Doxycycline (DOX) is a key antimalarial drug thought to kill Plasmodium parasites by blocking protein translation in the essential apicoplast organelle. Clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 µM serum concentrations. DOX concentrations >5 µM kill parasites with first-cycle activity but have been ascribed to off-target mechanisms outside the apicoplast. We report that 10 µM DOX blocks apicoplast biogenesis in the first cycle and is rescued by isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific mechanism. Exogenous iron rescues parasites and apicoplast biogenesis from first- but not second-cycle effects of 10 µM DOX, revealing that first-cycle activity involves a metal-dependent mechanism distinct from the delayed-death mechanism. These results critically expand the paradigm for understanding the fundamental antiparasitic mechanisms of DOX and suggest repurposing DOX as a faster-acting antimalarial at higher dosing whose multiple mechanisms would be expected to limit parasite resistance.
All data reported or described in this manuscript are available and included in the main and supplemental figures and in the microscopy source data file.
- Megan Okada
- Shai-anne Nalder
- Paul A Sigala
- Paul A Sigala
- Paul A Sigala
- Paul A Sigala
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Jon Clardy, Harvard Medical School, United States
© 2020, Okada et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.
Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.