

amplified in about 20% of L.donovani isolates. LDI is identified as an

important set of genes and the work done in this thesis relates to the expression

and characterization of two open reading frames (ort) of LDI namely ortF and

orfl. Before the discussion about the work done a brief review of the existing

literature related to Leishmania in general, chemotherapy and amplified DNAs

in Leishmania is presented below.

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2.0 Life Cycle:

The Life cycle of Leishmania includes transmission from the

Phlebotomine insects to its host i.e mammals (plate- I). The parasite lives

extracellularly in the alimentary tract of the sandfly in the form of flagellated

promastigotes. When the sandfly bites its mammalian host, the promastigotes

are transmitted to the host. The promastigotes then infect the macrophages and

differentiate into nonmotile, amastigote form. This intracellular parasitism

continues in a chronic form locally or spreads to either the mucocutaneous

tissues or the visceral organs. The reservoir of this parasite could be human,

dog, horse, etc.

3.0 Disease Incidence :

Leishmania is distributed world wide and 0.15 million people are

estimated to be infected. 40,000 new cases can be seen every year (1 ). In 1977,

Leishmaniasis was classified as one of the six major tropical diseases by World

Health Organisation (WHO). These include : Malaria, Schistosomiasis,

Filariasis (including Onchocerciasis), Trypanosomiasis (both African sleeping

sickness and the American Chagas disease), Leishmaniasis and Leprosy. Three

of these diseases, Leishmaniasis, Malaria and Trypanosomiasis are caused by

protozoans. Malaria is regarded as the most widely occurring protozoal disease

of man. Leishmaniasis is however, an added challenge since it presents much

greater problems in both diagnosis and treatment. As compared to four major

species of malaria, at least 19 different Leishmania species have been associated

with human Leishmaniasis, the majority of which unfortunately offer no cross

immunity (3, 4).

There are many reports of epidemics occurring in many countries at both

small and large scale. Some such epidemics are cited here. Venezuela had 13

thousand cases during 1955-1977 (5), Sudan had 7 thousand cases between

1956-57, Brazil had 63 thousand cases from 1971-86 (6), 50,000 cases were

reported from North Bihar (India) in 1978 (7) two thousand cases were reported

from Kenya from 1977-86 and an annual incidence of around 5% of the total

population was reported from Ethiopia (8).

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4.0 Geoi,!raphic Distribution:

The wide global distribution pattern of Visceral Leishmaniasis is depicted

in Platc-2 (2). The distribution of this disease is parallel to that of the sandfly

vector. The most prevalent areas arc India, Central and South America, Central

and South-east Asia, China, the Mediterranean region, Africa, the Arabian

Peninsula, Caspian littoral and Southern parts of Russia. Among these the most

severe foci are India, Africa (Kenya, Sudan, Ethiopia) and Latin America.

Some I 000 million people, a large proportion of world population, are at

potential risk of Leishmania infection and continuously newer disease foci are

coming up (I).

For more than I 00 years, Kala~azar has ravaged the eastern region of

India. This disease first appeared as an epidemic form in India in 1857 affecting

people of the Hoogly district of West Bengal and subsequently in I 962 in district

Burdwan (The Burdwan Fever). In 1868 Kala-azar appeared in the hilly areas

of Eastern India (Garo hills in Assam) and later between I 890 and I 900, it

developed into a full scale epidemic in the valley. Sir William Leishman in 1890

observed the parasite in spleen smear of a soldier who died of "Dum Dum fever"

or Kala-azar in Durn-Dum, West Bengal and reported his findings from London

in I 903. In the same year, Donovan also observed the same parasite in spleen

smear of a patient from Madras. Hence the name Leishmania donovani.

The disease incidence declined rapidly after 1950 and almost totally

disappeared within a decade. However the epidemic hit again in 1975-76 in

North Bihar. The most severe recent epidemic of Visceral Leishmaniasis

occurred in Bihar in I978 with 50,000 cases. The epidemic spread to West

Bengal and approximately 7500 cases were reported from the area in the first 8

months of I 982.

Currently endemic areas are: 30 districts of Bihar, 8 districts of West

Bengal and 2 districts of Uttar Pradesh; sporadic cases were noted in north-west,

in the states of Himachal Pradesh, Punjab, foothills of Himalayas, Jammu and

Kashmir. Sporadic cases were also reported from Tamil Nadu.

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5.0 Clinical Manifestation:

The intracellular parasitism of Leishmania occurs locally (Cutaneous

Leishmaniasis) or spreads to mucocutaneous tissue or visceral organs. Thus

clinically Leishmania species are divided into groups that cause (a) Visceral (b)

Cutaneous and (c) Mucocutaneous Leishmaniasis (Piate-3). The clinical

manifestations of these three groups also differ.

5.1 Visceral Leishmaniasis or Kala - azar:

Visceral Leishmanisis is caused by Leishmania donovani and is fatal if

allowed to go untreated. The intracellular amastigotes of L.donovani reside and

replicate in the mononuclear phagocytic cells of spleen, liver, lymph glands and

bone marrow and produce a chronic disease which is usually fatal.

