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Critical Role of a K Channel in Plasmodium Berghei Transmission

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Critical Role of a K Channel in Plasmodium Berghei Transmission Critical role of a K؉ channel in Plasmodium berghei transmission revealed by targeted gene disruption Peter Ellekvist*†, Jorge Maciel‡, Godfree Mlambo‡, Christina H. Ricke*, Hanne Colding§, Dan A. Klaerke¶, and Nirbhay Kumar†‡ *Department of Cellular and Molecular Medicine, Faculty of Health Sciences, and §Department of International Health, Immunology, and Microbiology, University of Copenhagen, DK-2200 Copenhagen, Denmark; ‡Malaria Research Institute, Molecular Microbiology and Immunology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205; and ¶Department of Physiology and Biochemistry, IBHV, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg, Denmark Communicated by Peter C. Agre, Duke University, Durham, NC, March 12, 2008 (received for review January 8, 2008) Regulated K؉ transport across the plasma membrane is of vital function in malaria parasites. We identified the orthologue of the importance for the survival of most cells. Two K؉ channels have P. falciparum Kϩ channel PfKch1 in the P. berghei genome by been identified in the Plasmodium falciparum genome; however, BLAST analysis, and we generated PbKch1-null parasites. Phys- their functional significance during parasite life cycle in the verte- iological and functional studies with these null parasites suggest brate host and during transmission through the mosquito vector that PbKch1 not only mediates Kϩ uptake in the erythrocytic remains unknown. We hypothesize that these two K؉ channels stages of the parasite but also is critical for the development of mediate the transport of K؉ in the parasites, and thus are impor- the mosquito midgut oocyst stage of the parasite, thus directly tant for parasite survival. To test this hypothesis, we identified implicating its functional significance during the malaria trans- .the orthologue of one of the P. falciparum K؉ channels, PfKch1, in mission process the rodent malaria parasite P. berghei (PbKch1) and examined the biological role by performing a targeted disruption of the gene Results and Discussion encoding PbKch1. The deduced amino acid sequence of the six Identification of the Orthologue of PfKch1 in P. berghei. PbKch1, the transmembrane domains of PfKch1 and PbKch1 share 82% identity, P. berghei orthologue of PfKch1, was identified by BLAST search and in particular the pore regions are completely identical. The of the P. berghei genome database (www.plasmodb.org). The PbKch1-null parasites were viable despite a marked reduction in amino acid identity was 82% between the putative six trans- the uptake of the K؉ congener 86Rb؉, and mice infected with membrane domains of the two orthologues (90% similarity), and PbKch1-null parasites survived slightly longer than mice infected in particular the pore loops were completely identical (amino with WT parasites. However, the most striking feature of the acid sequence N-DFVYFGVITMSTVGYGDYTP-C) (Fig. 1A). phenotype was the virtually complete inhibition of the develop- Kϩ channel proteins with identical pore-loop sequences were ment of PbKch1-null parasites in Anopheles stephensi mosquitoes. identified by BLAST search in several other Plasmodium species, In conclusion, these studies demonstrate that PbKch1 contributes and they all shared Ϸ90% amino acid sequence similarity with to the transport of K؉ in P. berghei parasites and supports the respect to the putative six transmembrane domains of PfKch1 growth of the parasites, in particular the development of oocysts (data not shown). There were no close homologues to PfKch1 in the mosquito midgut. K؉ channels therefore may constitute a outside of the Plasmodium genus, which indicates that this potential antimalarial drug target. particular Kϩ channel is unique to Plasmodium species. Many Kϩ channel blockers bind in or near the outer pore malaria ͉ pathogenesis ͉ mosquito ͉ drug target mouth, which is mainly contributed by the pore loop. The amino acid composition of the pore loop, therefore, constitutes a major ϩ he mosquito-borne parasite Plasmodium falciparum is the molecular determinant for pharmacological K channel inhibi- tion. The pore loop of PfKch1 shared 70–80% amino acid Tcausative agent of the deadliest form of malaria, claiming an ϩ estimated 1 million to 2 million deaths annually. The spread of sequence identity with pore loops of putative K channels from resistance to almost all of the currently available antimalarial other primitive eukaryotic organisms (Fig. 1B). The pore loops ϩ Ͻ drugs necessitates the development of new drugs. Putative drug of different human K channels all shared 60% sequence targets in P. falciparum are the parasite-encoded transport identity with the