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Originally published In Press as doi:10.1074/jbc.M610251200 on December 28, 2006

J. Biol. Chem., Vol. 282, Issue 9, 6388-6397, March 2, 2007
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15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum*

T. N. C. Ramya{ddagger}1, Krishanpal Karmodiya§, Avadhesha Surolia, Recipient of a grant from the Center of Excellence, DBT{ddagger}2, and Namita Surolia§3

From the {ddagger}Indian Institute of Science, Bangalore 560012, India, the §Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India, and the National Institute of Immunology, New Delhi 110067, India

Received for publication, November 2, 2006 , and in revised form, December 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2{alpha}. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
15-Deoxyspergualin (DSG)4 (Fig. 1), an analog of spergualin isolated from the culture broth of Bacillus laterosporus, inhibits the growth of the malaria parasite, Plasmodium, in vitro and in vivo, presumably by depleting its polyamine reserves (1, 2). However, other known inhibitors of polyamine biosynthesis, methylglyoxal-bis-(guanylhydrazone) analog (MGBCP), irinotecan hydrochloride (CPT11), and {alpha}-difluoromethyl ornithine (DFMO) are known to be incapable of suppressing malaria in infected mice (2). This led us to question the speculated mechanism of antimalarial action of DSG.

Despite the uncertainty in the actual mechanism of action of DSG, DSG is known to bind cellular chaperones, Hsp70 and Hsp90, potently, via their regulatory C-terminal motif EEVD (3, 4). DSG does not inhibit their chaperone activity (3–5). However, it selectively enhances the ATPase activity of heat shock proteins that have the C-terminal EEVD motif, and this could be the basis of a specific modulation of the function of proteins that bind the EEVD motif of heat shock proteins (5).

One of the cellular milieus in which heat shock proteins are speculated to be involved is translational regulation. Translation of an mRNA requires the formation of a ternary complex of the methionyl-tRNA, the mRNA and the 40 S subunit of the ribosome, a feat achieved through the intervention of the GTP-bound form of the initiation factor eIF2 (6). Phosphorylation of eIF2{alpha} at Ser51 blocks protein synthesis by preventing eIF2 recycling and depleting the cell of its pool of eIF2-GTP required for translation initiation (7). Several conditions of cellular stress trigger the phosphorylation of eIF2{alpha} via the activation of four different eIF2{alpha} kinases: PERK (PKR-like endoplasmic reticulum-associated kinase), GCN-2 (general control non-derepressing kinase 2), PKR (double-stranded RNA-activated protein kinase), and HRI (heme-regulated inhibitor or heme-regulated eukaryotic initiation factor 2{alpha} kinase) (8). The heme-regulated eIF2{alpha} kinase, HRI, is normally maintained in an inactive, unphosphorylated form by association with Hsp70 (9), and we recently demonstrated that sequestration of heat shock proteins by DSG in mammalian cells leads to protein synthesis inhibition and cell death through autophosphorylation of HRI and subsequent phosphorylation of eIF2{alpha} (10).

A heme-regulated eIF2{alpha} kinase akin to the mammalian HRI is known to exist in Plasmodium falciparum, and Surolia and Padmanaban have earlier demonstrated that the autophosphorylation of this eIF2{alpha} kinase and the subsequent phosphorylation of eIF2{alpha} in P. falciparum can bring about protein synthesis inhibition and parasite death (11, 12). We therefore decided to elucidate the cascade of events involved in protein synthesis initiation with respect to eIF2{alpha} in P. falciparum on addition of DSG. However, DSG did not bring about either protein synthesis inhibition or polyamine depletion at the submicromolar concentrations at which it was effective as an antimalarial agent. Our investigations directed us to another, and in retrospect, a far more exciting process, viz the disruption of the trafficking of nuclear-encoded apicoplast-targeted (NEAT) proteins to the apicoplast by this molecule, which we describe here.


Figure 1
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  		FIGURE 1.
Structural formula of DSG.

 
Plasmodium and other members of the protozoan group Apicomplexa are characterized by the presence of a four-membrane relict plastid, the apicoplast acquired by secondary endosymbiosis (13–15). Though the 35-kb apicoplast genome encodes almost exclusively only for genes involved in its translation, bioinformatic methods and experiments using fluorescent fusion proteins have demonstrated that the meager apicoplast proteome is supplemented with ~550 nucleus-encoded proteins that are targeted to the apicoplast (16–18). These NEAT proteins include enzymes involved in fatty acid synthesis, heme synthesis, and isoprenoid synthesis (19); metabolic processes which are apicoplast-resident (19–22) and which, because of their prokaryotic nature, are incidentally our best bet while designing antimalarial agents. The N termini of NEAT proteins have an N-terminal bipartite plastid targeting sequence (PTS) comprising a classic signal peptide with a hydrophobic domain that directs cotranslational insertion into the endomembrane system (including ER), and a 24–100-amino acid long transit peptide harboring Hsp70 binding sites and highly enriched in lysine and asparagine residues that sort proteins for apicoplast targeting within the secretory system (17, 23).

In situ experiments in the malaria parasite using PTS-GFP fusion proteins are compliant with a protein trafficking model, wherein all proteins entering the endomembrane system are cleaved of their signal peptide and bundled into endomembrane-derived vesicles, which subsequently dock at the apicoplast (17). Only proteins bearing transit peptides, which are maintained in an unfolded conformation by bound Hsp70 molecules (17, 24), are expected to be diverted into the apicoplast, probably through interaction with a duplicated set of Toc proteins in the second outermost membrane. The remaining proteins are thought to be packaged into vesicles that bud from the outer membrane of the apicoplast for transport to further destinations in the secretory pathway, culminating in secretion into the parasitophorous vacuole. Toc and Tic complexes are believed to mediate protein import across the two innermost membranes of the apicoplast in an ATP-dependent fashion (25). Following protein import, the transit peptide is cleaved by a stromal processing peptidase homologue in the apicoplast (26), so that following its targeting to the apicoplast, the processed NEAT protein is of lower molecular mass than the preprocessed form.