Infection leads to a fever which is typically irregular and intermittent at

first but later it becomes remittent. Enlargement of spleen is usually followed by

enlargement of the liver in this visceral infection. More than I 9 out of 20 deaths

arc caused by secondary infections mainly of the gut and respiratory tract.

Bacillary and amoebic dysentery, diarrhoea are common secondary infections of

Visceral Leishmaniasis. Some treated Kala-azar cases develop Post Kala-azar

Dermal Leishmaniasis (PKDL) in. a years time (9), which is characterized by the

appearance of depigmented patches on the skin that progress to diffuse nodular

lesions all over the body. PKDL is generally incurable but not fatal and

constitutes a residual reservoir.

5.2 Cutaneous Leishmaniasis or Oriental Sore:

The causative agents are L. tropica, L. aethiopica and L. major and

· several subspecies of L. mexicana. These parasites invade and replicate in

dermal histocytes and produce selflimiting skin lesions. Simple Cutaneous

Leishmaniasis in humans is usually self curative (2), but some exceptions are

there. This disease includes several symptomatic categories. It may be single

lesion non ulcerating ("dry") or multiple exudative ("wet") lesions or diffused

lesions. Frequent relapsing of lesions is also reported.

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5.3 Mucocutaneous Leishmaniasis:

This is caused by L. braziliensis. There is a relapse where the disease

may metastasize · many years later from a single cured lesion to the

mucocutenous area of the nose and mouth and patient may die due to secondary

infections of the affected area.

6.0 Commonly Used Anti-Leishmanial Drugs and Their

Mechanism of Action :

There are two problems associated with drug research in Leishmania

the parasite is intracellular and i't induces immunosuppression. However, there

was no specific treatment for Leishmaniasis before the "Wonder drug" was

introduced by Dr. U.N. Brahmachari in 1922. The drug was a soluble form of

the pentavalent antimonial, urea stibamine (I 0). Organic pentavalent antimonials

arc still the first line of defence against Visceral Leishmaniasis (VL), though

urea stibamine has been replaced by less toxic synthetic compounds e.g: Sodium

stibogluconatc solution (Pcntostam, Wellcomc Foundation, UK) and

antimony-N-methyl glutamine (Giucantime, Rhone Poulenc, France). However,

due to the limited use of antimony for VL and the development of resistant

strains, other drugs are also used as anti-leishmania! agents.

6.1 Pentavalent Antimonials:

Unresponsiveness of the Leishmania parasite to antimonials ts an

increasing problem. For instance in India, the wide spread use of antimonials

during the prolonged epidemic of YL has led to the emergence of primary

resistance (11). Antimonials are expensive, toxic when used for prolonged

periods and have limited usefulness for cutaneous and mucosal forms of

Leishmaniasis. Thus several other compounds and approaches are being tried to

combat this disease.

6.1.1 Treatment of Visceral Leishmaniasis:

When sodium stibogluconate was introduced, the recommended dose was

10 to 15 MKD (mg/kg body wt./day) for 6-8 days. But slowly higher doses for

prolonged period of time were administered to cure patients. World Health

Organization (WHO) recommended that YL be treated with four week course of

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20 MKD of antimony to a maximum of 850 mg Sb (12). Though this dose did

not produce satisfactory results in many cases studied. It was found that II% of

relapsed VL patients had become unresponsive and about I to 20% of previously

untreated patients showed primary unresponsiveness to antimonials (13).

6.1.2. Treatment of Cutaneous (CL) and Mucocutaneous

Leishmaniasis (ML);-

In case of CL, many spectes of parasites are involved and there are

increasing evidences to suggest innate difference in responsiveness to

antimonials. Usually CL is less responsive to antimonials. For example, 40

days treatment cured all patients with VL in Bihar, while I 20 days seems

necessary to cure patients with post Kala-azar dermal Leishmaniasis (14).

ML is almost exclusively prevalent in South America and the causative

organasm ts usually L. brazilensis, which showed relatively poor response to

antimonials.

6.1.3. Antimony toxicity and mode of action:

About 66-80% of the injected dose is excreted out through the kidney

within 6 h which is a drawback of pentavalent antimony, but a blessing too as it

helps to avoid acute toxicity. But cumulative toxicity occurs in patients,

probably due to the production of slowly excreted trivalent metabolites which

increase in proportio_n to dose and duration of treatment ( 15). Pentostam and

Glucantime, which are the least toxic of all antileishmanials, showed a large

variety of side effects. The most common side-effects are myalgia or arthralgia

and anorexia. High doses may cause fatal arrhythmia and liver enzymes may be

disturbed.

