Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin V.

Alexander J Polino,Daniel E Goldberg,Jacquin C Niles,Armiyaw S Nasamu

doi:10.1021/acsinfecdis.9b00460

Alexander J Polino, Daniel E Goldberg + Show 2 more

Open Access

https://doi.org/10.1021/acsinfecdis.9b00460

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Abstract

Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This protein export program is essential for parasite survival. Hence export-related proteins could be potential drug targets. One essential enzyme in this pathway is plasmepsin V (PMV), an aspartic protease that processes export-destined proteins in the parasite endoplasmic reticulum (ER) at the Plasmodium export element (PEXEL) motif. Despite long-standing interest in this enzyme, functional studies have been hindered by the inability of previous technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a system for stringent post-transcriptional regulation allowing a tightly controlled, tunable knockdown of PMV. Using this system, we found that PMV must be dramatically depleted to affect parasite growth, suggesting the parasite maintains this enzyme in substantial excess. Surprisingly, depletion of PMV arrested parasite growth immediately after RBC invasion, significantly before the death from exported protein deficit that has previously been described. The data suggest that PMV inhibitors can halt parasite growth at two distinct points in the parasite life cycle. However, overcoming the functional excess of PMV in the parasite may require inhibitor concentrations far beyond the enzyme’s IC50.

Highlights

Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the red blood cells (RBCs) cytosol
Cleavage occurs at the conserved amino acid motif RxLxE/Q/D, termed the Plasmodium export element (PEXEL).[8−12] plasmepsin V (PMV) is highly specific for RxL in the PEXEL and cleaves after the leucine.[10,13,14]
This system has previously been used to manipulate large [A+T]-rich genomic fragments, including those derived from the rodent malaria parasite, P. berghei.[20,21]

Summary

Introduction

Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. The most robust knockdown described used the glmS ribozyme system, reducing PMV levels 10-fold with no measurable effect on parasite growth or PEXEL processing.[7,16] Here, we sought to apply the recently described TetR-DOZI aptamer system for stringent and tunable regulation of PMV.[19] This system can deplete a reporter gene 45 to 70-fold when aptamers are installed in both the 5′ and 3′ untranslated regions (UTRs) of the target gene.[19] cloning such a construct in traditional plasmid systems requires the assembly and maintenance of large circular plasmids that are prone to deletions and vector rearrangements during propagation in E. coli.[20] To overcome this technical challenge, we assembled a number of tools for genetic manipulation onto a previously described linear vector. We found that PMV-depleted parasites die immediately after invasion in a manner distinct from that of disruptions of other protein export machinery, suggesting PMV may have additional roles beyond those previously described

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Conclusion