The apicoplast is essential to parasite survival (27), and its elimination by pharmacological or molecular genetic manipulation has been demonstrated to evoke distinctive delayed death kinetics, an intriguing biological phenomenon characterized by normal parasite growth in the host cell immediately following apicoplast perturbation, but parasite death subsequent to the invasion of a new host cell (28, 29). This phenomenon has been rationalized as the consequence of the generation of daughter cells devoid of apicoplast following missegregation of the apicoplast because of inhibition of an apicoplast function (28). Various groups have, in the last decade, assiduously investigated several apicoplast processes and their inhibition by specific molecules. However, the process of trafficking of NEAT proteins to the apicoplast has been hitherto unexplored as a drug target in the malaria parasite, and details of the trafficking process are yet to be worked out completely. Our somewhat serendipitous finding that a widely used small molecule, 15-deoxyspergualin, kills the malaria parasite by disrupting targeting of NEAT proteins to the apicoplast, provides new opportunities to study this interesting biological process as well as opens up possibilities of developing inhibitors of this process as antimalarial agents.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—15-Deoxyspergualin was a kind gift from Nippon Kayaku Co., Tokyo, Japan. Anti-actin antibody was from Sigma; anti-Hsp70 monoclonal antibody and polyclonal antibodies to eIF2{alpha} and Ser-phosphorylated eIF2{alpha} were from Cell Signaling. We raised antibodies to purified recombinant proteins PfTufA and PfFabG. The radioisotopes, [35S]methionine and [35S]cysteine labeling mix, [32P]ATP, [3H]hypoxanthine, [3H]spermidine, and [14C]acetate were from PerkinElmer Life Sciences. 1,4-[14C]Putrescine and [3H]putrescine were from Amersham Biosciences. The peptide EEVD was custom-synthesized from Genscript. Triclosan 5000 was obtained from Kumar Organic Products Ltd., Bangalore, India. MitoTracker Red CM-H2Xros as well as Alexa fluor 488 and Alexa fluor 568 were purchased from Molecular Probes and DAPI from Sigma.

Intraerythrocytic P. falciparum Cultures—The FCK2 parasite strain was cultured in O+ human red blood cells in RPMI medium supplemented with human serum by the candle jar method of Trager and Jenson (30). Cultures were synchronized by 5% sorbitol treatment (31), and parasites were observed for viability and changes in morphology by standard Giemsa staining.

Cloning, Expression, and Purification of Heat Shock Proteins—Parasite heat shock proteins PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif: PlasmodB PF08_0054) and PfHsp70-2 (an Hsp70 protein without the EEVD motif: PlasmodB PF11_0351) were cloned, overexpressed, and the recombinant proteins characterized as previously (5).

In Vitro Phosphorylation of Proteins in Parasite Lysate—Parasites were released from the red blood cells by 0.15% saponin. Free parasites were lysed with four volumes of ice-cold lysis buffer (100 mM KCl/7 mM Mg(OAc)2/380 mM sucrose/6.5 mM 2-mercaptoethanol/50 mM Tris-HCl, pH 7.4/0.14% Triton X-100/15 µM leupeptin). The suspension was then subjected to centrifugation at 15,000 x g for 30 min. The resulting supernatant was used for the phosphorylation reaction. The phosphorylation status of proteins was examined by following a previously reported protocol (10). Briefly, the reaction was carried out in 25 µl containing 20 mM HEPES pH 7.5, [{gamma}-32P]ATP (25 µCi, 3000 Ci/mmol; Amersham Biosciences), 0.1 mM GTP, 5 mM creatine phosphate, 45 units of creatine phosphokinase per ml, 0.5 mM glucose 6-phosphate, 7 mM Mg(0Ac)2, 110 mM KAc, 0.375 mM spermidine, 2.5 mM dithiothreitol, 2 µg wheat germ tRNA, and 40 µM each of the 20 amino acids. The reaction mixture was incubated at 30 °C for 5 min before [{gamma}-32P]ATP addition and was terminated after 2–3 min by the addition of freshly prepared reducing buffer with SDS. The samples were analyzed on polyacrylamide gels and subjected to Western analysis with specific antibodies and autoradiography. To examine the effect of heat shock proteins on phosphorylation, purified and concentrated recombinant heat shock protein (50 µl of a 50 µM solution) was incubated with or without DSG (added to a final concentration of 50 µM) on ice, the solution was subjected to centrifugation through a protein-concentrating device (MWCO 10 kDa Centricon), the filtrate was lyophilized to a convenient small volume (2.5 µl), and added to the in vitro phosphorylation assay (25 µl), so that the effective concentration of DSG or heat shock proteins would be 100 µM. With heat shock proteins incubated in the presence of DSG, the concentration of DSG in the phosphorylation reaction would be lowered because of its sequestration by the heat shock proteins and hence its inability to pass through the protein concentrating device. The peptide EEVD, like DSG, was added to a final concentration of 100 µM in the phosphorylation reaction.

