Studies hint at new drug targets and suggest the modified parasites could be used as attenuated vaccine.

Scientists claim to have identified the only compound that is essential for the Plasmodium parasite to survive without a functional apicoplast organelle, at least in culture. A duo of researchers from Stanford Medical School and the University of California, San Francisco (UCSF), has found that blood-cultured Plasmodium parasites treated with antibiotics that cause apicoplast dysfunction, and eventually loss of the organelle, can survive and grow indefinitely as long as they are supplemented with isopentenyl pyrophosphate (IPP). 

Stanford’s Ellen Yeh, M.D., and UCSF’s Joseph L. DeRisi, Ph.D., claim their findings demonstrate that production of isoprenoid precursors is the only absolutely critical function of the apicoplast during asexual blood-stage P. falciparum growth. Reporting in PLoS Biology, they admit that “the survival of apicoplast-minus P. falciparum invokes a slew of intriguing questions.”

Nevertheless, the parasites will also provide a useful tool for investigating apicoplast biology, protein trafficking, and drug targets, and could also represent “an ideal candidate for an attenuated blood-stage vaccine.” Drs. Yeh and DeRisi describe their findings in a paper titled “Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage P. falciparum.”

Over 500 proteins are predicted to localize to Plasmodium’s apicoplast, and the organelle, which is prokaryotic in origin, is a key target for antimalarial drug development, the researchers report. Several prokaryotic biochemical pathways have identified in the apicoplast, and research has shown that its function is necessary for both intraerythrocytic and intrahepatic development in the human host. 

Treating blood-stage Plasmodium with certain antibiotics initially inhibits apicoplast genome expression, translation, and protein processing in the first life cycle, but in the second life cycle failure of apicoplast genome replication means that daughter cells don’t inherit the organelle, and die. Unfortunately, this process can be too slow to be of significant therapeutic utility.

Moreover, the researchers add, despite its promise as Plasmodium’s Achilles heel, the function of the apicoplast has eluded researchers over the nearly 20 years since its discovery. “Without knowledge of specific proteins or pathways suitable as drug targets, particularly during the clinically symptomatic blood stage, efforts to develop apicoplast-directed therapies (beyond known antibiotics) have been stymied.”

In fact, although some 5–10% of Plasmodium’s nuclear genome is predicted to code for products destined for transport to the apicoplast or relating to apicoplast function, some 70% of the apicoplast gene products are of unknown function. The small section of genome retained in the apicoplast itself appears to encode largely for housekeeping genes. 

Among the apicoplast pathways that have been annotated, the prokaryotic MEP/DOXP/nonmevalonate pathway is used for synthesizing isoprenoid precursors. Plasmodium relies on this pathway rather than the canonical mevalonate pathway used by most other eukaryotes and all mammals. Although both routes lead to the production of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as the final products, they operate via a different set of enzymes and chemical intermediates.

Fosmidomycin, meanwhile, is an inhibitor of the MEP pathway, kills blood-stage parasites, and has been tested in clinical trials as an antimalarial. Inhibition by fosmidomycin suggests that isoprenoid precursor biosynthesis is essential in blood-stage infection, so to test this further, Drs. Yeh and DeRisi used a drug inhibition/chemical rescue approach—equivalent to genetic deletion/complementation—to establish whether the pathway was truly essential to apicoplast function and parasite survival.

What they found was that supplementing fosmidomycin-treated blood-stage P. falciparum W2 strain with IPP (but not DMAPP) was sufficient to completely reverse growth inhibition. Importantly, IPP supplementation didn’t rescue parasites treated with chloroquine, which doesn’t target isoprenoid precursor biosynthesis.

To determine whether rescue of the isoprenoid precursor biosynthesis pathway could even reverse delayed death due to antibiotics, P. falciparum W2 parasites were then treated with chloramphenicol, clindamycin, or doxycycline, in the presence of IPP. The addition of IPP reversed apicoplast-specific inhibition by the drugs, and even prevented death of parasite progeny in the second life cycle. Similar results were obtained with a separate strain, P. falciparum D10, suggesting that reversal of antibiotic inhibition by IPP is not strain-specific.

Blood-stage parasites were then taken through several life cycles with simultaneous antibiotic treatment and IPP supplementation, to evaluate whether there were any significant growth defects and whether the surviving parasites still needed the exogenous IPP to survive after antibiotic withdrawal. The results showed that removal of IPP rapidly led to parasite death, while continuing IPP supplementation allowed the parasites to survive through numerous life cycles whether the antibiotic was present or not. “These results show that antibiotic-treated parasites rescued by IPP supplementation have no gross growth defect but are entirely dependent on exogenous IPP for continued growth,” the authors write.

Genome analyses confirmed  that the apicoplast genome (but not mitochondrial or nuclear genomes) was gradually lost in antibiotic-treated, IPP-rescued parasites, and eventually became undetectable. To evaluate what was also occurring in terms of protein processing within the apicoplast during antibiotic therapy and IPP supplementation, the researchers used a transgenic D10 strain expressing green fluorescent protein fused to an apicoplast targeting sequence. Studies showed that while untreated parasites were capable of processing the GFP-containing protein, those treated with doxycycline began to lose apicoplast protein processing function during the second life cycle. Surviving parasites supplemented with IPP still showed successive reduction in protein-processing capacity, indicating a loss of the critical protein import function of the apicoplast. 

Because previous studies have shown that antibiotic treatment does, indeed, lead to failure of apicoplast replication and segregation and thus loss of the organelle in progeny, the team looked to see where the apicoplast-targeted GFP was localizing. As expected, in untreated parasites the GFP localized to a discrete structure. In contrast, in parasites that had been rescued from antibiotic death, GFP lost its discrete apicoplast localization and became diffuse. Confocal imaging studies showed that a number of GFP foci were scattered throughout the cytoplasm, and some of these measured over 200 nm, suggesting that they may represent vesicles containing protein.

“Combined with the absence of the apicoplast genome and protein import function, the loss of GFP localization indicates the absence of the apicoplast itself to which it is normally targeted,” the researchers state.

The team claims results from the chemical rescue experiments will help in the search for new Plasmodium drug targets and compounds. “The potential pathway for killing parasites without interfering with human cells is the reason the apicoplast has been a major focus for drug development, Dr. Yeh notes. “Now we have a way to specifically look for drugs that target its function and discover a whole new class of desperately needed antimalarials.”

IPP-dependent apicoplast-minus strains could in addition form the basis of an attenuated blood-stage vaccine, the researchers add. “Unlike irradiated or drug-treated whole parasite vaccines, apicoplast-minus parasites would continue to develop in blood at most one cycle, including a single erythrocyte rupture and reinvasion, thereby stimulating a stronger immune response. However, judging by the effects of IPP withdrawal in culture, they would be unable to develop further in the absence of exogenous IPP.” And because the chemical attenuation approach is relatively simple and doesn’t require genetic manipulation, the authors continue, this method would facilitate the generation of attenuated virus vaccines from potentially any circulating strain.

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