Transgenic fluorescent Plasmodium cynomolgi liver stages enable live imaging and purification of Malaria hypnozoite-forms.
A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.
Biomedical Primate Research Centre, Department of Parasitology, Rijswijk, The Netherlands.
Annemarie
A
Voorberg-van der Wel
Anne-Marie
AM
Zeeman
Sandra M
SM
van Amsterdam
Alexander
A
van den Berg
Els J
EJ
Klooster
Shiroh
S
Iwanaga
Chris J
CJ
Janse
Geert-Jan
GJ
van Gemert
Robert
R
Sauerwein
Niels
N
Beenhakker
Gerrit
G
Koopman
Alan W
AW
Thomas
Clemens H M
CH
Kocken
D000818:Animals; D030801:Animals, Genetically Modified; D000962:Antimalarials / Q000494:pharmacology*; D053626:Atovaquone / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D008099:Liver / Q000469:parasitology*; D016789:Plasmodium cynomolgi / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D011319:Primaquine / Q000494:pharmacology
D000962:Antimalarials; D011319:Primaquine; D053626:Atovaquone