Metabolic Labeling of Parasite Proteins in Situ—Synchronized parasites (~10% parasitemia) pretreated with varying concentrations of DSG for 3 h were incubated with [35S]methionine (specific activity >1000 Ci/mmol, added to a final concentration of 180 µCi/ml of culture) for 3 h. The parasites were then released by saponin lysis, lysed in hypotonic buffer, and subjected to analysis by SDS-PAGE and autoradiography or spotted on to Whatman 3 filter discs presoaked in 10% trichloroacetic acid, washed with hot and then cold trichloroacetic acid, and the radioactivity measured in a liquid scintillation counter (11).

Polyamine Biosynthesis in the Malaria Parasite—Polyamine synthesis in the malaria parasite was monitored by the autoradiography of TLC resolved dansylated polyamines following the metabolic labeling of the parasite polyamines using [14C]putrescine (107 mCi/mmol) (see Ref. 32). Briefly, P. falciparum-infected erythrocytes (~10 ml) synchronized with 5% sorbitol were incubated with [14C]putrescine (50 µCi; 100 mCi/mmol) to a final concentration of 0.2 mM for 6 h. They were then washed and resuspended in RPMI without [14C]putrescine for 18 h. The parasites were then isolated from the red blood cells by saponin lysis, and the polyamines in the parasites dansylated by the procedure that follows. The cell pellet was resuspended in 5% trichloroacetic acid to precipitate proteins. The pH of the supernatant was brought to ~9 with saturated sodium carbonate, and then an equal volume of 10 mg/ml dansyl chloride in acetone was added to it. The reaction mix was vortexed and allowed to incubate in the dark at 60 °C for 60 min. The reaction was stopped with 100 mg/ml of L-alanine, the acetone was evaporated, the reaction mix cooled to 4 °C, and dansylated polyamines extracted with an equal volume of toluene. The upper toluene phase was transferred to a fresh tube, concentrated to ~50 µl, and spotted on a Whatman silica gel 60 TLC plate along with standard dansylated polyamines (also prepared in a similar fashion). The dansylated polyamines were developed with hexane/ethyl acetate:: 6:4 (see Ref. 33) and visualized under UV illumination. The identification of the polyamines was also confirmed by comigration of the dansylated polyamines with the dansylated standards in a different solvent system (chloroform:triethylamine::5:1) previously reported by Bhatnagar et al. (34). Similar separation was obtained with this system also (data not shown).

Effect of DSG on Hypusination of PfeIF5A—Polyamines in P. falciparum-infected red blood cells were labeled with [14C]putrescine as mentioned earlier, the proteins precipitated with 5% trichloroacetic acid, and analyzed by SDS-PAGE and autoradiography. eIF5A, the only known protein with a post-translational modification derived from a polyamine, was the only radiolabeled band on the autoradiogram and was identified by its molecular size.

Polyamine Transport in Red Blood Cells—Putrescine/spermidine transport into uninfected or infected red blood cells was assayed by a previously reported protocol (32). Briefly, 900 µl of uninfected or P. falciparum-infected red blood cells (3 x 107 cells) were incubated in phosphate-buffered saline with 100 µl of 25 µCi/ml [3H]putrescine (26 Ci/mmol), 14C-putrescine (107 mCi/mmol), or [3H]spermidine (22.5 Ci/mmol) at 37 °C. Transport was terminated at 0, 5, 10, 15, 30, and 60 min time points by rapidly separating the parasites from the other assay components of a 150-µl aliquot by centrifugation through 800 µl of dibutyl phthalate (specific gravity 1.04). The upper aqueous layer was discarded, the oil layer washed twice with cold phosphate-buffered saline and removed, and the red blood cell pellet lysed with water. The proteins in the lysate were precipitated with 10% trichloroacetic acid and the supernatant blotted on to Whatman 3 filters. The filters were dried and placed in scintillation fluid, and the radioactivity counted in a liquid scintillation counter (Hewlett-Packard).

Inhibition of Parasite Growth by DSG—[3H]Hypoxanthine uptake by parasites was used to assess their sensitivity to DSG (see Ref. 35). Briefly, synchronized parasites were cultured in 96-well plates (Nunc, Copenhagen, Denmark) at 2–3% hematocrit and at an initial parasitemia of 1–2%, with varying concentrations of DSG, and addition of DSG in fresh medium every 24 h for 48 h. To follow death kinetics, parasites synchronized at the ring stage were either cultured in the presence of [3H]hypoxanthine and varying concentrations of DSG for 48 h and harvested, or cultured in the presence of varying concentrations of DSG for the first 48 h and then incubated with [3H]hypoxanthine for the next 48 h and harvested. Parasites were harvested with a Nunc cell harvester (Nunc) and the radioactivity counted in a liquid scintillation counter. Parasites were also observed microscopically in Giemsa-stained blood smears.

Quantitative Competitive PCR to Quantify Apicoplast: Nuclear DNA Ratio in DSG-treated Parasites—Using quantitative competitive PCR (36), we quantified the nuclear gene fabI (nuclear chromosomal gene coding for the protein enoyl-PfACP reductase) (37) and the plastid gene eftu (apicoplast gene coding for the elongation factor tu) (38).

Incorporation of [14C]Acetate into Fatty Acids of Parasites— Red blood cells infected with the malaria parasite (in the asexual stage of interest, ~10% parasitemia), 25 ml, treated with triclosan (10 µM) or 15-deoxyspergualin (1 µM) were resuspended in 5 ml of complete medium while retaining the same concentration of the inhibitor. 1,2[14C]Acetate (50 µCi/ml sodium acetate; 60 mCi/mmol) was added, and the incorporation of [14C]acetate into fatty acids measured (19).


