15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum

Abstract

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2␣. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process. 15-Deoxyspergualin (DSG)4 ( Fig. 1), an analog of spergualin isolated from the culture broth of Bacillus laterosporus, inhibits the growth of the malaria parasite, Plasmodium, in vitro and in vivo, presumably by depleting its polyamine reserves (1, 2). However, other known inhibitors of polyamine biosynthesis, methylglyoxal-bis-(guanylhydrazone) analog (MGBCP), irinotecan hydrochloride (CPT11), and ␣-difluoromethyl ornithine (DFMO) are known to be incapable of suppressing malaria in infected mice (2). This led us to question the speculated mechanism of antimalarial action of DSG.Despite the uncertainty in the actual mechanism of action of DSG, DSG is known to bind cellular chaperones, Hsp70 and Hsp90, potently, via their regulatory C-terminal motif EEVD (3, 4). DSG does not inhibit their chaperone activity (3-5). However, it selectively enhances the ATPase activity of heat shock proteins that have the C-terminal EEVD motif, and this could be the basis of a specific modulation of the function of proteins that bind the EEVD motif of heat shock proteins (5).One of the cellular milieus in which heat shock proteins are speculated to be involved is translational regulation. Translation of an mRNA requires the formation of a ternary complex of the methionyl-tRNA, the mRNA and the 40 S subunit of the ribosome, a feat achieved through the intervention of the GTPbound form of the initiation factor eIF2 (6). Phosphorylation of eIF2␣ at Ser 51 blocks protein synthesis by preventing eIF2 recycling and depleting the cell of its pool of eIF2-GTP required for translation initiation (7). Several conditions of cellular stress trigger the phosphorylation of eIF2␣ via the activation of four different eIF2␣ kinases: PERK (PKR-like endoplasmic reticulum-associated kina...

Multiple Antibiotics Exert Delayed Effects against the Plasmodium falciparum Apicoplast

“…We now report that, at clinically achievable doses, clindamycin, azithromycin, and ciprofloxacin exerted effects similar to those of doxycycline when cultured with malaria parasites. Our results agree with a recent report that apicoplast segregation was unaffected by clindamycin or tetracycline (20) but disagree with another report (34), suggesting that clindamycin blocked apicoplast segregation. We did observe abnormal apicoplasts following treatment with high doses of antibiotics (twice the IC 50 at 48 h), but these parasites did not produce progeny and were grossly abnormal, suggesting that at these doses antibiotics were interfering with multiple targets in addition to the apicoplast (data not shown).…”

The Plastid-Like Organelle of Apicomplexan Parasites as Drug Target

“…In accordance with that hypothesis, a pronounced delayed kill effect was also observed with the immunosuppressive agent 15-deoxyspergualin 17, which was found to inhibit trafficking of proteins into the apicoplast of P. falciparum by an unknown mechanism ( Fig. 4) [43]. In contrast to those classical antibiotics, no delayed kill effect is observed with compounds which directly inhibit enzymes involved in metabolic functions of the apicoplast, such as isoprenoid and fatty acid synthesis [37,44].…”

Antibiotics in Malaria Therapy and their Effect on the Parasite Apicoplast

“…Inhibition of the apicoplast protein synthesis, however, could prevent the formation of apicoplast-encoded proteins required for import and for processing of nuclear-encoded proteins in the second cycle. Indeed it has been shown that under the influence of clindamycin the nuclear-encoded protein PfFabG is not transported into the apicoplast during the second cycle, but remains in the cytoplasm [70]. The lack of proteins required for metabolic functions consequently leads to non-functional apicoplasts in the second progeny.…”

“…(3)) [70]. DSG inhibits trafficking of NEAT proteins during the first asexual cycle, potentially by interfering with binding of HSP70 to the transit domain and thus preventing this domain from remaining in an unfolded conformation that is essential for apicoplast import [70].…”