Pentavalent antimony inhibits glycolytic enzymes and fatty acid oxidation

in Leishmania amastigotes (20). It has been speculated that glycolytic enzymes,

especially phosphofructokinase, may be adversely affected (17). However, a

relatively recent report indicated that the phosphofructokinase of L. mexicana

was not inhibited by low concentration of Pentostam (18). Pentostam has also

been shown to interfere with fatty acid oxidation in Leishmania amastigotes

(16). Pentostam has also been shown to interfere with nucleic acid and protein

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synthesis in L. mexicana, a process which may be attributable to decreased

nucleoside triphosphate formation ( 19). Being a heavy metal antimonials may

well have other modes of action.

6.2 Pentamidine:

It is a used as a second line drug in case of antimony resistance. Until

recently it was the drug of choice for diffused CL and ML caused by L.

aethiopica.

6.2.1. Toxicity and mode of action:

Unlike antimony, Pentamidine is excreted slowly by the liver and kidney :

50% of a dose over five days. Its coinmon side effects are early hypotension

and late adverse reactions eg. hypoglycemia, diabetes, nephrotoxicity, abscess

formation and even death. Thus, it should be used in patients who show

unresponsiveness to antimony.

Pentamidine's mode of action is poorly understood, although it is known

to da·mage the kinetoplast-DNA-mitochondrial complex (20).

6.3 Amphotericin U; Amphotericin B dioxycholate is a polyene antibiotic,

used as colloidal suspension. It is upto 400 times as potent as ·sodium

stibo?luconate in hamsters and monkeys infected with L. donovani. (21). It is

highly toxic and thus has never been considered satisfactory as a first line drug

for Leishmaniasis.

6.3.1 Toxicity and Mode of Action :

Its usefulness is limited by adverse reactions including an aphylaxis,

thrombocytopenia, flushing, generalized pain, convulsion, chills, fever,

phlebitis, anemia, anorexia, decreased renal tubular and glomerular functions

and lypokalaemia (15).

Amphotericin B binds preferentially to 24 substituted sterols such as

ergosterol, which is a major cell membrane sterol of Leishmania and of fungus,

but not of mammalian cell membrane (16). It also binds to cholesterol in

mammalian membranes and causes toxicity. To reduce the toxicity, other

compositions have been tried, eg. lipid associated ·amphotericin B, Liposomal

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amphotericin B, amphotericin B cholesterol sulfate etc; but none of them have

enough appeal to be recommended for wide field practice (20).

6.4. Allopurinol:

Like other haemoflagellates, Leishmania are unable to synthesize purines

de novo and therefore salvage nucleosides and bases. Allopurinol, a

hypoxanthine analogue hydrolysis to allopurinol riboside which is an analogue of

inosine. These allopurinol ribosides incorporate into the RNA of Leishmania

instead of adenine and hamper protein synthesis, thus killing the parasites (16).

No wide spread use of this compound has been seen.

6.5 Other Antileishmanial A~:ents:

Inhibitors of the biosynthetic pathway of ergosterol from acetate eg:

azoles, allylamines and morpholines have shown some anti-leishmania! activity

(16). Polyamine biosynthctic pathway inhibitors have also been shown to be

potent antileishmanial agent (22-24).

Arninosidine (Paromomycin), an antibiotic of the amino-glycoside family,

is a potent anti-leishmania! agent. Preliminary trials showed satisfactory results.

The practical value of these trials has been to place aminosidine as a safe

tolerable . effective "first-line" alternative to pentavalent antimony for the

treatment of naive and unresponsive VL (20).

7.0 Dru~: Resistance: A General Account:

Chemicals as insecticides, herbicides and chemotherapeutic agents are

being increasingly employed to treat cancer, bacterial, viral and other parasitic

infections. The term "drug" is now more generalized to describe all foreign

chemicals that are used by man either as chemotherapeutic agents, (e.g:

antibiotics, antiviral, antiparasitic and anticancer agents etc), as herbicide,

insecticides or as byproducts of chemical industries.

The repeated use of chemicals often leads to their becoming ineffective

due to onset of resistance or tolerance by the target cells or organisms. This

serves as a major challenge to the pharmaceutical industries. It is thus of great

interest to know about the rnechanism(s) of the type of resistance.

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7.1 Types of Dru2 Resistance:-

Drug resistance can be classified as (i) intrinsic or(ii) acquired

Intrinsic: Where an organism or cell possesses a characteristic "feature" which

allows all normal members of the species or all-type to tolerate a particular drug.

This is also called natural or de novo resistance. This property of the cells or

organisms has arisen through the process of evolution.

Acquired: Where a resistant strain or cell-line emerges from a population that

was previously drug sensitive. In addition a cell showing resistance to a

particular chemical may also show resistance to other drugs. This acquired form.

of resistance may arise by several different mechanisms.

Drug resistance in parasitic protozoa is a major barrier to the treatment

and control of parasitic diseases (25-29). Leishmania spp, the causative agents

of Leishmaniasis have served as a useful paradigm for analyzing mechanisms of

drug resistance in parasitic protozoa, since this genus of parasites can be

cultivated continuously in defined growth media. The substantial increase in the

cases of Leishmaniasis refractory to antimonial treatment necessitates an urgent·

need for alternative chemotherapy. Designing of an effective chemotherapeutic

strategy for the treatment of Leishmaniasis or any other parasitic diseases

depends on fundamental understanding of the mechanism by which the parasite

becomes resistant to drug(s).

