Despite the concerted efforts of researchers, policy makers and public health workers worldwide, malaria persists as a significant disease threat for nearly half the world’s population. Recent advances in vector control measures, diagnostics and antimalarial drug therapies have contributed greatly to reducing the incidence of clinical disease, and by extension, the number of deaths attributable to malaria in the past two decades; however, the latter remains high—over 400,000 people die each year from malaria, the vast majority of these being children under the age of five. Our ability to rapidly and effectively treat malaria has been a cornerstone of efforts to control and eradicate this devastating disease. Nonetheless, the constant evolution and spread of drug-resistant forms of the Plasmodium parasites that cause malaria—particularly the most virulent of these, Plasmodium falciparum—have historically greatly hindered these efforts, compromising the efficacy of every previous first-line treatment.
Today, treatment of P. falciparum malaria relies on artemisinin derivatives, an exquisitely potent and fast-acting class of antimalarials that are deployed ubiquitously in artemisinin-based combination therapies, or ACTs. Now, emerging resistance to ACTs threatens to once again reverse the hard-fought advances made in the global fight against malaria. Resistance to artemisinin itself was first documented in western Cambodia and northwest Thailand in 2009 and has continued to spread throughout Southeast Asia at alarming rates. Reports of resistance to ACTs followed soon thereafter. Artemisinin resistance has also emerged de novo in other parts of the world. The major concern is that it will spread to Africa, where the disease burden is highest.
Previous studies have provided compelling evidence that resistance to artemisinin results primarily from specific point mutations in the C-terminal Kelch propeller domain of the P. falciparum protein K13. Here, we have addressed two central aims regarding the role of this protein in mediating resistance to artemisinin. The first was to genetically dissect the contribution of a panel of K13 polymorphisms to artemisinin resistance and parasite fitness as assessed in vitro, with the latter being a key factor impacting the spread of resistance-conferring alleles in high-transmission settings. These experiments were conducted by CRISPR-Cas9-mediated gene editing, which allowed us to successfully engineer K13 mutations into a variety of strain backgrounds, including, for the first time, recently culture-adapted African parasites. These experiments clearly show that there is no genetic obstacle to the acquisition of artemisinin resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may counter-select against the spread of resistance.
The second aim relating to K13 was to investigate the underlying biology of this protein. To this end, we raised monoclonal antibodies to recombinant K13 and generated transgenic lines expressing tagged versions of the protein. Using these tools, we describe the subcellular localization of K13 in wild-type and mutant parasites in the presence and absence of drug pressure, and identify potential K13-associated proteins. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13, suggesting that mutations result in a loss of protein function.
In order to overcome K13-mediated artemisinin resistance, novel therapeutics with distinct modes of action will be required. In our last aim, we characterize inhibitors of a particularly promising new antimalarial drug target, the proteasome. We report that these covalent peptide vinyl sulfone inhibitors are highly potent against genetically diverse parasites, including K13-mutant, artemisinin-resistant lines. Moreover, we observe that parasites do not readily acquire resistance to these compounds, nor do related compounds select for cross-resistance to one another. We also observe strong synergy between artemisinin and related compounds with these inhibitors in both K13 mutant and wild-type parasites. These results highlight the potential for targeting the Plasmodium proteasome as a means of overcoming artemisinin-resistant malaria.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-t7gf-x240 |
| Date | January 2020 |
| Creators | Stokes, Barbara |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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