Table of contents 1. Figure S1: ClustalW alignment of glutathione

Table of contents 1. Figure S1: ClustalW alignment of glutathione reductase of P. falciparum and P. berghei. 2. Figure S2: Generation of parasites lacking expression of GR (Δgr3 parasites) without drugselectable marker in its genome. 3. Figure S3: Reduction of GSH and GSSG in the presence of different DTE concentrations.

Figure S1: ClustalW alignment of glutathione reductase of P. falciparum and P. berghei. Dotted box indicates the 3 amino acids deletion in the GR of P. berghei. The 34 amino acids insertion of the Plasmodium GR is indicated by a box.

A (i) construct pL1377 3‘gr

eef1aa 5‘gr 3‘dhfr

hdhfr

yfcu

amp

3‘dhfr

(ii) wild type gr

(iii) Δgr integration after positive selection 4.7kb S

5‘gr

K 3‘gr

eef1aa

3‘dhfr

hdhfr

yfcu

3‘dhfr

(iv) Δgr3 integration after negative selection 2.1kb S

K 5‘gr

3‘gr Δgr3 after neg. selec.

Δgr3 before neg selec. Chr. 10 -

wt

B

3‘dhfr

cl1 cl2

kb ~10 ~7 -

1 2 3

7-

3-

2.1 -

75 50 -

58 -

Δgr 3

Δgr 2

M

Δgr 1

kDa

Wt 1

C

Wt 2

Probe: 3’dhfr

Probe: 3’dhfr

An#‐PfGR  An#‐PbB5 

Figure S2: Generation of parasites lacking expression of GR (∆gr3 parasites) without drug-selectable marker in its genome. A. Schematic representation of the disruption vector pL1377 (i), the wt gr locus (ii), the genomic locus of parasites after integration of pL1377 and positive selection, (iii) and (iv) the genomic locus of parasites after integration of pL1377 and negative selection. Negative selection with the drug 5-FC results in selection of parasites with the positive/negative selectable marker cassette (hdfr-yfcu) excised from the integrated construct by a recombination event between the two 3′dhfr sequences. Restriction sites (S, ScaI and K, KpnI) and size of restriction fragments used for diagnostic Southerns (see B) and the hdhfr-yfcu selectable marker are indicated. The pL1377 construct deletes part of exon 3 similar to disruption vector pL1282 as shown in Figure 1A. B. Southern analysis of separated chromosomes (left panel) and ScaI/KpnI digested genomic DNA (right panel) confirming correct disruption of gr and correct removal of the drug-selectable cassette in ∆gr 3 parasites. Hybridization of ∆gr chromosomes with the 3’-dhfr probe recognizes the construct integrated into gr on chromosome 10, the endogenous dhft-ts gene on chromosome 7 and the integrated GFP construct on chromosome 3. Hybridization of digested DNA recognizes the expected DNA fragment (2.1kb) indicated in A after removal of the drug-selectable marker and 2 fragments of 7 and 10 kb from the endogenous dhft-ts gene on chromosome 7 and the integrated GFP construct on chromosome 3 respectively. C. Western analysis of expression of GR. Protein extracts of blood stages were probed with a rabbit polyclonal antiserum against P. falciparum GR. In wt the GR protein of 56 KDa is detected which is absent in the ∆gr 3 parasites (see also Fig. 1). Antibody PbB5 is used as a loading control.

Fluorescence /min

Fluorescence /min

A. 

GSH 36µM

DTE (mM)

DTE (mM)

B. 

GSSG 4µM

GSH (µM)

Fluorescence /min

C. 

DTE (mM)

DTE (mM)

Figure S3. Reduction of GSH and GSSG in the presence of different DTE concentrations. A) Samples containing 36 µM GSH and 4 µM GSSG were treated with a range of DTE concentrations (0 25 mM). Based on these results, a DTE concentration of 6.25 mM was chosen for all glutathione measurements. B) The same range of DTE concentrations (0-25 mM) was used to treat samples of P. berghei wild type (ANKA) extracts. C) The GSH concentration was determined for the same parasite extract treated with two different concentrations of DTE (1.6 mM and 6.25 mM).