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A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates.

Ganesan K, Ponmee N, Jiang L, Fowble JW, White J, Kamchonwongpaisan S, Yuthavong Y, Wilairat P, Rathod PK - PLoS Pathog. (2008)

Bottom Line: In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS).No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h.We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Global Health, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

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Related in: MedlinePlus

The antifolate WR99210 rapidly triggers commitment to death but fails to shut down metabolism or development.While 10 nM WR99210 is sufficient to commit 50% of the cells to lethality within 6 h, the parasites continue to obey normal metabolic pattern for hypoxanthine uptake and incorporation into DNA (albeit at a lower amplitude) and continue to develop to schizogony for upto 24 h. (A) Rapid decrease in clonal viability of WR99210 exposed Dd2 cells. Trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Washed cells were diluted and plated in 96-well plates (see methods). Control diluted cells revealed about 20 positive colonies starting at 12–16 days. (B) Continued incorporation of hypoxanthine in WR99210-treated cells. Young trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Cells were pulsed directly with radioactive hypoxanthine for 1 h. Incorporation of radioactive hypoxanthine into DNA was measured by precipitation of nucleic acids on glass fiber filters. Maximum incorporations was seen at 4–8 h into trophozoite development. •; Solvent-treated cells, ▪; WR99210-treated cells. (C) Synchronized Dd2 cells at early trophozoite stage were treated with 10 nM WR99210 or 0.1% DMSO (control) for 48 h. Parasites were visualized by light microscopy of Giemsa-stained blood smears. Images at 6 h, 12 h, 24 h, and 30 h of WR99210 treatment are shown. Based on microscopy, the parasites followed normal development up to about 24 h after WR99210 treatment.
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ppat-1000214-g001: The antifolate WR99210 rapidly triggers commitment to death but fails to shut down metabolism or development.While 10 nM WR99210 is sufficient to commit 50% of the cells to lethality within 6 h, the parasites continue to obey normal metabolic pattern for hypoxanthine uptake and incorporation into DNA (albeit at a lower amplitude) and continue to develop to schizogony for upto 24 h. (A) Rapid decrease in clonal viability of WR99210 exposed Dd2 cells. Trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Washed cells were diluted and plated in 96-well plates (see methods). Control diluted cells revealed about 20 positive colonies starting at 12–16 days. (B) Continued incorporation of hypoxanthine in WR99210-treated cells. Young trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Cells were pulsed directly with radioactive hypoxanthine for 1 h. Incorporation of radioactive hypoxanthine into DNA was measured by precipitation of nucleic acids on glass fiber filters. Maximum incorporations was seen at 4–8 h into trophozoite development. •; Solvent-treated cells, ▪; WR99210-treated cells. (C) Synchronized Dd2 cells at early trophozoite stage were treated with 10 nM WR99210 or 0.1% DMSO (control) for 48 h. Parasites were visualized by light microscopy of Giemsa-stained blood smears. Images at 6 h, 12 h, 24 h, and 30 h of WR99210 treatment are shown. Based on microscopy, the parasites followed normal development up to about 24 h after WR99210 treatment.

Mentions: A comparison of biochemical changes, morphological alterations and loss of cell viability in WR99210-treated Dd2 provided the first indication that malaria parasites resisted broad metabolic or developmental arrests in response to specific lethal perturbations. Using clonal viability as a measure of drug-induced death [31], 50% of P. falciparum trophozoites became less viable after as little as 6 h of exposure to 10 nM WR99210 (p<0.01) and practically all parasite cells were non-viable after 12 h of drug exposure (Figure 1A). However, even after 24 h of WR99210 treatment, trophozoites continued to follow a preordained metabolic program for converting short pulses of radioactive hypoxanthine into DNA, albeit with a lower amplitude (Figure 1B). Microscopic examination of WR99210-treated trophozoite forms of the parasite failed to show morphological changes until about 24 h after treatment when the schizonts appeared unhealthy (Figure 1C). At subsequent hours, control cells released merozoites and generated healthy rings but the WR99210-treated parasites remained as ill schizonts. In parallel assays, WR99210-resistant B1G9 cells behaved like untreated Dd2 (data not shown).


A genetically hard-wired metabolic transcriptome in Plasmodium falciparum fails to mount protective responses to lethal antifolates.

