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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I
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