wtKelch13 and endogenous Kelch13C580Y)ren-
dered these resistant parasites fully sensitive
to dihydroartemisinin (DHA) again (Fig. 5E).
To test whether this was due to an additional
property held by wtKelch13 that is lacking
in Kelch13C580Y, we episomally expressed
Kelch13C580Yon the resistant background
(resulting in parasites expressing episomal
Kelch13C580Yand endogenous Kelch13C580Y,in
effect only raising the abundance of Kelch13C580Y).
This also reverted the parasites from resistant
to sensitive again (Fig. 5E), demonstrating
that reduced protein levels (or reduced activ-
ity) alone explains resistance, congruent with
the finding that partially inactivating Kelch13
has a similar effect (Fig. 4E). In contrast, Kelch13
missing thePlasmodium-specific region (PSR),
a modification that led to a loss of the correct
location of this construct (Fig. 5E), did not re-
vert the resistance phenotype when added on
top of thekelch13C580Ybackground (Fig. 5E).
This demonstrated the specificity of the effect
with episomal Kelch13C580Yand indicated that
the BTB and Kelch13 domain without the PSR
are not functional.
Overall, these results show that reduced ac-
tivity of Kelch13 is the most likely mechanism
of how Kelch13 mutation causes resistance
and that this reduced activity, at least in part,
is due to decreased Kelch13 levels in resistant
parasites. A generally reduced function also
explains why inactivation of several Kelch13
compartment proteins renders parasites resist-
ant, as each will reduce the output of a common
pathway that, based on our functional data, is
endocytosis.
Discussion
Here, we show that Kelch13 defines an endo-
cytosis pathway required for the uptake of
host cell hemoglobin and that this pathway
is critical for ART resistance. Although it is
well established that ARTs are activated by
the parasite’s digestion products of hemoglo-
bin and that this is a prerequisite for ART
action ( 37 – 39 ), this process was not consi-
dered to be involved in resistance in parasites
with Kelch13 mutations ( 15 , 16 ). Instead, po-
tential roles of Kelch13 downstream of ART
activation, e.g., in mitigating the ART-induced
cellular stress response, are the current target
of interest and central to hypotheses on the
mechanism of resistance ( 15 , 16 ). However, our
data indicate that Kelch13 and its compart-
ment proteins mediate resistance upstream of
both, drug activation and action. We propose
a model where Kelch13 and its compartment
proteins control endocytosis levels, thereby
influencing the amount of hemoglobin avail-
able for degradation and hence the concentra-
tion of active drug (Fig. 5F). This mechanism
explains the slowed development of ring stages
observed in ART-resistant parasites ( 9 , 44 ), as
the reduction in hemoglobin endocytosis in
rings diminishes the supply of amino acids.
This agrees with the finding that ART-resistant
parasites depend more on exogenous amino
acids to mature from rings to trophozoites than
wild-type parasites ( 45 ) and that removing func-
tions needed for amino acid access of the
parasite prolongs the ring stage ( 46 ). Reduced
levels of amino acids could also account for
some of the changes observed in ART-resistant
parasites, such as elevated cellular stress re-
sponses ( 9 , 15 ). It also indicates that the fitness
cost incurred by resistance-conferring Kelch13
mutations ( 47 , 48 ) is a direct result of the re-
sistance mechanism. Hence, there is a trade-
off between resistance and growth levels in
ring stages (Fig. 5F), creating a pressure for
compensatory adaptations that may explain
the importance of the genetic background in
parasite resistance and fitness ( 8 , 48 ).
Altered endocytosis as a mechanism of ART
resistance reconciles aspects of previous find-
ings relating to ART resistance, e.g., a role of
phosphatidylinositol 3-phosphate (and its ge-
nerating kinase) ( 12 ), a membrane signature
specific for endosomes ( 49 ) that is also pre-
sent on the endolysosomal system of malaria
parasites ( 36 , 50 ), or of coronin ( 51 )thatcould
influence endocytosis via actin. We also vali-
dated the suspected role of AP-2min resistance
( 30 , 31 ) and show that, as in other organisms
( 24 ),this protein is involved in endocytosis.
This indicates that Kelch13 marks a clathrin-
independent endocytosis pathway that un-
expectedly still contains the typical clathrin
adaptor AP-2m. Besides Eps15 and AP-2m,KIC4
shows homology to alpha adaptins [according
to HHPred ( 52 )], adding an additional protein
typical for endocytosis in a generally highly
derived pathway.
In contrast to its interactors, inactivation of
Kelch13 impaired endocytosis only in rings,
not in trophozoites. This likely is the reason
why Kelch13, among all the proteins influenc-
ing endocytosis, is the one frequently found
mutated in resistant parasite isolates ( 53 ). Com-
plete inactivation of Kelch13 arrests growth in
ring stages ( 17 ), and a similar phenotype was
here observed with KIC7 and UBP1. The stage-
specific effects of inactivating Kelch13 are
observed in a milder form in ART-resistant
parasites that display a prolonged ring stage
followed by an accelerated development in
later stages ( 9 , 44 ), and this observation was
here recapitulated by partial inactivation
of Kelch13. This indicates that resistance-
conferring mutations partially reduce Kelch13
function, a hypothesis supported by our find-
ing that partial inactivation of Kelch13 in rings
induces parasite resistance and that addi-
tional expression of Kelch13C580Yon a resistant
background with the same mutation is suffi-
cient to render parasites fully sensitive again.
As resistant parasite field isolates harbor less
Kelch13 ( 43 ), which we also found in our re-sistant laboratory line here, but transcript
levels appear to be unchanged ( 9 ), it can be
assumed that resistance-conferring mutations
alter Kelch13 protein stability.
We envisage that the mechanism of ART re-
sistance indicated by this work will aid in find-
ing ways to antagonize it. It may also inform
the choice of ART partner drugs, particularly as
hemoglobin digestive processes are the target
of existing drugs. Finally, the here-identified
proteins of the resistance pathway are candi-
dates for novel parasite markers influencing
ART resistance that now can be assessed in
population studies, as illustrated by the iden-
tification of a resistance-conferring mutation
in UBP1. It should, however, be noted that
resistance-causing mutations are most likely
to occur in proteins that have the least impact
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