GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Stanway, Rebecca R.; Witt, Tina; Zobiak, Bernd; Aepfelbacher, Martin; Heussler, Volker

doi:10.1042/bc20080202

Cited by 81 publications

(99 citation statements)

References 43 publications

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“…Figure 2A shows that apicoplast-targeted FabI was strongly expressed in the day 9 oocyst and was colocalized with the known apicoplast marker, ACP. The extremely branched structure of the apicoplast at this time point is in agreement with apicoplast-targeted fluorophore expression in the rodent malaria P. berghei oocyst (43). The colocalized expression of FabI and ACP was punctate in the mature day 12 oocyst (Fig.…”

Section: Resultssupporting

confidence: 80%

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Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

et al. 2014

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The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

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Section: Resultssupporting

confidence: 80%

“…4A) to day 5 ( Fig. 4B) was mirrored by an increasingly complex apicoplast structure, as has been shown previously for liver-stage apicoplast development in P. berghei (43). Liver-stage maturation results in the release of thousands of exoerythrocytic merozoites, and Fig.…”

Section: Resultssupporting

confidence: 76%

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

et al. 2014

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“…Here we showed activity against both male and female gametocytes, as expected for a compound targeting such universal biological pathways. Interestingly, the male gametocyte is 15 times more sensitive to thiostrepton than the female (and 4 times more sensitive than asexual parasites [3]), even though the apicoplast remains in the residual body of the gametocyte and is not present in the newly formed male gametes (42). The role of the proteasome in exflagellation has never been investigated; however, another proteasome inhibitor, epoxomycin, has been shown to have potent activity against "late-stage" gametocytes (5).…”

Section: Discussionmentioning

confidence: 99%

Male and Female Plasmodium falciparum Mature Gametocytes Show Different Responses to Antimalarial Drugs

Delves

Ruecker

Straschil

et al. 2013

Antimicrob Agents Chemother

175

217

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It is the mature gametocytes of Plasmodium that are solely responsible for parasite transmission from the mammalian host to the mosquito. They are therefore a logical target for transmission-blocking antimalarial interventions, which aim to break the cycle of reinfection and reduce the prevalence of malaria cases. Gametocytes, however, are not a homogeneous cell population. They are sexually dimorphic, and both males and females are required for parasite transmission. Using two bioassays, we explored the effects of 20 antimalarials on the functional viability of both male and female mature gametocytes of Plasmodium falciparum. We show that mature male gametocytes (as reported by their ability to produce male gametes, i.e., to exflagellate) are sensitive to antifolates, some endoperoxides, methylene blue, and thiostrepton, with submicromolar 50% inhibitory concentrations (IC 50 s), whereas female gametocytes (as reported by their ability to activate and form gametes expressing the marker Pfs25) are much less sensitive to antimalarial intervention, with only methylene blue and thiostrepton showing any significant activity. These findings show firstly that the antimalarial responses of male and female gametocytes differ and secondly that the mature male gametocyte should be considered a more vulnerable target than the female gametocyte for transmission-blocking drugs. Given the female-biased sex ratio of Plasmodium falciparum (ϳ3 to 5 females:1 male), current gametocyte assays without a sexspecific readout are unlikely to identify male-targeted compounds and prioritize them for further development. Both assays reported here are being scaled up to at least medium throughput and will permit identification of key transmission-blocking molecules that have been overlooked by other screening campaigns. Malaria is a disease of devastating economic and health burdens, with 216 million cases and 655,000 fatalities per year, among which most are either pregnant women or children of less than 5 years of age (1). The recent appreciation that local elimination and global eradication of malaria will require interventions that prevent parasite transmission from the human host to the vector (2) has revitalized the search for transmission-blocking drugs (3-7). One target of such drugs is the gametocyte, which is the parasite stage uniquely responsible for Plasmodium transmission to the mosquito.Plasmodium asexual parasites form gametocytes at a low frequency (0.2 to 1%) (8), with sexually committed merozoites from one precommitted schizont all forming gametocytes of the same sex (9). In Plasmodium falciparum, gametocytes develop over a period of 12 days, during which they are initially susceptible to schizonticidal antimalarials (stages I to III), but for the final part of their maturation process (stages IV and V), they become broadly insensitive to most antimalarial drugs, except for primaquine and methylene blue (6, 10-13). Mature stage V gametocytes, when considered as a single population, are developmentally arrested, and curr...

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“…Due to its prokaryotic origin and evolution as a secondary plastid, it contains pathways that have no counterpart in the human host (3,4). The apicoplast in Plasmodium is essential for both intraerythrocytic and intrahepatic development in the human host (5,6).…”

mentioning

confidence: 99%

A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Herrera

Ebert

et al. 2015

Antimicrob Agents Chemother

109

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The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC 50 ) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-D-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC 50 ) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. Despite encouraging progress over the past decade, malaria caused by Plasmodium parasites continues to pose an enormous disease burden (1). New antimalarials with novel mechanisms of action are needed to circumvent existing or emerging drug resistance (2). The apicoplast is a plastid organelle unique to Plasmodium spp. (and other pathogenic Apicomplexa parasites) and is a key target for development of new antimalarials. Due to its prokaryotic origin and evolution as a secondary plastid, it contains pathways that have no counterpart in the human host (3, 4). The apicoplast in Plasmodium is essential for both intraerythrocytic and intrahepatic development in the human host (5, 6).Despite efforts to develop inhibitors of apicoplast function, to date, there have been no primary agents for treatment of acute malaria whose mechanism of action targets this unusual plastid organelle. Antibiotics that inhibit prokaryotic transcription and translation, such as doxycycline and clindamycin, block expression of the apicoplast genome and are active against Plasmodium parasites (5). Unfortunately, these drugs show a "delayed death" phenotype, in which growth inhibition occurs only after 2 replication cycles (96 h). The slow kinetics limit the use of doxycycline and clindamycin to chemoprophylaxis or as partner drugs in combination therapies with faster-acting compounds. Fosmidomycin, which inhibits the enzyme DoxR/IspC for MEP (methyl-D-erythritol-4-phosphate) isoprenoid precursor biosynthesis in the apicoplast, has immediate onset but shows high recrudescence rates clinically when used as monotherapy (7,8). The efficacy of fosmidomycin-based combination therapy is currently being evaluated, with mixe...

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GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Cited by 81 publications

References 43 publications

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

Male and Female Plasmodium falciparum Mature Gametocytes Show Different Responses to Antimalarial Drugs

A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

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