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The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.

Vega-Rodríguez J, Franke-Fayard B, Dinglasan RR, Janse CJ, Pastrana-Mena R, Waters AP, Coppens I, Rodríguez-Orengo JF, Srinivasan P, Jacobs-Lorena M, Serrano AE - PLoS Pathog. (2009)

Bottom Line: Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type.In contrast, a dramatic effect on development of the parasites in the mosquito was observed.These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Medical Zoology, University of Puerto Rico, School of Medicine, San Juan, Puerto Rico.

ABSTRACT
Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

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Genetic complementation of the pbggcs− parasites with the wild type pbgccs gene.(A) Diagrammatic representation of the construct (pbggcs-comp) used for complementation (top), the dssurrna genomic locus used for targeted integration of the construct (center), and the locus after integration of the construct by single cross-over recombination (bottom). The pbggcs-comp vector contains the hdhfr selectable marker and the dssurrna targeting sequence. Dashed lines represent the probes used for Southern analysis (see B). (B) Southern analysis of CHEF separated chromosomes from pbggcs−, pbggcs-comp, and pbggcs-comp* parasites (asterisk indicates complemented parasites after mosquito passage). Chromosomes were hybridized to different probes showing correct integration of the pbggcs-comp plasmid in chromosome 5, which contains the target dssurrna locus. (C) Reverse transcriptase PCR analysis showing the presence of pbggcs transcripts (345 bp fragments) in wild type and in pbggcs-comp parasites and the absence of pbggcs transcripts in pbggcs− parasites. For the PCR reaction, the specific pbggcs primers PIY-FM/DamR were used (see A). cDNA was synthesized from total mRNA and control reactions described in Fig. 1C. (D) Presence of maturing oocysts (stained with mercurochrome) in midguts from mosquitoes infected with pbggcs-comp parasites. Note the internal structure developing inside the oocysts, indicative of segmentation during sporogony and formation of sporozoites.
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ppat-1000302-g008: Genetic complementation of the pbggcs− parasites with the wild type pbgccs gene.(A) Diagrammatic representation of the construct (pbggcs-comp) used for complementation (top), the dssurrna genomic locus used for targeted integration of the construct (center), and the locus after integration of the construct by single cross-over recombination (bottom). The pbggcs-comp vector contains the hdhfr selectable marker and the dssurrna targeting sequence. Dashed lines represent the probes used for Southern analysis (see B). (B) Southern analysis of CHEF separated chromosomes from pbggcs−, pbggcs-comp, and pbggcs-comp* parasites (asterisk indicates complemented parasites after mosquito passage). Chromosomes were hybridized to different probes showing correct integration of the pbggcs-comp plasmid in chromosome 5, which contains the target dssurrna locus. (C) Reverse transcriptase PCR analysis showing the presence of pbggcs transcripts (345 bp fragments) in wild type and in pbggcs-comp parasites and the absence of pbggcs transcripts in pbggcs− parasites. For the PCR reaction, the specific pbggcs primers PIY-FM/DamR were used (see A). cDNA was synthesized from total mRNA and control reactions described in Fig. 1C. (D) Presence of maturing oocysts (stained with mercurochrome) in midguts from mosquitoes infected with pbggcs-comp parasites. Note the internal structure developing inside the oocysts, indicative of segmentation during sporogony and formation of sporozoites.

Mentions: We used a reverse genetics approach to complement pbggcs− parasites with a wild type copy of pbggcs. The pbggcs− parasites (pbggcs−2) were transfected using a construct that contains the pbggcs gene under the control of the strong, constitutive eef1a promoter (Fig. 8A) [26]. In addition, this plasmid contains the human dihydrofolate reductase (hdhfr) selection cassette and the D-type small subunit rRNA (dssurrna) as targeting sequence for integration into the parasite genome by single cross-over recombination. Resistant parasites were selected by treatment of mice with WR99210 [27]. Correct integration of the complementation plasmid into the dssurrna genomic locus of these parasites (pbggcs-comp parasites) was confirmed by Southern analysis of contour clamped homogeneous electric field (CHEF) separated chromosomes (Fig. 8B). Detection of pbggcs mRNA by PCR analysis of cDNA from pbggcs-comp parasites confirmed successful expression of the introduced pbggcs gene in blood stage forms (Fig. 8C).


The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.