The main molecular mechanisms involved in drug resistance are changes

in the membranes, resulting in decreased influx (30) or increased efflux of the

drug molecules, and qualitative and quantitative modification of their target

(31-33). Understanding of the molecular mechanism of resistance is important.

One mechanism by which Leishmania spp becomes refractory to drugs in vitro is

gene amplification.

8.0. Amplified DNAs in Leishmania:-

The Leishmania genome appears to be diploid (34,35) and consists of at

least 36 distinct chromosomes that range in size from 200 Kb to several

megabase pairs (36). Multi-copy chromosomes occur frequently in strains

selected for drug resistance in the laboratory and also in natural isolates. Several

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sets of genes have been reported to be amplified in response to drug selection

upon exposure to drug pressure in the laboratory eg: G-DNA, H-DNA, R-DNA

etc. In addition there are DNA amplification which occur in field isolates and

are not due to intentional selection pressure e.g: D-DNA, T-DNA and LD I. A

description of different amplified DNAs in Leishmania is reviewed below and

shown in Table I.

8.1 G-DNA:

A 63 Kb circular DNA amplified during selection against tunicamycin

(TM). TM is an antibiotic which inhibits the transfer of N-acetylglycosamine to

dolichol phosphate, the first step in the lipid . pathway for protein

N-glycosylation (35), catalyzed by N-acetyl glucosomine-1-phosphate transferase

(NAGT). Twelve additional Leishmania spp, when subjected to TM pressure

have also been found to contain circular amplicons, varying in size in different

species ranging from 30 Kb to 70 Kb, but all share a consensus region of 20 Kb

(36). Later on it was found that a 4.6 Kb region of 63 Kb amp! icon confers TM

resistance and complete sequencing of this 4.6 Kb region revealed that a 1.4 Kb

gene was homologous to NAGT {37).

8.2 I-I-DNA

It is a closed circular DNA that occurs as a dimeric inverted repeat of an

?pproximately 35 Kb sequence each separated by a 5 Kb sequence and also as a

tetramer of the 35 Kb sequence. Thus the total size of the H-DNA ranges from

68 Kb to 170 kb. H-DNA was found to be amplified in stocks selected for

resistance to methotrexate (inhibitor of dihydrofolate reductase), arsenate,

primaquine and terbinafine (38). In methotrexate resistant L. tarentola (a

Leishmania which infects lizards) monomeric H~circles predominate over

dimeric and tetrameric forms (36). In some stocks, H-DNA amplification

confers multiple drug resistance. This may be due to the expression of

P-glycoprotein related gene: P-glycoprotein is encoded by the inammalian

multidrug resistant element, a large plasma membrane protein known to extrude

out lipophilic drugs from mammalian cells. The 5.5 Kb transcript of the

P-glycoprotcin related gene (ltpgpA) in Leishmania proportionally increases with

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amplification of H-circles.. The methotrexate resistant stocks which amplify

H-circles proportional to 5.5 Kb ltpgpA transcript were not cross resistant to any

of the drugs extruded by mammalian multi-drug resistant cells. Transport

studies indicate that there is no clear relationship between H-circles

amplification and methotrexate efflux in L. major. It is therefore not clear

whether there is link between ltpgpA and methotrexate resistance (39). H-ONA

also occurs in stocks that have not been selected for drug resistance (39).

8.3 R-DNA

The first observed and most studied the 30 Kb R-ONA circles, occur m

independently derived resistant stocks of Leishmania against inhibitors of

dihydrofolate reductase (OHFR) or thymidylate synthase (TS). L. major

promastigotes when selected for resistance against methotrexate, over produced

the bifunctional protein thymidylate synthase-dihydrofolate reductase

(TS-OHFR) with amplification at R-ONA which contains the gene for TS-DHFR

(38). Another amplified DNA (H-DNA) was also found, when L. major was

selected for methotrexate resistance. L. major promastigotes when selected for

resistance against I 0-propagryl - 5,8- dieazofolate (CB 3717), an inhibitor of

thymidylate synthase showed over produdion ofTS-DHFR by amplification of

R-ONA. However, there was no amplification of H-DNA. TS and OHFR exist

as distinct and readily separable proteins in bacteriophage, bacteria, yeast and

vertebrates. In contrast, TS and DHFR have been shown to exist as a

bifunctional protein in a number of protozoa (42). In this bifunctional protein

DHFR is located at the N-terminal and TS at the C-terminal (40).

