Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Parasitology Section
In the human malaria parasite Plasmodium falciparum the
glutathione level is well balanced and possibly involved
in chloroquine resistance
Zusammenfassung
Der mit Plasmodium falciparum-infizierte Erythrozyt
ist einer hohen oxidativen Belastung ausgesetzt, verursacht
durch endogene metabolische Prozesse wie
Hämoglobinabbau im Parasiten, aber auch hervorgerufen
durch reaktive Sauerstoffspezies der humanen
Immunantwort. Parasit und Wirtszelle verfügen
über komplexe antioxidative Systeme, wobei dem
Glutathion eine entscheidende Rolle zukommt.
Glutathiongehalt und GSH/GSSG-Gleichgewicht
werden durch GSSG-Reduktion und Efflux sowie
durch die γ-Glutamylcystein-Synthetase, ein stress-induzierbares
Schrittmacherenzym im GSH-Syntheseweg,
kontrolliert. Plasmodienstämme können diese
Regulationsmechanismen in unterschiedlichem
Maße nutzen, wie am Beispiel zweier P. falciparum-
Stämme untersucht wurde, von denen Pf3D7 chloroquin-sensitiv
und PfDd2 chloroquin-resistent ist.
Summary
In the malaria parasite glutathione is not only involved
in maintaining an adequate intracellular redox environment
and protects the cell against oxidative stress, but
it also was shown that it degrades non-polymerised ferriprotoporphyrin
IX and is thus implicated in the development
of chloroquine (CQ) resistance. Glutathione levels
in Plasmodium falciparum infected erythrocytes are
regulated by GSH synthesis, GSSG reduction and efflux,
respectively. Here we studied the effects of drugs
that interfere with these processes to establish possible
differences in the regulation of the glutathione metabolism
of the respective CQ sensitive and resistant strains
CQS 3D7 and CQR Dd2. The results clearly indicate
that maintenance of intracellular glutathione in the CQ
resistant strain is mainly dependent on glutathione synthesis
via a stress-inducible γ-glutamylcysteine synthetase
(γ-GCS), whereas in the sensitive strain it is regulated
via glutathione reductase. Generally, CQ resistant
P. falciparum appear to be able to sustain their intracellular
glutathione more efficiently. In agreement
with these findings is the differential susceptibility to
oxidative stress of both parasite strains.
Introduction
The spread of drug resistance to CQ and other drugs
available against malaria poses a serious health problem
and emphasises the need for the elucidation of the
mechanisms of drug resistance and the discovery of
modulators potentially reverting drug resistance.
CQ prevents the polymerisation of toxic FP IX that is released
as a result of haemoglobin digestion by the parasite,
and if not polymerised, leads to damage of the
32
cell’s membranes and to cell death. Ginsburg and colleagues
have previously reported that glutathione degrades
free FP IX and that this degradation process is inhibited
by CQ (Ginsburg et al. 1998, Biochem Pharmacol
56: 1305-1313; Famin et al. 1999, Biochem Pharmacol
58: 59-68). Further, it has been reported that the
glutathione levels in CQR and CQS strains of P. berghei
correlate with the hypothesis that CQ prevents glutathione
mediated FP IX degradation and that drugs that
alter the intracellular glutathione concentrations such
as D, L-buthionine-(S,R)-sulphoximine (BSO) change
the susceptibility of P. falciparum to CQ after administration
(Dubois et al. 1995, Exp Parasitol 81: 117-124).
Project Description and Results
The parasite’s intracellular glutathione concentration is
regulated via glutathione synthesis, GSSG reduction
and efflux. Measuring the GSSG efflux and using BSO
as a tool to block glutathione synthesis and methylene
blue (MB) to inhibit glutathione reductase (GR), we
aimed in identifying differences in the regulatory mechanisms
in erythrocytes infected with CQS 3D7 and the
Figure 1: Growth inhibition curves of CQR PfDd2- and CQS
Pf3D7-infected RBC incubated with (A) BSO and (B) MB. The
drug screening was performed for 48 h. IC50 values result from
4 to 5 independent determinations.