Figure 2
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  		FIGURE 2.
Effect of DSG on protein synthesis in P. falciparum in situ. A, inhibition of overall protein synthesis in parasites by cycloheximide, chloroquine, and DSG as assessed by [35S]methionine incorporation. Synchronized parasites (5% parasitemia, early trophozoites stage) pretreated with various concentration of DSG for 3 h were incubated with [35S]methionine (specific activity >1000 Ci/mmol, added to a final concentration of 180 µCi/ml of culture) for 3 h. The parasites were then released by saponin lysis, lysed in hypotonic buffer and subjected to analysis by SDS-PAGE and autoradiography. B, status of phosphorylation of eIF2{alpha} in the parasite lysate. Phosphorylation of proteins in the parasite lysate was done using [{gamma}-32P]ATP in the presence of added PfHsp70-2, PfHsp70-1, PfHsp70-1-EEVD deletion mutant, peptide EEVD and DSG. The effective concentration of the heat shock proteins, EEVD and DSG was 100 µM. The proteins in the lysate were analyzed by SDS-PAGE and Western blotting followed by autoradiography. The upper panel is the autoradiogram, and the lower panel is the Western blot probed with anti-eIF2{alpha} antibody. C, IC50 of DSG for inhibition of parasite protein synthesis as determined by [35S]methionine incorporation into proteins in situ was found to be 443 µM whereas (inset in E) DSG inhibited parasite growth with an IC50 of 148 nM, as estimated by [3H]hypoxanthine uptake.

 
Determination of Levels of Apicoplast Proteins in DSG-treated Parasites—Synchronized infected red blood cells, untreated or pretreated with various concentrations of DSG for 3 h were labeled in situ with [35S]methionine (180 µCi/ml culture) for 3 h. Saponin-released parasites were lysed in hypotonic buffer (1 mM Tris, pH 7.4). Levels of actin, PfTufA, and PfFabG were examined by Western analysis and autoradiography.


Figure 3
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  		FIGURE 3.
Effect of DSG on polyamine biosynthesis and transport. A, effect of DSG on polyamine biosynthesis in the malaria parasite. P. falciparum-infected erythrocytes (~10 ml) synchronized with 5% sorbitol were incubated with [14C]putrescine (50 µCi; 100 mCi/mmol) to a final concentration of 0.2 mM for 6 h. They were then washed and resuspended in RPMI without [14C]putrescine for 18 h. The parasites were then isolated from the red blood cells by saponin lysis, and the polyamines in the parasites dansylated and resolved by thin layer chromatography as described under "Experimental Procedures." Lanes 1–4 of the TLC are dansylated polyamines from parasites treated with varying concentrations of DSG, and lanes 5–7 are dansylated polyamine standards. B, P. falciparum-infected erythrocytes were cultured in the presence or absence of exogenously added spermidine to a final concentration of 500 µM (established prior to the experiment to be the highest concentration of spermidine not detrimental to parasite growth). The effect of DSG on parasite growth was not reversed by exogenous addition of spermidine to the infected red blood cells. C, effect of DSG on hypusination of PfeIF5A. Polyamines in P. falciparum-infected red blood cells were labeled with [14C]putrescine as mentioned earlier, the proteins precipitated with 5% trichloroacetic acid and analyzed by SDS-PAGE and autoradiography. eIF5A, the only known protein with a post-translational modification derived from a polyamine, was the only radiolabeled band on the autoradiogram and was identified by its molecular size. D, IC50 of DSG for putrescine transport in uninfected and infected RBCs. Putrescine transport was monitored using [3H]putrescine (26 Ci/mmol) or [14C]putrescine (107 mCi/mmol). For each concentration of DSG, putrescine transport was monitored at 0, 5, 10, 15, 30, and 60 min time points by rapidly separating the parasites from the other assay components by centrifugation through dibutyl phthalate. The proteins were precipitated with 5% trichloroacetic acid, and the radioactivity in the supernatant measured using a liquid scintillation counter. The rate of transport calculated was plotted versus the DSG concentration. E, IC50 of DSG for spermidine transport in uninfected and infected red blood cells. Spermidine transport was monitored using [3H]spermidine (22.5 Ci/mmol). For each concentration of DSG, transport was monitored at 0, 5, 10, 15, 30, and 60 min time points by rapidly separating the parasites from the other assay components by centrifugation through dibutyl phthalate. The proteins were precipitated with 5% trichloroacetic acid and the radioactivity in the supernatant measured using a liquid scintillation counter. The rate of transport calculated was plotted versus the DSG concentration.

 
Pulse-Chase and Immunoprecipitation of Native PfFabG— Synchronized ring stage parasite cultures (18 ml, ~10% parasitemia) were untreated or treated with 500 nM clindamycin or 1 µM DSG for 24, 48, or 72 h. The cultures were then washed with methionine-free RPMI twice, each suspended in 3-ml volume, and labeled in situ with [35S]methionine/[35S]cysteine mix (300 µCi/ml of culture) for 1 h, after which the label was removed by extensive washes with complete RPMI. Parasites (from 1 ml of resuspended culture each) were harvested at 0, 2, and 4 h, and released from the erythrocytes by saponin lysis. The lysates were subjected to immunoprecipitation with anti-FabG or anti-TufA antibodies. Briefly, the parasites were lysed in i.e. cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 15 µM leupeptin) on ice for 30 min. Following centrifugation at 15,000 x g, the supernatant was precleared with 50 µl of protein A-Sepharose for 2 h at 4 °C. The precleared lysate was then incubated with 5 µg of anti-FabG or anti-TufA antibody and protein A-Sepharose for 2 h at 4 °C. The protein A-Sepharose beads were then washed twice with buffer containing 50 mM Tris, pH 7.4, 500 mM NaCl, and 0.1% Triton X-100, once with buffer containing 3 mM bovine serum albumin, 50 mM Tris, pH 7.4, and 200 mM NaCl and once with buffer containing 50 mM Tris, pH 7.4 and 150 mM NaCl. The immunoprecipitates were then eluted by resuspending the beads in nonreducing SDS lysis buffer, and analyzed by SDS-PAGE and autoradiography.


