Drug resistance mechanisms in clinical isolates of Leishmania donovani.

Drug resistance mechanisms in clinical isolates of
Leishmania donovani
Neeloo Singh
Central Drug Research Institute, Lucknow, India
Received March 7, 2005
Leishmania are protozoan parasites distributed worldwide. About 1.5-2.0 million cases are reported
in the world annually from this disease and the death toll is estimated to be 57,000. Along with
Brazil, Sudan and Bangladesh, India contributes to 90 per cent of the global burden of visceral
leishmaniasis (VL). The absence of effective vaccines and vector control programmes, makes
chemotherapy the most widely used tool against leishmaniasis. Chemotherapy based on pentavalent
antimonials has been used for more than 50 years and remains the mainstay for treatment of
leishmaniasis. Clinical resistance to pentavalent antimonials, in the form of sodium antimony
gluconate (SAG), has become a major problem in the treatment of kala-azar (visceral leishmaniasis)
in India. The mechanism of resistance is unclear in these clinical isolates although a lot of work has
been carried out with Leishmania mutants selected in vitro by step-wise increasing drug
concentration using the antimony related metal arsenic and more recently sodium antimony
gluconate. We for the first time, investigated the molecular aspect of drug resistance in clinically
confirmed sodium antimony gluconate resistant field isolates and found that the parasite evaded
cytotoxic effects of therapy by enhanced efflux of drugs through overexpressed membrane proteins
belonging to the superfamily of ABC (ATP-binding cassette) transporters. Additionally, our study
also points towards cell surface changes in resistant isolates.
Key words Clinical isolates - drug resistance - Leishmania donovani - molecular mechanism - sodium antimony gluconate
Leishmania protozoan parasite, belongs to the
family of trypanosomatids and is responsible for a
group of diseases whose symptoms range from mild
cutaneous lesions to fatal visceral involvement.
Today, the leishmaniases are endemic in 88 countries
with an estimated 350 million people at risk. It has
been estimated that 12 million people are affected
by this group of diseases with around 1.5 to 2 million
new cases occurring annually; and this number is
rising1. The group of diseases caused by Leishmania
parasites is transmitted by the bite of sandflies. In
humans, the disease occurs in four forms; life
threatening visceral leishmaniasis (VL), commonly
known as kala-azar; mucosal leishmaniasis (MCL),
self-healing cutaneous leishmaniasis (CL), and post-
kala-azar dermal leishmaniasis (PKDL). Visceral
leishmaniasis is fatal, if left untreated. In 1999, there
were 57,000 deaths reported in India due to VL, but
the real number is thought to be significantly higher.
Ninety per cent of those afflicted by VL live in five
Indian J Med Res 123, March 2006, pp 411-422
411
Review Article

developing countries namely, India (especially
Bihar), Bangladesh, Nepal, northeastern Brazil and
Sudan.
Current treatment options for patients with
leishmaniasis
Pentavalent antimony, the most widely prescribed
drug to treat leishmaniasis patients, was discovered
a century ago, has serious side effects, requires a
prolonged course of treatment and is losing its
efficacy in some regions due to increasing parasite
resistance. Although newer treatments exist, they are
not optimal due to problems of toxicity, high price
or difficulty in administration. Co-infection with HIV
poses an additional challenge. In northern Bihar,
resistance levels of up to 65 per cent have been
recorded in parasites and other regions might also
follow2,3. Research over the past decade has identified
a number of drugs and formulations that offer
improved treatment for this disease4,5. The main
alternative currently available in the area is
amphotericin B (AmB). Although highly efficacious,
AmB treatment is associated with serious side-
effects, and can only be given in hospital settings.
Other alternatives exist to treat VL6 but these remain
largely inaccessible to patients because they are too
expensive (AmBisome, miltefosine which is
teratogenic, only registered in India and expensive)
or not registered (paromomycin). Given the problems
of toxicity, need for hospitalization, growing
resistance, and high costs associated with the
currently available drugs for leishmaniasis, it is clear
that patients urgently need new and improved
treatments to replace or complement these drugs.
Excellent recent reviews deal with Leishmania and
anti-leishmanial drugs available or under
development7,8.
Antimony treatment
It is quite remarkable that even after 50 years of
clinical use, the mode of action of antimony is
unknown, but there is a general belief that to be
active, pentavalent form Sb(V) needs to be reduced
to the trivalent form9. Evidence for reduction inside
the parasites has been described10. An alternative
view is that the metal is reduced in the macrophage
of the host11. Reduction could either occur
enzymatically, as in yeast12, or by parasite or host
derived thiols13. Parasite-specific thiols such as
trypanothione as well as macrophage-specific thiols
such as glycylcysteine, can reduce SbV to SbIII non
enzymatically. Recently, a parasite-specific enzyme
thiol dependent reductase (TDR1), that contains
domains with similarities to omega glutathione
transferases, was shown to catalyze the conversion
of SbV to SbIII using glutathione as a reductant14. A
new antimoniate reductase, ACR2, was characterized
in Leishmania and was shown to reduce SbV and to
increase the sensitivity of Leishmania cells to SbV15.
Recent data suggest that antimony compromises the
thiol redox potential of the cell by inducing the efflux
of intracellular thiols and by inhibiting trypanothione
reductase16. It is possible that more than one
mechanism are responsible for drug activation.
Antimonials are thought to act directly by targeting
important biological features of the parasite. In
macrophage infection models, SbV is leishmanicidal,
but in an animal infection model its mode of action
is dependent on a number of factors including T cell
subsets and cytokines17. Stibogluconate was found
to be a potent inhibitor of protein tyrosine
phosphatases, leading to an increase in cytokine
responses18. These results suggest that SbV may kill
the parasites by both direct and indirect mechanisms,
the host response being implicated in the activity of
SbV. It has been shown that both SbIII and SbV
mediate DNA fragmentation in Leishmania species,
suggesting that antimony kills the parasite by a
process reminiscent of apoptosis19,20. The routes of
entry of antimonials into Leishmania (or into
macrophages) are not known, although pentavalent
arsenate, a metal related to SbV, is known to enter
via phosphate transporters21. The accumulation of
SbV is measured with radioactive isotopes22, and
lately using mass spectrometric approaches23.
Potential mechanisms of drug resistance
Potential mechanism of drug resistance include:
(i) conversion of the drug to an inactive form by an
enzyme; (ii) modification of a drug sensitive site;
(iii) increased efflux or decreased influx; (iv)
alternative pathway to bypass inhibited reaction; (v)
increased production of drug sensitive enzymes; (vi)
412 INDIAN J MED RES, MARCH 2006

