The Glutathione Biosynthetic Pathway of Plasmodium Is Essential for Mosquito Transmission

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 (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-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.

Inhibition of Glutathione Biosynthesis Sensitizes Plasmodium berghei to Antifolates

“…In this study, we confirmed that ␥-gcs can be knocked out in P. berghei, similar to a previous report (29). Interestingly, ␥-gcs cannot be knocked out in P. falciparum (32).…”

“…3A), indicating that loss of ␥-gcs, and not the integration of the Tgdhfr-ts cassette, was responsible for the growth retardation. These results confirm that GSH biosynthesis was important for optimal growth of P. berghei ANKA in vivo (29).…”

“…As ␥-gcs disruption leads to a malaria parasite growth defect (29) and sensitivity to antifolate, GSH biosynthesis may be involved in DNA synthesis or repair. GSH deficiency in other organisms, such as Escherichia coli, Saccharomyces cerevisiae, and Acinetobacter baylyi, leads to hypersensitivity to mutagens, similar to that in organisms with deficiency in DNA replication and repair (39)(40)(41).…”

Sterile Protective Immunity to Malaria is Associated with a Panel of Novel P. falciparum Antigens

“…Notably, gammaglutamylcysteine synthase (␥GCS, PFI0925w), an antigen on our Top 20 list, is the rate-limiting enzyme in the synthesis of GSH. The disruption of this biosynthetic pathway caused a minor decrease in parasite growth at the blood stage (possibly because of availability of host GSH) but had severe effects for the parasite at the mosquito stage with arrested development of oocysts and no sporozoite production (66). Pf ␥GCS is expressed in P. berghei sporozoites (31) and host ␥GCS is highly induced in P. yoelii-infected livers (67).…”

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

“…Pictures of oocysts were processed using Adobe Photoshop CS2. Sporozoite counts were performed by salivary gland dissection at day 17 postfeeding based on the work of Vega-Rodriguez et al (31), with the following modifications. Salivary glands from 5 mosquitoes were pooled into 1.5-ml tubes containing 100 l of PBS.…”