MALARIA
A Kelch13-defined endocytosis pathway mediates
artemisinin resistance in malaria parasites
Jakob Birnbaum^1 *†, Sarah Scharf^1 †, Sabine Schmidt^1 , Ernst Jonscher^1 ,
Wieteke Anna Maria Hoeijmakers^2 , Sven Flemming^1 ‡, Christa Geeke Toenhake^2 , Marius Schmitt^1 ,
Ricarda Sabitzki^1 , Bärbel Bergmann^1 , Ulrike Fröhlke^1 , Paolo Mesén-Ramírez^1 ,
Alexandra Blancke Soares^1 , Hendrik Herrmann^1 §, Richárd Bártfai^2 , Tobias Spielmann^1 ¶
Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance
is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite’s Kelch13
protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified
proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including
Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional
analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell.
Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced
hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced
activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation,
resulting in parasite resistance.
L
ife-threatening malaria is caused by
Plasmodium falciparumparasites con-
tinuously multiplying in human red blood
cells. Artemisinin and its derivatives
(ARTs) are the recommended first-line
drugs against malaria ( 1 ), but their usefulness
is threatened by“resistance”( 2 ), defined as
reduced clearance of parasites in ART-treated
malaria patients ( 3 ). ART resistance mani-
fests as a decreased susceptibility of young
ring-stage parasites to a short pulse of ART,
a property linked to delayed clearance of para-
sites and recrudescence of disease in patients
( 4 – 6 ).Thiscanbemeasuredinvitrousinga
ring-stage survival assay (RSA) ( 4 ).
ART resistance is primarily associated with
point mutations in the parasite’s Kelch pro-
peller protein Kelch13 ( 7 , 8 ). At present, there
is no coherent picture of the mechanism of
ART resistance in the parasite, which, among
others, has been connected to increased cell-
ular stress, an activated unfolded protein re-
sponse, reduced protein translation, altered
DNA replication, and increased levels of phos-
phatidylinositol 3-phosphate ( 9 – 16 ). Condi-
tional inactivation of Kelch13 causes an arrest
of parasites in ring stages ( 17 ). However, the
cellular function of Kelch13 and its involvement
in ART resistance remain enigmatic. Here, we
study Kelch13 and identify its interactors and
their function. This reveals an entire pathway
in ART resistance and a Kelch13-dependent
mechanism explaining the reduced suscepti-
bility to ART in resistant parasites.The Kelch13 compartment and associated
proteins defined by a novel BioID approach
We previously found endogenous Kelch13
tagged with green fluorescent protein (GFP)
(i.e., expressed from itsgenomic locus) at foci
in close proximity to the parasite’s food vacuole
(FV) ( 17 ), a lysosome-like compartment where
parasites digest endocytosed host cell cytosol
(predominantly hemoglobin). To better define
the cellular location of Kelch13, we colocalized
it with a series of markers (Fig. 1A and fig. S1).
This confirmed the proximity of Kelch13 foci
to the FV. Although Kelch13 foci rarely over-
lapped with secretory pathway markers [endo-
plasmic reticulum (ER) and Golgi], they were
frequently in the vicinity of parts of the ER that
were near the FV. Kelch13 foci were also found
close to the apicoplast, which itself is located
in proximity of the FV. Notably, the resistance-
conferring form of Kelch13 (Kelch13C580Y;
Cys^580 →Tyr) fully colocalized with episomally
expressed wild-type Kelch13 (wtKelch13) (Fig.
1A, fig. S1, and table S1), indicating that re-
sistance does not involve an altered cellular
distribution. Kelch13C580Yalso colocalized with
episomally expressed hemagglutinin (HA)–
tagged Kelch13 (fig. S1). Overall, this analysis
suggests that Kelch13 is located at an unknown
compartment or cellular structure (henceforth,
Kelch13 compartment) not identified by gen-
eral markers of the secretory system.
