trans

DRUG RESISTANCE IN KINETOPLASTID PARASITES 415
more than a single event. It is generally agreed
that SbV is not toxic to Leishmania but that it
must be reduced to the trivalent form SbIII. The
site of reduction is unclear; some data are highly
suggestive of the host macrophages while
other data have highlighted a stage-specific
amastigote intracellular SbV-reducing activity.
If reduction of SbV to SbIII is rate-limiting,
and provided that the enzyme does not have
another essential role, inactivation of this
enzyme would lead to SbV resistance. Interestingly,
axenic L. donovani amastigotes selected
for in vitro SbV resistance have lower SbV
reductase activity than wild-type cells. The isolation
of the putative reductase should help in
determining whether reduction occurs predominantly
within the parasite or the host,
and measuring its exact role in drug resistance.
Work on metal resistance induced in
Leishmania promastigotes has led to a coherent
model of SbV resistance (Figure 16.6).
Trypanothione (TSH), N 1 , N 8 -bis-glutathionyl
spermidine, the major reduced thiol of Trypanosomatidae,
is increased in several Leishmania
promastigote species selected for metal
resistance. This increase in TSH is due to the
amplification or overexpression of genes coding
for -glutamylcysteine synthase (-GCS) or
ornithine decarboxylase (ODC), the respective
rate limiting steps in glutathione and spermidine
biosynthesis (Figure 16.6). Reducing
the level of TSH by using the glutathione of
spermidine biosynthesis inhibitors buthionine
sulfoximine (BSO) and difluoromethylornithine
(DFMO) was shown to revert resistance.
Although high levels of TSH appear essential for
metal resistance in Leishmania promastigotes,
they are not sufficient, and the activity of
membrane transporters that recognize SbIII
conjugated to TSH is also modulated in drugresistant
mutants (Figure 16.6). One transporter
is the intracellular ABC transporter PGPA that
possibly confers resistance by sequestration of
the metal conjugate inside vesicular elements;
another transporter is an ATP-thiol-X efflux
system located in the plasma membrane, that
confers resistance by extruding the drug outside
the cell (Figure 16.6). PGPA is part of a
large gene family, and it is possible that one
of these members corresponds to this plasma
membrane efflux pump. In mammalian cells,
resistance mediated by the MRP pump can
depend on a high level of glutathione, high
activity of the pump, and increased glutathione
S-transferase activity. By analogy to the MRP
pump, it is possible that a trypanothione S-
transferase is required for achieving high-level
metal resistance in Leishmania.
Additional work has allowed the isolation of
novel antimony resistance genes that do not
appear to be part of the resistance pathway
shown in Figure 16.6. For example, a gene coding
for a protein of 770 amino acids was found
to be amplified in Pentostam-selected Leishmania,
while overexpression of a leucine-rich
repeat protein was shown to contribute to
SbIII resistance in both the promastigote and
amastigote stage of the parasite. Some antimony-resistant
cells were also shown to have
increased expression of the heat-shock protein
HSP 70. In the coming years it should be
possible to test whether any of the resistance
mechanisms described above plays a role in
clinical resistance to SbV in Leishmania.
Pentamidine
Pentamidine is a second-line drug against various
Leishmania infections and was used successfully
to treat infections refractory to SbV.
With time, however, even by using higher drug
concentrations, the efficacy of pentamidine
rapidly declined in India, suggesting that
parasites were becoming resistant. The cellular
target of pentamidine is unknown, although
it causes competitive inhibition of polyamine
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