Figure 4
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  		FIGURE 4.
DSG and delayed death. A, synchronized parasites (5% parasitemia) were cultured in 96-well plates at 2–3% hematocrit and at an initial parasitemia of 1–2%, with 1 µM DSG, and addition of DSG in fresh medium every 24 h for 48 h. Parasite growth inhibition was assessed by microscopy of Giemsa-stained smears at 48 and 96 h. Parasites were killed by DSG only at 96 h. B, death kinetics invoked by DSG as examined by hypoxanthine uptake. Parasites synchronized at the ring stage were either cultured in the presence of [3H]hypoxanthine and varying concentrations of DSG for 48 h and harvested, or cultured in the presence of varying concentrations of DSG for the first 48 h and then incubated with [3H]hypoxanthine for the next 48 h and harvested. C, apicoplast copy numbers of parasites treated with 100 nM clindamycin (CLN), 100 nM chloroquine (CQ), and 1 µM DSG were determined by competitive PCR using PffabI and Pfeftu probes (37).

 
Immunofluorescence—Synchronized parasite cultures were untreated or treated with 500 nM clindamycin or 1 µM DSG for 24, 48, or 72 h. They were then processed for immunofluorescence of native PfFabG and PfTufA following the protocol reported by Tonkin et al. (39). Immunofluorescence was carried out with rabbit anti-PfFabG antibody conjugated with Alexa fluor 488 and rabbit anti-PfTufA antibody conjugated with Alexa fluor 568. Nuclei were stained with DAPI. The various asexual stages were identified as follows. A parasite with a single nucleus and no hemozoin was considered a ring (0–24 h of the asexual cycle), a parasite with a nucleus and hemozoin was considered a trophozoite (24–36 h of the asexual cycle), and a parasites with multiple nuclei and hemozoin was considered a schizont (36–48 h of the asexual cycle) Images were captured using confocal microscopy and overlay images obtained using Adobe Photoshop 6.0.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of DSG on Parasite Protein Synthesis—DSG abrogated protein synthesis in malaria parasites with an IC50 of 443 µM, as assessed by in situ incorporation of [35S]methionine into parasite proteins (Fig. 2A). DSG, like chloroquine and heme deficiency (11), inhibited in vitro protein synthesis in the parasite lysate through the autophosphorylation of eIF2{alpha} kinase (data not shown) and phosphorylation of eIF2{alpha} (Fig. 2B). This DSG-induced eIF2{alpha} phosphorylation could be averted by addition of tetrapeptide EEVD or purified PfHsp70-1 protein containing the C-terminal EEVD motif (annotated in PlasmodB as PF08_0054) (Fig. 2B). Incubation with PfHsp70-2 (annotated in PlasmodB as PF11_0351) or the C-terminal deletion mutant, PfHsp70-1-EEVD, both of which lack the C-terminal EEVD motif, could not reverse DSG-induced phosphorylation of eIF2{alpha} (Fig. 2B). This suggested that in the malaria parasite, like in the mammalian system (10), the interaction of DSG with the EEVD motif of Hsp70 is responsible for the autophosphorylation of eIF2{alpha} kinase and subsequent protein synthesis inhibition. However, DSG inhibited the growth of P. falciparum cultures in erythrocytes with an IC50 of 148 nM as estimated by [3H]hypoxanthine uptake, an IC50 ~1000-fold lower than that required for the inhibition of parasite protein synthesis (Fig. 2C), suggesting that yet another target in the parasite is responsible for the potent inhibitory activity of DSG.


Figure 5
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  		FIGURE 5.
Effect of DSG on apicoplast functions. A–C, effect of DSG on apicoplast protein synthesis was determined by probing blots of parasite lysate with antibodies to a cytosolic protein (actin) (A), an apicoplast protein (PfTufA) (B), and a NEAT protein (C)(PfFabG - the upper and lower panels represent the preprocessed and processed (following targeting to the apicoplast) forms of the protein). D, pulse-chase experiment demonstrating the post-translational processing of a NEAT protein PfFabG in untreated (control) parasites (lanes 1–3); clindamycin-treated parasites, first asexual cycle (lanes 4–6); DSG-treated parasites, first asexual cycle (lanes 7–9); DSG-treated parasites, second asexual cycle (lanes 10–12); clindamycin-treated parasites, second asexual cycle (lanes 13–15). PfFabG was immunoprecipitated following 0, 2, and 4 h of metabolic labeling of parasites with [35S]methionine. The upper and lower arrows point to the preprocessed and processed forms of the protein, respectively. E, immunoprecipitation of PfTufA following metabolic labeling of untreated parasites (lanes 1–3), DSG-treated parasites, first asexual cycle (lanes 4–6), clindamycin-treated parasites, first asexual cycle (lanes 7–9), DSG-treated parasites, second asexual cycle (lanes 10–12), and clindamycin-treated parasites, second asexual cycle (lanes 13–15). Synthesis of PfTufA was inhibited in clindamycin-treated parasites, but not in the DSG-treated parasites in the first asexual cycle. In the second asexual cycle, no PfTufA could be immunoprecipitated from either DSG or clindamycin-treated parasites, indicating the absence of apicoplast (and consequently PfTufA). The arrow points to PfTufA. No degradation of PfTufA was seen until 4 h after metabolic labeling.