8.4 V-DNA

A mutant strain of L. donovani was generated by virtue of its ability to

proliferate in medium containing increasing concentration of vinblastine, an

inhibitor of microtubule assembly, which showed cross resistance to puromycin

and anthracylines, unrelated drugs to vinblastine. PCR amplification from

n1utant genome and subsequent screening of genomic library identified Ldmdrl,

a mdr-like gene (43). Later, when L. enrieuii was selected in increasing

concentration of vinblastine, resistant clones were isolated which grew in

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concentration 5-30 times the IC50 (30mg/ml) of parental cells. These lines

shared resistance to puromycin, which inhibits protein synthesis. These mutant

lines were found to amplify mdr-like gene as an extrachromosomal circle of

35-40 Kb. The mdr-like gene Lemdr/ was cloned and found to be 1280 amino

acid long with molecular weight of 140 KD. This Lemdrl is structurally similar

to P-glycoprotein which contains 12 transmembrane domains and two ATP

binding sites arranged in two similar half-molecules. This Lemdrl showed

significant homology (37%) in amino acid sequence with human mdrl and 83%

with L. donovimi Ldmdrl gene (44).

A vinblastine resistant L. amazonensis cell line which exhibits

cross-resistance to the unrelated drug adriamycin, was demonstrated to over

express a mdr-like gene (Lemdrl) within 27Kb extrachromosomal circular DNA

(V-DNA). This Lamdrl is homologous to Ldmdrl and Lemdrl (45).

8.5. D-DNA and T-DNA:-

Multicqpy circular DNAs arc also detected in different stocks of

Leishmania, which were not intentionally subjected to drug pressure. These

include D-ONA in L. tropica and T-DNA (19kb) in L. tarentolae. A random

sample of II f!Uthenticated Leishmania strains were obtained among which L.

tropica WR 455 cells contained an amplified DNA, referred to as D-ONA which

migrated distinct from H-DNA on Pulse Field Gel Electroph_oresis (PFGE).

Electron Microscopy (EM) study revealed the mean contour length of this

D-ONA is around 75 Kb which contain a large inverted repeat structure. This

D-ONA originates from a 2Mb chromosome that migrates the compression zone

of PFGE. This D-DNA was found to be a multicopy around 20 copies/cells

(46).

8.6. ODC70-C and ODC140-L:-

L. donovani D 1700 promastigotes when selected against

a~difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase

(ODC) showed over expression of ODC activity. This mutant cell showed over

expression of kinetically similar ODC, the first enzyme of the polyamine

biosynthetic pathway. These mutant promastigotes were also cross-resistant to

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a-rnethylornithine. a-mono-tluoromethyl-3, 4-dehydroornithine methyl ester

and a-methyl-acetylenic putrecine are three other inhibitors of ODC (32). The 3

DFMO resistant L. donovani promastigotes were found to amplify one 140Kb

linear DNA (ODCI40-L) on which all amplified copies of ODC gene were

located and another 70 Kb circular DNA (ODC70-C) containing an inverted

repeat but lacking ODC gene. Both ODCI40-L and ODC70-C were derived

from a pre-existing wild-type chromosome from which amplification occurred in

resistant cells. Both the 140 Kb and 70 Kb elements were not only found to

disappear but also coincided with decrease in ODC activity when grown in the

absence of DFMO (47). ODC70-C is somewhat similar to H-DNA, D-ONA and

LD I in the sense that all four multi copy circular DNAs encompass two unique

sequences flanked by duplicated regions arranged in opposite orientation. The

amplification of ODC 140-L can be correlated with the over expression of the

ODC gene which counteracts its inhibitors, but the function of ODC70-C is not

known.

8. 7 IMPDI-1-280:

A mutant strain (MPA-100) of L.donovani was generated from wild type

01700 when given selection pressure against mycophenolic acid (MPA), an

._ inhibitor of IMP dehydrogenase (IMPDH). The mutant line showed 20 fold

higher enzyme activity, 10-20 fold greater levels of 3.0 Kb mRNA expression

and showed cross resistance to ribavarin, another inhibitor of IMPDH. The 514

amino acid long IMPDH gene showed 52.5% identity with that of

corresponding htunan IMPDH. The 15 fold amplified IMPDH gene copy

number in mutant promastigotes was revealed on 280 Kb extrachromosomal

DNA (IMPDH~I80), confirmed by PFGE. IMPDH-280 was found to be a

linear chromosome which was recognized by a telomere probe and susceptible to

lambda-exonuclease digestion. PFGE Southern blot when hybridized with

IMPDH probe showed that this gene is present on a 700 Kb wild type

chromosome. In addition to these a 740 Kb chromosome also recognized by

IMPDH probe in the mutant genome. Unlike ODCI40-L, the IMPDH-280 and

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drug resistant phenotype remained stable even when the mutant cells were

propagated in the absence of drug for two years (48, 49).

8.8 Amplified LDl circular DNA:

This is another multicopy dimeric circular DNA of L. donovani, L.

infant urn and L. amazonensis. Without any selection pressure, LD I circles

occur as an inverted repeat of two 27.5 Kb sequence. Circular LDI from five

independent isolates have indistinguishable restriction fragment patterns (50).

The LD I sequence is found on a 2.2 Mb source chromosome in all Leishmania

stocks.