pore loop of PfKch1, which suggests that it may be possible to identify specific pharmacological inhibitors of the proteins, which mediate the uptake of nutrients and disposal of ϩ waste products across the parasite’s plasma membrane (1). Plasmodium K channel 1 homologue. Several of the parasite’s transport proteins have been cloned and functionally characterized (2–6), and some of them may be Targeted Disruption of the PbKch1 Gene. To study the biological potential drug targets (7–9). role of PbKch1, we generated P. berghei parasites in which the Kϩ channels constitute the largest and most diverse of ion PbKch1 locus was disrupted by homologous recombination (Fig. Ј Ј channel families and are involved in Kϩ transport, cell volume 2A). Two fragments from the 5 and 3 flanking regions of the control, and regulation of membrane potential. Two putative Kϩ putative six transmembrane domains of PbKch1 were cloned on channel-encoding genes have been found in the P. falciparum either side of the Toxoplasma gondii dihydrofolate reductase genome (10–11), but it remains to be established whether they (TgDHFR) cassette in the vector pB3D. The resultant plasmid are functional and whether they are important for parasite (pB3DPbKch1) was introduced into mature schizonts of P. survival. So far attempts to express and functionally characterize Plasmodium Kϩ channels in heterologous cell systems have been Author contributions: P.E., J.M., G.M., D.A.K., and N.K. designed research; P.E., J.M., G.M., unsuccessful. Targeted gene disruption by homologous recom- C.H.R., and H.C. performed research; P.E., J.M., G.M., D.A.K., and N.K. analyzed data; bination has provided a valuable approach for functional char- and P.E., D.A.K., and N.K. wrote the paper. acterization of gene products in the Plasmodium genus. In The authors declare no conflict of interest. contrast to P. falciparum, the Plasmodium berghei genome can be †To whom correspondence may be addressed. E-mail: ellekvist@mfi.ku.dk or nkumar@ manipulated with relative ease (12), and in that regard P. berghei jhsph.edu. serves as an excellent model organism for the study of gene © 2008 by The National Academy of Sciences of the USA 6398–6402 ͉ PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802384105 Downloaded by guest on September 24, 2021 A B Pore loop B. bovis FSGMMYLLE-SPRSDADFHTP-----FDFIYFGVATMGTVGYGDFTPRTFMGRLMSILLICTCISLG C. hominis FSGAMMIIE-SPK--PNFTTL-----FDYFYFTIITISTVGYGDYTPSNFVSRLICIILIIFTIIYV T. annulata FSGLLFLLE-APKNDINFSKP-----FDFIYFSVATMATVGYGDFSPVTLAGRILCVLFIVLCVTIA PfKch1 FSGIMYILE-APDIERDFVKP-----LDFVYFGVITMSTVGYGDYTPVTKAGKFLTMFIIITCISFV PbKch1 FSGIMYILE-APDIERQFISP-----LDFVYFGVITMSTVGYGDYTPVTPAGKCLTMFIIVTCFTFV nBK GAGLVHLLENSGDFFKGFINPHRITYADSVYFVLVTMSTVGYGDIYCTTLCGRLFMIFFILFGLAMF hBK AAGFIHLVENSGDPWENFQNNQALTYWECVYLLMVTMSTVGYGDVYAKTTLGRLFMVFFILGGLAMF hShaker LFSSAVYFAEADDPTSGFSSIP-----DAFWWAVVTMTTVGYGDMHPVTIGGKIVGSLCAIAGVLTI hKCNQ1 FSSFLVYLV-EKDANKEFSTY-----ADALWWGTITLTTIGYGDKTPLTWLGRLLSAGFALLGISFF Fig. 1. Sequence analysis of the Plasmodium Kϩ channels. (A) Alignment of the P. berghei putative Kϩ channel PbKch1 and the orthologue P. falciparum Kϩ channel PfKch1. The ORFs of PbKch1 and PfKch1 encode 986 and 1,940 aa, respectively. Identical or similar amino acids are colored red. The channels belong to the superfamily of six transmembrane-segment Kϩ channels. Except for the S4 segment, the membrane topology of the channel was predicted by the TMHMM algorithm. The S4 segment of six transmembrane-segment Kϩ channels is notoriously difficult to predict because of a number of charged residues, usually arginines, and this particular segment was therefore identified by eye. The amino acid identity in the parts of the proteins comprising the six transmembrane domains (indicated by gray boxes) is 82%. The amino acids of the pore regions are identical. (B) Alignment of the S5–S6 linker of PfKch1 and PbKch1 with corresponding linkers from different hypothetical and known Kϩ channels. The pore loop contains the canonical Kϩ channel signature sequence ((T/S)XXTXGYG). B. bovis, C. hominis, and T. annulata: Hypothetical proteins from Babesia bovis [Protein Data Bank (PDB) ID code XP࿝001610013], Cryptosporidium hominis (PDB ID code XP࿝668687), and Theileria annulata (PDB ID code XP࿝952459). nBK, Caenorhabditis elegans BK channel (PDB ID code NP࿝001024261); hBK, human BK channel (PDB ID code NP࿝001014797); hShaker, human voltage-gated shaker-related Kϩ channel (PDB ID code NP࿝002223); hKCNQ1, human cardiac KCNQ channel (PDB ID code CAO03369). Amino acids in the Kϩ channel signature sequence are highlighted in bold; amino acid identity with PfKch1 is colored gray. berghei by the Amaxa electroporation procedure. Transfected from the parasite’s cytoplasm (13, 14). The thereby generated parasites were immediately introduced into
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