Vega-Rodríguez J, Franke-Fayard B, Dinglasan RR, Janse CJ, Pastrana-Mena R, Waters AP, Coppens I, Rodríguez-Orengo JF, Srinivasan P, Jacobs-Lorena M, Serrano AE - PLoS Pathog. (2009)

Genetic complementation of the pbggcs− parasites with the wild type pbgccs gene.(A) Diagrammatic representation of the construct (pbggcs-comp) used for complementation (top), the dssurrna genomic locus used for targeted integration of the construct (center), and the locus after integration of the construct by single cross-over recombination (bottom). The pbggcs-comp vector contains the hdhfr selectable marker and the dssurrna targeting sequence. Dashed lines represent the probes used for Southern analysis (see B). (B) Southern analysis of CHEF separated chromosomes from pbggcs−, pbggcs-comp, and pbggcs-comp* parasites (asterisk indicates complemented parasites after mosquito passage). Chromosomes were hybridized to different probes showing correct integration of the pbggcs-comp plasmid in chromosome 5, which contains the target dssurrna locus. (C) Reverse transcriptase PCR analysis showing the presence of pbggcs transcripts (345 bp fragments) in wild type and in pbggcs-comp parasites and the absence of pbggcs transcripts in pbggcs− parasites. For the PCR reaction, the specific pbggcs primers PIY-FM/DamR were used (see A). cDNA was synthesized from total mRNA and control reactions described in Fig. 1C. (D) Presence of maturing oocysts (stained with mercurochrome) in midguts from mosquitoes infected with pbggcs-comp parasites. Note the internal structure developing inside the oocysts, indicative of segmentation during sporogony and formation of sporozoites.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636896&req=5

ppat-1000302-g008: Genetic complementation of the pbggcs− parasites with the wild type pbgccs gene.(A) Diagrammatic representation of the construct (pbggcs-comp) used for complementation (top), the dssurrna genomic locus used for targeted integration of the construct (center), and the locus after integration of the construct by single cross-over recombination (bottom). The pbggcs-comp vector contains the hdhfr selectable marker and the dssurrna targeting sequence. Dashed lines represent the probes used for Southern analysis (see B). (B) Southern analysis of CHEF separated chromosomes from pbggcs−, pbggcs-comp, and pbggcs-comp* parasites (asterisk indicates complemented parasites after mosquito passage). Chromosomes were hybridized to different probes showing correct integration of the pbggcs-comp plasmid in chromosome 5, which contains the target dssurrna locus. (C) Reverse transcriptase PCR analysis showing the presence of pbggcs transcripts (345 bp fragments) in wild type and in pbggcs-comp parasites and the absence of pbggcs transcripts in pbggcs− parasites. For the PCR reaction, the specific pbggcs primers PIY-FM/DamR were used (see A). cDNA was synthesized from total mRNA and control reactions described in Fig. 1C. (D) Presence of maturing oocysts (stained with mercurochrome) in midguts from mosquitoes infected with pbggcs-comp parasites. Note the internal structure developing inside the oocysts, indicative of segmentation during sporogony and formation of sporozoites.
Mentions: We used a reverse genetics approach to complement pbggcs− parasites with a wild type copy of pbggcs. The pbggcs− parasites (pbggcs−2) were transfected using a construct that contains the pbggcs gene under the control of the strong, constitutive eef1a promoter (Fig. 8A) [26]. In addition, this plasmid contains the human dihydrofolate reductase (hdhfr) selection cassette and the D-type small subunit rRNA (dssurrna) as targeting sequence for integration into the parasite genome by single cross-over recombination. Resistant parasites were selected by treatment of mice with WR99210 [27]. Correct integration of the complementation plasmid into the dssurrna genomic locus of these parasites (pbggcs-comp parasites) was confirmed by Southern analysis of contour clamped homogeneous electric field (CHEF) separated chromosomes (Fig. 8B). Detection of pbggcs mRNA by PCR analysis of cDNA from pbggcs-comp parasites confirmed successful expression of the introduced pbggcs gene in blood stage forms (Fig. 8C).

Bottom Line: Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type.In contrast, a dramatic effect on development of the parasites in the mosquito was observed.These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Medical Zoology, University of Puerto Rico, School of Medicine, San Juan, Puerto Rico.

ABSTRACT
Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

Show MeSH
Related in: MedlinePlus