8.9 Amplified LDl as small linear chromosomal DNA:

LD I not only occurs in source chromosomes and circular DNAs, but is

also· amplified as small linear chromosomal DNA. LD I has been found in

200-550 Kb small linear chromosomal DNAs, some with high copy number (in

12 stocks) {50).

LDl is amplified in about 20% of L. donovani isolates, specially in those

from India and also in isolates from Visceral Leishmaniasis patients. Gene

amplification provides a survival advantage to the parasite and thus LD I

identifies as important sets of genes and an exploration of the potential of this

locus could prove advantageous against this dreaded disease. The existing

literature on LD I locus is reviewed below.

9.0 What is LDl?

The genome of Leishmania appears to be plastic in the sense that the

segments of genome are often amplified as circular DNAs or small linear

chromosomes. LD I was originally observed on a multicopy 250 Kb linear

chromosome (37). But it has subsequently been defined as the 27.5 Kb DNA

sequence which occurs as a dimeric inverted repeat within the 55 Kb multicopy

circular episomal element in L. infantum (MHOM/BL/671 ITMAP263) (plate

4A) (50). LD l occurs in all Leishmania stocks on megabase size chromosome

and is present (at least in part) in Crirhidia fasciculara, Tlypanosoma cruzi and

T. lewisi, but is absent in African trypanosomes and Plasmodium spp. (50).

When circular DNA of L. injallfli/11 ITMAP 263 was enriched by modified

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alkaline ly:sis (51) and run on a 0,7% agarose gel three major DNA species were

observed : one which migrates at an apparent size of 27.5 kb, another with a

slower mOJbility and the third which was unresolved DNA stuck in the welL The

restricti<?n enzyme digestion pattern of gel purified 27.5 Kb DNA and total

alkaline lysed DNA appeared to be same except for some minor differences.

These results along with digestion of alkaline lysed enriched DNA and digestion

of circular LDl molecules from isopycnic CsCl/EtBr gradients with various

restriction enzyme results in fragments that add up to different apparent total

size, supports the inverted dimeric repeat nature of 27.5 Kb sequence in circular

LDI of L. infantum ITMAP263. The variation adds up to different apparent

total size and is due to the restriction fragments spanning the junctions between

inverted repeats, since these junction sequence represented only once per

molecule whereas other fragments occur twice (50).

When gel purified 27.5 Kb fTagment from alkaline lysed enriched DNA

was hybridized ,with Southern blot of PFGE of different stocks of Leishmania,

all 91 stocks examined contained LD 1 sequence, but the genomic organization

varied among stocks. All stocks contained LD I on megabase size chromosome.

5 stocks (representing 3 different species) contain LD 1 as a multi copy circular

molecule and 12 stocks (representing at least 6 species) also contain LDl within

small chromosomes. But circular and small chromosomal ampJifications are

found to be mutually exclusive (50). Later it was found that LDl is present on a

2.2 Mb chromosome and the size of the small chromosomes on which LD 1

amplifies in some stocks varies from 200-550 kb. This was confirmed by

hybridizing clamped heterogeneous electric field gel electrophoretically·

separated chromosomes of Leishmania (52).

The 27.5 Kb band found from alkaline lysed method was the 27.5 Kb

double-stranded fold back molecule formed from nicked or cleaved circular 55

Kb. LDl during denaturing alkaline treatment. The denatured two inverted

repeats within single DNA strand of 55 Kb reannealed to form double stranded

fold back molecule which migrated as 27.5 Kb linear DNA. This foldback

molecule was resistant to Sl, Exolll and Bal31 nucleases which substantiates

17

their unusual, non-linear. structure and confirms the absence of umque

intervening sequence which would be otherwise looped out and be sensitive to SI

nuclease (53).

The DNA purified from L. infantum lTMAP263 was digested with EcoRJ,

BamHI: Sail, Hindi/! or Kpnl and ligated into similarly restricted pBluscript SK

plasmid vector. By this method the total 27.5 Kb LDI was cloned in a series of

overlapping clones (53).

L. infantum ITMAP263 which has amplified circular LDI, showed 26 stable

transcripts of varying size (0.6 - 15 kb) and abundance covering both. inverted

repeat unit of 55 Kb LD I molecule. Nine adjacent transcripts, ranging in size

from 0.6 - 8.4 kb, mapping to Strand l of 27.5 Kb inverted repeat were more

abundant and thus thought to be mRNAs (plate 4B). Northern blot analysis

using poly A+ RNA showed these transcripts are polyadenylated (53).

The inverted repeat nature of the circular LD I molecule made it difficult

to determine whether the other strand (Strand II) of the molecule also transcribed

(in the opposite direction), since both sets of transcripts would appear identical.

But a number of less abundant overlapping transcripts, mapped through out the

55 Kb molecule which are larger in size and may represent processing

precursors and intermediates.