To gain insight into the Kelch13 compart-
ment, we used a novel version of BioID ( 18 ),
which we call dimerization-induced quantita-
tive BioID (DiQ-BioID), to identify Kelch13 in-
teractors and compartment neighbors in the
living cell. In DiQ-BioID, the biotin ligase BirA*is not directly fused to the target protein but is
expressed separately (the biotinylizer) and con-
ditionally recruited to the target (using rapa-
log), where it biotinylates interaction partners
(Fig. 1B). The same parasite culture grown
without rapalog (i.e., BirA* not on the target)
serves as a control (Fig. 1B), permitting highly
specific identification of interactors by quan-
titative mass spectrometry. We used this ap-
proach in parasites expressing FK506 binding
protein (FKBP)–tagged Kelch13 from the ge-
nomic locus ( 17 ). Induction of dimerization
with rapalog efficientlyrecruited the biotinyl-
izer to the Kelch13 foci (Fig. 1C), and quan-
titative mass spectrometry of asynchronous
parasites resulted in a list of proteins enriched
with very high confidence over control (Fig. 1D,
fig. S2, and data S1). Kelch13 was the top hit,
confirming that the biotinylizer was success-
fully recruited to Kelch13, and a band consistent
in size with the tagged Kelch13 was detected
in anti-biotin blots (fig. S2B). The other hits
[designated as Kelch13 interaction candidates
(KICs), unless already otherwise named] were
predominantly proteins of unknown function.
These included proteins that had previously
been suspected in ART resistance or were in
that context highlighted in genome-wide as-
sociation studies—for example, an Eps15-like
protein (PF3D7_1025000, originally annotated
as Formin2) ( 19 ); ubiquitin carboxyl-terminal
hydrolase 1 (UBP1) ( 19 – 23 ); KIC6 (PF3D7_
0609700) ( 20 ); and MyosinC (MyoC) ( 19 ) (Fig.
1D, fig. S2, and data S1)—suggesting that the
interactome identified a pathway that may be
relevant for ART resistance. Modification of
the genomic locus to express the endogenous
protein fused to GFP (fig. S3) showed that the
top hit of these previously highlighted pro-
teins, the Eps15-like protein (henceforth Eps15),
colocalized with Kelch13 (Fig. 1E), and coim-
munoprecipitation confirmed that Eps15 in-
teracts with Kelch13 (Fig. 1F and fig. S4).
To further validate the Kelch13 DiQ-BioID
results and the method per se, we carried out
a reverse DiQ-BioID with the endogenously
tagged Eps15 (Fig. 1G and fig. S5). This resulted
in a list of high-confidence hits that extensively
overlapped with the Kelch13 DiQ-BioID (all 16
top hits were also found in the high-confidence
hits of the K13 DiQ-BioID), including Kelch13
and UBP1 (Figs. 1G and 2C and data S1). This
suggested that the hits are indeed interactors
or compartment neighbors of the Kelch13-
Eps15 complex and that the DiQ-BioID ex-
periments reached a high depth of coverage.
This was confirmed by tagging the corre-
sponding endogenous genes with the sequence
encoding GFP (fig. S3), which showed that, be-
sides Eps15, 10 of 12 tested Kelch13 hits colo-
calize with Kelch13 compartment foci (Fig. 1H,
fig. S6, and table S1), demonstrating the power
of the method. Only PFK9 and KIC10 did
not show an overlap with Kelch13. Of theRESEARCH
Birnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 1of9
(^1) Bernhard Nocht Institute for Tropical Medicine, Bernhard
Nocht Str. 74, 20359 Hamburg, Germany.^2 Department
of Molecular Biology, Radboud University, Geert Grooteplein
26-28, 6525 GA Nijmegen, Netherlands.
*Present address: Medical Research Council Laboratory of
Molecular Biology, Francis Crick Avenue, Cambridge, UK.†These
authors contributed equally to this work.‡Present address:
Institute of Physics and Astronomy, Karl-Liebknecht-Straße 24/25,
University of Potsdam, 14476 Potsdam, Germany. §Deceased
¶Corresponding author. Email: [email protected]