 
Effect of DSG on Polyamine Biosynthesis and Transport— With protein synthesis no longer the primary pathway targeted by DSG in the malaria parasites, we looked into the previously implicated pathway, synthesis of polyamines (1, 2). Normal erythrocytes are devoid of polyamine biosynthetic machinery, and contain traces of putrescine, spermidine, and spermine. However, a functional polyamine biosynthesis pathway exists in P. falciparum-infected erythrocytes (32), and depleting the malaria parasite of polyamines could be deleterious to its growth. Putrescine conversion to spermidine and spermine was abrogated only at high micomolar concentrations of DSG in malaria parasite infected red blood cells (Fig. 3A). Moreover, DSG inhibition of parasite growth was not reversed by exogenous addition of spermidine (Fig. 3B), suggesting that DSG does not compromise the growth of the malaria parasite through inhibition of polyamine biosynthesis. DSG has earlier been demonstrated to inhibit formation of active eIF5A, which is generated by modification with hypusine, a derivative of spermidine, in mouse mammary carcinoma FM3A cells (40). However, the amount of hypusinated eIF5A in the malaria parasite was reduced only upon polyamine depletion at high micromolar concentrations of DSG (Fig. 3C). Hence, neither polyamine biosynthesis nor eIF5A formation was the primary target of DSG in P. falciparum.

Not only does P. falciparum possess a functional polyamine synthesis pathway, but infected red blood cells also salvage polyamines through new polyamine transporters (32). Because the inhibitory effect of DSG on parasite growth could not be reversed by the exogenous addition of polyamines (Fig. 3B), we wondered if DSG, by virtue of its structural similarity to polyamines, inhibits their transport. Using radiolabeled putrescine and spermidine, we studied the effect of DSG on the new putrescine and spermidine transporters in infected red blood cells (Fig. 3, D and E). However, DSG did not potently inhibit either putrescine or spermidine transport. The IC50 of DSG for inhibition of putrescine transport in uninfected and infected red blood cells were 924 ± 3.1 µM and 1118 ± 4.7 µM, respectively, and while 371.5 ± 4.13 µM DSG inhibited spermidine transport in uninfected RBCs, DSG did not inhibit spermidine transport in infected red blood cells up to the highest concentration tested (1 mM) (Fig. 3, D and E).

Effect of DSG on Apicoplast Processes—While assaying DSG for its antimalarial potential, we noticed that DSG induced delayed death; growth inhibition of the parasite was manifested only after 72 h, i.e. in the second asexual cycle or the cycle following new host cell invasion (each asexual cycle of the malaria parasite, P. falciparum, takes 48 h to complete) (Fig. 4, A and B). Moreover, using quantitative competitive PCR, we determined an apicoplast copy number of 0.13 in DSG-treated parasites as opposed to 1.0 in untreated (control) parasites, and 0.4 in parasites treated with clindamycin, an agent already known to invoke delayed death by inhibiting prokaryotic protein synthesis in the apicoplast (Fig. 4C). This demonstration of apicoplast loss confirmed delayed death kinetics in DSG-treated parasites.


Figure 6
Figure 6
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  		FIGURE 6.
Immunofluorescence of native PfFAbG and PfTufA in malaria parasites. Synchronized parasites untreated or treated with 500 nM clindamycin or 1 µM DSG for 24 h (to see the trophozoite stage of the first asexual cycle), 48 h (to see the schizont stage of the first asexual cycle), or 72 h (to see the trophozoite stage of the second asexual cycle), were processed for immunofluorescence of native PfFabG and PfTufA using rabbit anti-PfFabG antibody conjugated with Alexa fluor 488 and rabbit anti-PfTufA antibody conjugated with Alexa fluor 568. Nuclei were stained with DAPI. Arrows point to the parasitophorous vacuole (PV), parasite cytosol (PC), red blood cell (RBC), and a daughter merozoite in the schizont. The red scale bar is 5 micrometers. A, untreated parasite, trophozoite stage. Both PfFabG and PfTufA are localized to the apicoplast. B, untreated parasite, schizont stage. Both PfFabG and PfTufA are localized to the apicoplast, and every daughter merozoite has its apicoplast. C and D, DSG-treated parasite, first asexual cycle, trophozoite stage. PfTufA is localized to the apicoplast while PfFabG is localized to the PV or PC. E, DSG-treated parasite, first asexual cycle, schizont stage. Only one daughter has received an apicoplast as seen by PfTufA localization. F and G, DSG-treated parasite, second asexual cycle. There is no apicoplast as noted by the absence of PfTufA. PfFabG is localized to the PV or PC. H, clindamycin-treated parasite, first asexual cycle, trophozoite stage. There is no PfTufA because of the inhibition of prokaryotic protein synthesis in the apicoplast by clindamycin. PfFabG is localized to the apicoplast. I, clindamycin-treated parasite, first asexual cycle, schizont stage. Only one daughter merozoite has an apicoplast as noted by PfFabG localization. J and K, clindamycin-treated parasite, second asexual cycle. PfFabG is localized to the PV or PC following apicoplast loss. L, reversibility of DSG action was followed by incubation with DSG for the first 2, 4, 6, 12, 18, or 24 h of the first asexual cycle followed by its removal and the addition of cycloheximide (500 nM) to inhibit further protein synthesis. This is a representative plot of 24 h. PfFabG was seen in the parasitophorous vacuole, indicating that once mistargeted, FabG cannot be re-targeted to the apicoplast.