These results suggest that the sequence of both the strands of inverted

repeats are transcribed. Indeed some transcripts from strand II of the 55 Kb

elements with relatively high abundance were not detected in strains lacking

circular LD I (48). Some less abundant large transcripts that span sequence

encoding abundant mRNAs are also present, suggesting that transcription of LD I

is polycistronic (50). Of particular interest are the abundant 6.3 and 8.4 Kb

transcripts from the strand that is complementary to putative mRNA coding

strand. These RNAs are of the same size and map to similar positions as two

transcripts from the putative coding strand, but are transcribed from the opposite

strand and are thus "antisense" RNAs. Although the 6.3 and 8.4 Kb transcripts

are abundant, sequence analysis indicates they contain no sizable orfs, indicating

a role other than protein coding functions (53).

18

The entire LD 1 has been sequenced as a series of genomic clones (53)

(Piate-4C). The sequences were analysed by using DNASTAR (DNASTAR Inc.

Madison, W l), ESEE (54) and UWGCC (55) software. The overall results of

the analysis is shown in the table.

Transcript mapping and sequence analysis studies identified nine potential

mRNAs that map to adjacent positions on one LD1 strand, suggesting that LDI

encodes at least nine genes (Plate-4C). These nine potential open reading frames

are named orfA to orfl. A brief description of the open reading frames of LDl

is given below.

Qift1 is predicted to contain an epidermal growth factor motif.

Qrf11 encodes a ribosomal protein which is homologous to Rat, Human and

Yeast L37 protein. A four cysteine residue forming motif CX2CX 11CX2C,

which is similar to that found in the steroid receptor family of the zinc finger

domain was also identified in OrfB (58). Northern analysis showed a positive

correlation between PRL37 mRNA abundance and amplification of the LDt;

sequence. However the PRL37 gene is not always amplified with other LD I.

sequences and sequence analysis of LDI reveals another eight likely protein

coding genes which are amplified in L. infantum (ITMAP263 clone 10 i.e.· LSB

7.1)(58).

Qif.{;. encodes a protein which is widely conserved from prokaryotes to

eukaryotes and homologous to SjhB of E.coli. Mutation in SjhB gene causes

slow growth in E. coli and it is possible that OrfC gene product may have a

similar role in Leishmania. Amino acid sequence analysis also revealed that

orfC product contains a heme binding signature sequence:(CXXCH) (57).

Searches with amino acid sequence predicted from oifD, oifE and oifF failed to

show any significant similarities to protein sequence in the data base (57).

OifG is the most interesting among the nine orfs. Searches with orfG revealed

homology with a gene (ESAG I 0) from VSG expression site of Trypanosoma

brucei. This OrfG product is predicted to contain a 10-12 membrane spaning

domains which seems to be a transmembrane protein. The OrfG product also

contains one large hydrophobic region (17a.a) between domains 6 and 7 which

19

supports to their being involved in the formation of aqueous channels through the

lipid membrane (58); possibly functioning as a transporter.

Qd1l is predicted to be a small protein (IOKD) with PI 10.89 i.e. it is very basic

in nature. Both of these properties are the hall mark of a ribosomal protein (58).

Qd1 product is predicted to contain one ATP/GTP binding motif with the

sequence A/G XXXX GKS/T (58).

However, the common feature of these different LD I amplification events •

appear to be over expression of OrfF and OrfG at RNA level, suggesting that

one or both genes play an important role in the cellular physiology of ..

Leishmania and may provide the basis for selection of LD I amplification.

10.0 Aim of the Present Study:

The main objectives of the present work was chromosomal analysis of

LD I in different strains of Leishmania with special reference to Indian isolates.

The correlationship (if any) between drug resistance and LD I amplification has

been another thrust area of this work. Cloning, expression and characterization .

of two open reading frames of LD I namely orfF and orfl has been undertaken in

this work in order to throw light on the possible functions of these two loci of

LD I. Immunodiagnostic potential of recombinant orfF antigen with Visceral

Leishmaniasis (VL) and Cutaneous Leishmaniasis (CL) cases from India, Sudan ;

_and Turkey respectively is another highlight of this work.

·._.

20

Table - I

Salient features of lD1 Orfs

Orf Transcript Protein size PI Signature Homology Remark Ref.

Size (Kb) amino acid Mol. \It (KO) domain to known

gene

A 2.4 469 51.0 Epidermal 56

growth factor

8 0.6 83 9.8 11.85 cx2cx 11 cx2c L37 of 39 nt splice

Zinc finger Rat, Human leader Sequence

domain of yeast

steroid ribosomal

receptor protein

family.

c 3.0 741 80.1 8.02 CXXCH heme SfhB gene 57

binding of E.Coli

domain of

CytC family.