increase in the amount of an enzyme substrate
(i.e. , to compete with the drug); (vii) decrease
requirement for product of inhibited reaction; and
(viii) failure to activate the drug.
Many of these mechanisms have been observed
in parasites such as alteration in cell permeability,
modifications of drug sensitive sites and increased
quantities of the target enzyme. These modifications
may arise in a population of parasites by a number
of mechanisms such as (i) physiological adaptations;
(ii) differential selection of resistant individuals from
a mixed population of susceptible and resistant
individuals; (iii) spontaneous mutations followed by
selection; and (iv) changes in gene expression (gene
amplification).
It is believed that the genes which confer
resistance to a particular drug are already within the
parasite populations, but prior to exposure to the
drug, are present at low frequencies. Exposure to the
drug selects for these resistant individuals and their
frequency within the population increases rapidly.
The appearance of resistance within a population has
been observed to occur within 5-50 generations. One
factor which might cause the rapid acquisition of
resistance is the sub-optimal concentration of the
drug, resulting in more survivors.
Drug resistance in Leishmania
Resistance of Leishmania against a given drug
may be either natural, or may be acquired when the
parasites are exposed to sub-optimal drug doses24.
Resistant phenotypes selected in vitro are usually
obtained by culturing wild type parasites under step-
wise increasing drug concentration.
Leishmania has the potential to respond to drug
pressure in multiple ways. Most of the understanding
in this area is gained from work based on cells in
which resistance was selected in vitro . Toxic
metalloids such as arsenic and antimony have always
been an integral part of natural environment.
Metalloid containing drugs are used to combat
infectious diseases caused by pathogenic parasites,
as well as in anticancer therapy. To survive in such a
hostile habitat, it is crucial to develop strategies to
exclude toxic substances from the cell and to acquire
tolerance. Cells remove metalloids from the cytosol
either by active efflux or by sequestration in an
integral organelle. Controlling the influx appears to
be another way of maintaining a low intracellular
metalloid content. The presence of metalloids could
also activate transcription of various cellular defence
genes. The emergence of resistance to metalloid
containing drugs is a serious threat to effective
medical treatment. It is therefore, important to
identify the components that cause the resistance
phenomenon.
Leishmania cells have been selected in the past
for Sb(V) resistance, and some resistance
mechanisms were suggested, including reduced
accumulation25, gene amplification26, and loss of
reduction of the metal10. Since the active drug is likely
to be Sb(III), cells were also selected for Sb(III)
resistance27, and analysis of these mutants led to the
proposal of a model for resistance. This model was
derived mostly from work carried out while studying
resistance mechanisms to arsenite, a metal sharing
several characteristics with antimony, but seems to
hold true for Sb(III), at least in L. tarentolae
promastigotes27. Once Sb(III) is within the cell, it
would be conjugated to trypanothione9, the parasite
specific spermidine glutathione conjugate28.
Trypanothione is found to be increased in arsenite
and antimoniate resistant cells27,29. This Sb-
trypanothione conjugate could then be sequestered
inside a vacuole by the ATP binding cassette (ABC)
transporter PGPA30 or extruded from the cell by a
thiol-X pump31. Altered transport of metals appears
to be an important determinant for resistance25. An
altered membrane partition model for decreased drug
accumulation in drug resistant cells has been
described32. This model suggests that decreased drug
accumulation is the result of alterations in pH
gradients, electrical membrane potential and perhaps
other biophysical parameters, and is not necessarily
a direct result of drug trafficking32. Drug resistance
is also associated with changes in physiological
events such as parasite infectivity, incorporation of
metabolites, xenobiotics conjugation and traffic,
intracellular metabolism, host-parasite interaction,
parasite cell shape and promastigote-amastigote
differentiation33. An understanding of these
SINGH: DRUG RESISTANCE IN L. DONOVANI 413