 
Because delayed death is typically associated with inhibition of an apicoplast function, we examined the effect of DSG on a typical apicoplast function, apicoplast protein synthesis. Prokaryotic protein synthesis in the parasite apicoplast was followed as a function of DSG concentration in the culture medium by probing the lysate of the harvested parasites with antibodies specific for an apicoplast-encoded protein (PfTufA), as well as a nucleus-encoded cytosolic protein, actin, and a nucleus-encoded apicoplast-targeted protein, PfFabG. Interestingly, while levels of PfTufA and actin remained unchanged, implying that apicoplast and cytosolic protein synthesis were not affected by DSG, there was a decrease in the level of the processed form of the NEAT protein, PfFabG (Fig. 5, A–C). Our observation that the level of the processed form of NEAT protein PfFabG is reduced in DSG-treated parasites suggested that trafficking or processing of NEAT proteins could be a process inhibited by DSG. To confirm this effect of DSG on NEAT proteins, we conducted pulse chase experiments. Using [35S]methionine, proteins were metabolically labeled in situ in parasites untreated or treated with DSG or clindamycin, followed by immunoprecipitation and autoradiography for analyses of PfFabG and PfTufA. In untreated parasites, the labeled unprocessed PfFabG was chased into a processed form within 4 h (Fig. 5D). However, unprocessed PfFabG failed to get chased into the processed form of PfFabG in both the first and second asexual cycle of malaria parasites treated with DSG (Fig. 5D). This was in contrast to clindamycin-treated parasites where labeled PfFabG was chased into the processed form in the first asexual cycle, but not in the second asexual cycle (Fig. 5D). The synthesis of PfTufA was inhibited in clindamycin-treated parasites, but not in the DSG-treated parasites in the first asexual cycle (Fig. 5E). In the second asexual cycle, no PfTufA could be immunoprecipitated from either DSG or clindamycin-treated parasites, indicating the absence of apicoplast (and consequently PfTufA) (Fig. 5E).

To determine if it were trafficking or processing of NEAT proteins that is inhibited by DSG, we performed immunofluorescence of parasites untreated or treated with DSG or clindamycin using antibodies to PfFabG (NEAT protein) and PfTufA (apicoplast-encoded protein). Both anti-PfFabG and anti-PfTufA antibodies colocalized to the apicoplast in control parasites (Fig. 6, A and B). In DSG-treated parasites, in both the first and second asexual cycles, anti-PfFabG antibody localized to the parasitophorous vacuole, and sometimes in the cytosol as well (Fig. 6, C–G), indicating a defect in NEAT protein trafficking. Anti-PfTufA antibody localized to the apicoplast in the first cycle, suggesting apicoplast integrity (Fig. 6C). Further, it was present in only one of the daughter parasites in the dividing stage of the first cycle, indicating apicoplast missegregation (Fig. 6E). Anti-PfTufA antibody was not seen in the second cycle, implying the absence of an apicoplast (Fig. 6, F and G). In clindamycin-treated parasites, anti-PfFabG antibody localized to the apicoplast in the first cycle, indicating normal trafficking of NEAT proteins (Fig. 6H). In the dividing stage of the first cycle, however, anti-PfFabG antibody localized to only one of the daughter parasites of the schizont, suggesting apicoplast missegregation (Fig. 6I). In the second cycle, PfFabG was seen in the parasitophorous vacuole or sometimes in the cytosol, suggesting that it could not be trafficked in the absence of an apicoplast (Fig. 6, J and K). No fluorescence could be attributed to the anti-PfTufA antibody in the first or the second asexual cycle, as anticipated, because apicoplast protein synthesis is inhibited by clindamycin (Fig. 6, H–K). The reversibility of DSG-induced FabG mistargeting was also followed by incubation with DSG for 2, 4, 6, 12, 18, or 24 h of the first asexual cycle followed by its removal and the addition of cycloheximide (500 nM) to inhibit further protein synthesis. PfFabG was seen in the parasitophorous vacuole, indicating that once mistargeted, FabG cannot be re-targeted to the apicoplast (Fig. 6L).

The immunofluorescence data demonstrated a clear difference between the mechanisms of action of DSG and clindamycin, two agents inducing delayed death. It was clear that DSG inhibited trafficking of NEAT proteins (such as PfFabG) in the first asexual cycle, which led to apicoplast missegregation and a subsequent loss of apicoplast (and thereby apicoplast proteins such as PfTufA) in the second asexual cycle. Clindamycin, in contrast, by inhibiting synthesis of apicoplast proteins (such as PfTufA), brought about apicoplast missegregation and subsequent apicoplast loss, which in turn inhibited trafficking of NEAT proteins (such as PfFabG) in the second asexual cycle.

NEAT proteins, as mentioned earlier, are involved in anabolic pathways such as fatty acid synthesis, heme synthesis, or isoprenoid synthesis. In the event of inhibition of NEAT protein trafficking by DSG, one would expect the synthesis of fatty acids, heme, and isoprenoids also to be affected. We monitored the incorporation of radiolabeled acetate into fatty acids also in conjunction with NEAT protein trafficking in the various stages of malaria parasites treated with DSG (Fig. 7). There was no inhibition of fatty acid synthesis in the ring (~12 h) or the young trophozoite stage (~28 h). In the late trophozoite (~36 h) and schizont (~44 h) stages, however, fatty acid synthesis was inhibited (Fig. 7, A and B). These results were in corroboration with the observation of more inhibition of processing of NEAT proteins in these stages of DSG-treated parasites in the first asexual cycle (data not shown). Fatty acid synthesis as determined from [14C]acetate incorporation is known to actively occur only in the young trophozoite stage (19), and this is reflected in this experiment also. The low level of fatty acids being synthesized during the late trophozoite and schizont stages could account for the absence of overt effects arising from fatty acid synthesis inhibition by DSG treatment in the first cycle. There was no detectable incorporation of radiolabeled acetate into fatty acids in DSG treated parasites in the second asexual cycle, which is in accordance with our earlier results that imply the absence of an apicoplast in them.