D 2.6 720 81.4 5.63 57

E 2.0 370 40.0 6.32 57

F 1.1 361 39.7 6.32 57

G 3.5 627 68.9 7.20 ESAG10 of 10-12 membrane 58

VSG spanning

expression domain

site of

Trypano-

soma

brucei

H 0.9 87 10.0 10.89 Basic protein 58

1.4 306 33.6 6.42 A/GXXXX/GKS/T ~· 58

ATP/GTP ~-l binding

domain ·~ . -ts

Plate- 1

Leishmania) life cycle in sandfly and in mammalian host. (Adapted from Chang and Bray, 1985). 1. Delivery of promastigotes (proboscis form) into human skin by the bite of sandfly vector; 2. attachment and engulfment by phagocytosis of promastigotes by a macrophage; 3. fusion of phagosome containing a promastigote with lysosome in a macrophage; 4. differentiation of promastigote into amastigote in the phagolysosome of the infected macrophage; 5. multiplication of an amastigote in a parasite-containing or parasitophorous vacuole; 6-. ·formation of large parasitophorous vacuole and continuing replication of intravacuolar amastigotes; 7. rupture of heavily parasitized macrophage and release of amastigotes; 8. phagocytosis of released amastigotes by a macrophage; 9.ingestion of parasitized macrophage by sandfly after a blood meal taken from infected person or reservoir animal; 10. rupture of the ingested macrophage and release of amastigotes in the gut of sand fly; 11. replication of amastigotes and­their differentiation into promastigotes; 12. replication of promastigotes (termed neptomonads for Leishmania mexicana group) in the abdominal midgut and insertion of their flagella into microvilli of the gut epithelial cells; 13. replication of L. braziliensis group in the pylorus and ileum attached to the chitinous gut wall via hemi-dcsmosome; 14.forward movement of promastigotes to thoracic midgut as heptomonads with broad flagella attached to the chitinous gut wall; 15. sessile promastigotes with broad flagella attached to the chitinous wall of stomadeal valve; pharynx and buccal cavity (cibarium); 16. actively motile promastigotcs found in the proboscis or mouth part of sand fly (2).

PLATE- 1

Plate- 2 ·

The distribution of Viseral Leishmaniasis in the world (Shaded areas = endemic areas; dots = spordic cases) (7).

[ ___ _

1

'

'\ ~·' ~., r ~-.

'

D -,_ I

PLATE- 2·

Plate- 3

Pathology of Leishmaniasis . a> Autopsy of a Visceral Leishmaniasjs patient. Note enlarged liver

and spleen.

b > A man showing Post f<ala-azar Dermal Leishmaniasis.

c > A Venezuelan with diffused anergic Cutaneous Leishmaniasis . . .

d > A Brazilian with Mucocutaneous Leishmaniasis showing deformed nose and lips . ·

(Adapted from Pathology of Tropical and Extraordinary Disease, an Atlas, Vol . l)

-~-~~-~---~~------~-----~~------~ ~

a b

c d

PLATE-3

Plate- 4

A) Restrictio.n map of 55 Kb circular LDl molecule in L. infantum ITMAP263. The location of cloned LDI sequences is shown inside the circle. The junctions of the 27.5 Kb inverted repeats are demarcateq by dotted lines. For simplicity, all clones are shown derived from one of the repeats. Restriction enzyme abbreviation. B, BamHI; C, Clal; E, EcoRI; H, Hindii.I; K, Kpnl; N, Notl; S,Sall and Sf,Sfil (53).

p

PLATE- 4A

Plate- 4

B) Transcription map of the 55 Kb circular LD 1 molecule in L. irifantium ITMAP263. The dashed lines demarcate the inverted repeat units. The location, size and relative abundance (indicated by the thickness of the lines) of transcripts are shown. The location of clones used as probes are indicated by thin barred lines within the circle (53).

/

0 ~·

PLATE- 4B

Plate- 4

C) Linear map of LD I. The open reading frames and their transcripts are shown by shaded box and arrows respectively the positions of all the LD I clones are shown as thin lines.

LD1 ORFs, transcripts and genomic clones

0 10

~' ~~~-I~~~--~' ~~ 20 30

HJS c:• ====:I T15~

LT4._. T9-­no-

E13-­EC48 ....

C3-EC55..__.

I

830--------~--------------------~~ ss...-. BN1._.

88---------­BH11-

H8-­KH6...._

BH5._

HC1 ..

522• • xs 1 e-----illl

545 ..

H 2 --------------~N~H~1-~----

EB1-.. 510 e--il

K22.-------------~

K27• • EK1__...

NK1 .,.__.

SK12--

532II===::::J K12--­

K81:J

922-----------------e

HJO.--------------------------. 585--------------------------.

CB1•------• S21e-•-----• SX1..._. SN1...,._.

NS1 e---t1 CK1e-•--~•

~- ····· · ···"•' ' "'' ' ''"'""'' ' ' ' ''' ·~ ~~ ~-}:'~ ~ gs:;:(, ,---------~ s:.lrt.:.w a<!·· ······················ ············.;! Y:i.i·:: ·lil·····-------·--·- -----.;; x.:lH.2v ~-----*

:·~ :~~.; :~ !J ......... ...... - ... ........ -.-·-········ •·•''* :.> ~~ FL.~ n ~-· · · · · ······· · ·~

1 clones

! .__. LSI3-7.1 ORF ' ! ,, .• ---1'.1 L ')S 51.1 I

i T7 • • n +--direct repea ts L _______________________________ ~ !!,~~ ~

PLATE- 4C

I