The Glutathione Biosynthetic Pathway of Plasmodium Is Essential for Mosquito Transmission
Figure 8
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.