Figure 7
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  		FIGURE 7.
A, fatty acid synthesis in young trophozoites (~10% parasitemia) following treatment with various drugs. Parasites incubated with triclosan (10 µM) or DSG (1 µM) or clindamycin (500 nM) for 2 h were resuspended in 5 ml of complete medium while retaining the same concentration of the inhibitor. 1,2[14C]Acetate (50 µCi/ml sodium acetate; 60 mCi/mmol) was added, and the incorporation of [14C]acetate into fatty acids measured. DSG, like clindamycin (CLN), did not inhibit [14C]acetate incorporation into fatty acids in young trophozoites. Fatty acid synthesis was inhibited in young trophozoites treated with 10 µM triclosan (TRI). B, stage-specific fatty acid synthesis in DSG-treated parasites. R (ring), ET (early trophozoite), LT (late trophozoite), S (schizont), ET (2) (early trophozoite, second cycle). Fatty acid synthesis was not inhibited in rings and early trophozoites. However, fatty acid synthesis was inhibited in late trophozoites and schizonts of the first asexual cycle, as well as in DSG-treated parasites in the second asexual cycle.

 
Additionally, we looked at the reversibility of the DSG effect on the malaria parasites by incubating parasites with 1 µM DSG for time points (2, 4, 6, 12, 18, 24, 30, and 36 h), followed by removing the DSG by culture medium change, and monitoring the growth of the parasites by [3H]hypoxanthine uptake in the second asexual cycle. Incubation with DSG for more than 18 h of the first asexual cycle followed by its removal still resulted in the death of the parasites during the second asexual cycle. However, if the parasites were treated with DSG for 12 h or shorter time periods, there was no death of the parasites in the second asexual cycle (data not shown). This could be attributed to the reversibility of the effect of DSG at earlier time points.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first report of delayed death resulting from the inhibition of NEAT protein trafficking by a small molecule. No inhibitor capable of interfering with trafficking of NEAT proteins in the malaria parasite has been described yet. However, a study by He et al. demonstrated the elimination of the apicoplast and subsequent delayed death engineered through the expression of a recombinant poison; a fusion protein between a NEAT protein, GFP and a rhoptry protein; in a related apicomplexan, Toxoplasma gondii. The fusion protein thus harbored the N-terminal apicoplast signaling sequence as well as the C-terminal rhoptry retention sequence. Though this fusion protein localized in the apicoplast, it associated with the apicoplast membranes and blocked apicoplast segregation during mitosis in these parasites (28).

Our experiments demonstrate that DSG, at submicromolar concentrations, specifically inhibits trafficking of NEAT proteins in the first asexual cycle itself, without affecting apicoplast integrity. Consequently, however, the apicoplast is devoid of proteins essential to the functioning of various metabolic processes that occur within it, and the parasite dies a delayed death following apicoplast missegregation. Immunofluorescence as well as biochemical experiments not only demonstrate the mistargeting of NEAT proteins in DSG-treated parasites, but also draw a clear distinction between the effects of DSG and clindamycin, an archetypical delayed death-invoking agent.

How does DSG abolish NEAT protein trafficking to the apicoplast? One attractive hypothesis is that DSG interferes with Hsp70 binding to the transit peptide of the NEAT protein, preventing the transit peptide from remaining in an unfolded conformation that is essential to apicoplast targeting. DSG is known to bind the EEVD motif of heat shock proteins, and our initial biochemical experiments indeed demonstrate that micromolar concentrations of DSG inhibit eukaryotic protein synthesis in the malaria parasite following sequestration of heat shock proteins. The premise that DSG interferes with Hsp70 binding to the transit peptide of NEAT proteins, would however require that a heat shock protein with an EEVD motif (of parasite or host origin) be available in the secretory pathway. While several heat shock proteins are known to exist in the secretory pathway, the only two heat shock proteins containing the EEVD motif in the malaria parasite are an Hsp70 and an Hsp90 protein, both of which are localized in the cytosol. Alternately, DSG might interfere with an exquisitely specific charge-charge interaction between the transit peptide and Toc, which is speculated to be crucial for apicoplast protein import. DSG, being a highly positively charged small molecule, could effectively compete with the positively charged transit peptide for the negatively charged pores on the apicoplast membrane, and prevent NEAT protein entry.

Further studies will be required to dissect the mode of DSG inhibition of trafficking of NEAT proteins to the apicoplast. However, our study, while identifying the primary target of the immunosuppressant, DSG, in the malaria parasite, also uncovers a novel and extremely potent inhibition of a hitherto underexplored apicoplast function.


   FOOTNOTES
 
* This work was supported in part by the Dept. of Biotechnology (DBT), Government of India (to N. S. and A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a Council of Scientific and Industrial Research Senior Research Fellowship. Back

2 To whom correspondence may be addressed: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: 91-11-26717102; Fax: 91-11-26717104; E-mail: surolia{at}nii.res.in. 3 To whom correspondence may be addressed: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India. Tel.: 91-80-22082821; Fax: 91-80-22082766; E-mail: surolia{at}jncasr.ac.in.

4 The abbreviations used are: DSG, 15-deoxyspergualin; NEAT, nucleus-encoded apicoplast-targeted; eIF2{alpha}, eukaryotic initiation factor 2 alpha; Hsp70, heat shock protein 70; Hsp90, heat shock protein 90; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole. Back


   ACKNOWLEDGMENTS
 
We thank B. S. Suma at the confocal laser scanning microscopy facility at JNCASR, Bangalore, for expert assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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