Ganesan K, Ponmee N, Jiang L, Fowble JW, White J, Kamchonwongpaisan S, Yuthavong Y, Wilairat P, Rathod PK - PLoS Pathog. (2008)

The antifolate WR99210 rapidly triggers commitment to death but fails to shut down metabolism or development.While 10 nM WR99210 is sufficient to commit 50% of the cells to lethality within 6 h, the parasites continue to obey normal metabolic pattern for hypoxanthine uptake and incorporation into DNA (albeit at a lower amplitude) and continue to develop to schizogony for upto 24 h. (A) Rapid decrease in clonal viability of WR99210 exposed Dd2 cells. Trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Washed cells were diluted and plated in 96-well plates (see methods). Control diluted cells revealed about 20 positive colonies starting at 12–16 days. (B) Continued incorporation of hypoxanthine in WR99210-treated cells. Young trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Cells were pulsed directly with radioactive hypoxanthine for 1 h. Incorporation of radioactive hypoxanthine into DNA was measured by precipitation of nucleic acids on glass fiber filters. Maximum incorporations was seen at 4–8 h into trophozoite development. •; Solvent-treated cells, ▪; WR99210-treated cells. (C) Synchronized Dd2 cells at early trophozoite stage were treated with 10 nM WR99210 or 0.1% DMSO (control) for 48 h. Parasites were visualized by light microscopy of Giemsa-stained blood smears. Images at 6 h, 12 h, 24 h, and 30 h of WR99210 treatment are shown. Based on microscopy, the parasites followed normal development up to about 24 h after WR99210 treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2581438&req=5

ppat-1000214-g001: The antifolate WR99210 rapidly triggers commitment to death but fails to shut down metabolism or development.While 10 nM WR99210 is sufficient to commit 50% of the cells to lethality within 6 h, the parasites continue to obey normal metabolic pattern for hypoxanthine uptake and incorporation into DNA (albeit at a lower amplitude) and continue to develop to schizogony for upto 24 h. (A) Rapid decrease in clonal viability of WR99210 exposed Dd2 cells. Trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Washed cells were diluted and plated in 96-well plates (see methods). Control diluted cells revealed about 20 positive colonies starting at 12–16 days. (B) Continued incorporation of hypoxanthine in WR99210-treated cells. Young trophozoite forms of infected erythrocytes in 10 ml cultures were exposed to 10 nM WR99210 for varying periods. Cells were pulsed directly with radioactive hypoxanthine for 1 h. Incorporation of radioactive hypoxanthine into DNA was measured by precipitation of nucleic acids on glass fiber filters. Maximum incorporations was seen at 4–8 h into trophozoite development. •; Solvent-treated cells, ▪; WR99210-treated cells. (C) Synchronized Dd2 cells at early trophozoite stage were treated with 10 nM WR99210 or 0.1% DMSO (control) for 48 h. Parasites were visualized by light microscopy of Giemsa-stained blood smears. Images at 6 h, 12 h, 24 h, and 30 h of WR99210 treatment are shown. Based on microscopy, the parasites followed normal development up to about 24 h after WR99210 treatment.
Mentions: A comparison of biochemical changes, morphological alterations and loss of cell viability in WR99210-treated Dd2 provided the first indication that malaria parasites resisted broad metabolic or developmental arrests in response to specific lethal perturbations. Using clonal viability as a measure of drug-induced death [31], 50% of P. falciparum trophozoites became less viable after as little as 6 h of exposure to 10 nM WR99210 (p<0.01) and practically all parasite cells were non-viable after 12 h of drug exposure (Figure 1A). However, even after 24 h of WR99210 treatment, trophozoites continued to follow a preordained metabolic program for converting short pulses of radioactive hypoxanthine into DNA, albeit with a lower amplitude (Figure 1B). Microscopic examination of WR99210-treated trophozoite forms of the parasite failed to show morphological changes until about 24 h after treatment when the schizonts appeared unhealthy (Figure 1C). At subsequent hours, control cells released merozoites and generated healthy rings but the WR99210-treated parasites remained as ill schizonts. In parallel assays, WR99210-resistant B1G9 cells behaved like untreated Dd2 (data not shown).

Bottom Line: In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS).No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h.We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Global Health, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

Show MeSH
Related in: MedlinePlus