physiological events may be helpful for designing
chemotherapeutic approaches to multiple cellular
targets, identifying strategies to circumvent
Leishmania drug resistance. Transport studies of
Leishmania cells selected for SbIII resistance in vitro
(either as promastigotes or amastigotes) have
measured a reduction in accumulation of metals in
resistant Leishmania. Either reduced uptake or
increased efflux could explain the reduced
accumulation25,34. It has been shown that a metal
efflux pump is present in the Leishmania plasma
membrane31 which recognizes the metal conjugated
to thiols, such as glutathione or trypanothione29,31. In
mammalian cells, thiol associated efflux is mediated
by the ATP binding cassette (ABC) transporters of
the multidrug resistance protein (MRP) family35,36.
These are involved in multidrug resistance as well
as metal resistance37,38. Three different classes of
ABC transporters have been described in Leishmania
parasites. The first group is homologous to the human
multidrug resistance protein (MRP) subfamily of
transporters39. The second class of ABC proteins
identified in Leishmania parasites is transporters with
a higher similarity to mammalian P-glycoproteins
that confer a multidrug resistance (MDR) phenotype
similar to that observed in cancer cells40,41. The third
class of ABC transporters has been reported to show
high homology with members of the mammalian
ABCA subfamily with related gene been identified
in L. tropica42.
Drug resistance in clinical isolates of L. donovani
So far ours has been the only attempt to elucidate
the molecular mechanism of resistance to antimony
in field isolates. A lot of knowledge has been
generated on work with laboratory mutants but the
most pertinent question is whether this knowledge
can be translated to the field. A better understanding
of drug resistance mechanisms in the field will allow
the development of new diagnostic assays, such as
nucleic acid based tests, that could rapidly detect a
resistance gene.
The first task is to confirm whether the field isolates
are truly resistant to antimonial therapy or not?43. The
field isolates from drug unresponsive and responsive
VL patients were collected by us from disease endemic
areas of Muzaffarpur in Bihar and Varanasi between
1995-1998; 1999-2000; 2003-2004 and 2005. The
patients were admitted to the Kala-azar Medical
Research Centre of the Institute of Medical Sciences,
Banaras Hindu University, Varanasi, Uttar Pradesh
and also its affiliated hospital situated at Muzaffarpur,
Bihar. The criterion for diagnosis was the presence of
Leishman Donovan (LD) bodies in splenic aspirations
performed and graded according to standard criteria44.
After diagnosis, the patients were administered a
course of sodium antimony gluconate, 20 mg/kg body
weight intravenously once daily for 30 days. Response
to treatment was evaluated by a repeat splenic
aspiration on day 30 of treatment. Patients were
designated responsive based on the absence of fever,
clinical improvement with reduction in spleen size and
absence of parasites in the splenic aspirate while
patients who showed presence of parasites in splenic
aspirates were labelled as antimonial unresponsive.
These patients were subsequently treated successfully
with amphotericin B.
Splenic aspirates of responsive (S) and
unresponsive (R) kala-azar patients (VL) were
adapted in vitro culture as described43 and cultures
maintained at 260C. In order to determine whether
resistance is the intrinsic property of parasites and
not the host, antimonial drug sensitivity of these
isolates were evaluated in vitro43. A correlation
between clinical response and SAG sensitivity in
vitro was observed (Fig. 1). Isolate R1 was more
resistant than isolate R2 and R3, and isolate S in
comparison was drug responsive.
Fig. 1. Sodium antimony gluconate (SAG) sensitivity of Indian
Leishmania donovani isolates (S) sensitive; (R1-R3) resistant,
assayed as amastigotes in J774 macrophages.
414 INDIAN J MED RES, MARCH 2006
N
o
In
tra
ve
llu
la
r a
m
as
tig
ot
e
%
Co
nt
ro
l

Fig. 2 (A). A confocal image. Expression of green fluorescent protein (GFP) in electroporated Leishmania donovani field isolate.
Cells were viewed after washing and fixing in 3.8 per cent formaldehyde in phosphate buffer saline (PBS).
Fig. 2 (B). Confocal image of intracellular amastigotes expressing GFP from episomal vector.
SINGH: DRUG RESISTANCE IN L. DONOVANI 415