Critical role of a K؉ channel in berghei transmission revealed by targeted 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; ‡ 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 by been identified in the 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 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 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 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 have been found in the P. falciparum either side of the 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 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 bovis [Protein Data Bank (PDB) ID code XP࿝001610013], hominis (PDB ID code XP࿝668687), and 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 mice, and the mice highly negative membrane potential likely facilitates a net Kϩ were treated with pyrimethamine to select for recombinant uptake into the parasites (15), and we hypothesized that PbKch1, parasites, which were further cloned by limiting dilution. at least in part, mediates this Kϩ uptake. To determine whether the introduced plasmid had integrated To investigate whether disruption of PbKch1 affected Kϩ into the parasite genome, genomic DNA from the WT parasite transport, we compared the uptake of the Kϩ congener 86Rbϩ in and the cloned transfectant parasite were analyzed by PCR using PbKch1-null parasites with the uptake in WT parasites. 86Rbϩ integration-specific primers. Primers targeting the undisrupted added to a suspension of Plasmodium-infected red blood cells ORF produced bands with WT DNA, but no bands were visible enters the cytoplasm via the parasite-induced, with DNA from the PbKch1-null parasites (Fig. 2B). On the broad-specificity, new permeation pathways (16), and subse- other hand, primer pairs that targeted the DHFR ORF and quently accumulates in the parasites because of their high either the 5Ј or the 3Ј flanking regions produced bands with negative membrane potential (15, 17). In this study, infected DNA from the PbKch1-null parasites, but not from WT para- erythrocytes were treated with saponin to allow functional sites. These findings confirmed replacement of the targeted access to the parasite’s plasma membrane (18). The saponin- sequence of the PbKch1 ORF by insertion of the DHFR ORF isolated parasites were incubated with 86Rbϩ, and the intracel- in PbKch1-null parasites DNA. Lack of expression of PbchK1 in lular accumulation was followed for up to 30 min. The uptake the null parasites was also confirmed by RT-PCR using purified data were fitted to a one-phase exponential association, and our RNA from WT and PbKch1-null parasites (Fig. 2C). results show that the uptake in PbKch1-null parasites was significantly decreased compared with the uptake in WT para- ؉ ϩ 1 K Uptake Kinetics in WT and PbKch1-Null Parasites. In most eu- sites (Fig. 3A). K -uptake rates for the initial 1 ⁄2 min were karyotic organisms, Kϩ channels contribute to the maintenance calculated from 86Rbϩ uptake, and for WT parasites it was 5.5 Ϯ of the resting membrane potential. In Plasmodium parasites, the 1.2 ϫ 10Ϫ9 mol Kϩ/(109 cell ϫ min) [95% confidence interval membrane potential is probably created through the action of (CI): 4.3–6.7 ϫ 10Ϫ9]. This value is lower than an earlier reported the plasma membrane proton pump, which extrudes protons Kϩ-uptake rate for intact P. falciparum-infected erythrocytes MEDICAL SCIENCES

Ellekvist et al. PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 ͉ 6399 Downloaded by guest on September 24, 2021 PB_RP3798 554-3613 WT 50000 #2 A A #1

5’ 5’ Core 3’ 3’ 40000 1548-2032 3149-3602 cells

9 30000 PB_RP3798 554-3613 Disrupted

#3 #4 20000 5’ 5’ Tg -DHFR 3’ 3’ cpm per 10

1548-20324615 bp 3149-3602 10000 B 0 0 5 10 15 20 25 30 35 WT PbKch1-null t (min) St 1 2 3 4 5 6 7 8 St B 25000

2323 20000 1929

1371 cells 9 1264 15000

702 10000

cpm per 10 * * * * 5000 * *

0 l + l + o 2 e 2 tr EA in a EA T in B T inine T T Ba u contro l+ W W ll + l Qu C ll u u + WT PbKch1-null WT con u WT Q 1-n ull ch1-n n ch1-n Kch K K Pb Pb Pb Kch1- St 1 2 3 4 5 6 St b P

Fig. 3. Characterization of 86Rbϩ transport in the PbKch1-null parasite. (A) Uptake of 86Rbϩ in isolated P. berghei parasites. Erythrocytes from mice infected 2323 1929 with WT or PbKch1-null parasites were treated with saponin, thereby allowing 1371 functional access to the parasite’s plasma membrane. The saponin-treated par- 1264 asites were incubated with trace amounts of 86Rbϩ, and samples were taken at 702 the indicated time points. PbKch1-null parasites (triangles) showed decreased uptake compared to WT parasites (squares). Each point represents mean value Ϯ SEM (n ϭ 3). (B) Pharmacological characterization of 86Rbϩ uptake in isolated parasites. Uptake was assessed at a fixed time point (t ϭ 10 min) in the presence of either suspending buffer alone (control), 10 mM Ba2ϩ, 20 mM TEA, or 0.5 mM quinine. The height of each bar represents mean value Ϯ SEM (n ϭ 4). When ϩ ϩ Fig. 2. Disruption strategy for generation of PbKch1-null parasites. (A) denoted by *, 86Rb uptake was statistically different from 86Rb uptake in (Upper) The genomic locus. (Lower) The disrupted gene. The black line untreated WT parasites (P Ͻ 0.05, unpaired t test). represents the locus (PDB ID code PB࿝RP3798) containing the gene encod- ing PbKch1 (bar). The deduced six transmembrane domains (indicated by the small interconnected bars below the gene) of PbKch1 are encoded by (16). The difference is most likely because the values reported Ј Ј the core region. The 5 and 3 regions are the recombination sites used to here were determined for a mixed population of parasites, replace, by double crossover, the core region of the gene with the Tg-DHFR ORF (gray color). The Tg-DHFR confers resistance to the compound py- whereas the values reported in ref. 16 were assessed for late- stage parasites only. Erythrocytes infected with late-stage par- rimethamine, which can be used to select PbKch1-null parasites. (B) Vali- ϩ dation of the PbKch1-null genotype. Gene-specific primers amplify differ- asites are known to have a higher K -uptake rate than eryth- ϩ ent PCR products from DNA purified from WT P. berghei and PbKch1-null rocytes infected with early-stage parasites (16). The K -uptake Ϫ ϩ P. berghei. Fragments 1 and 2 (same as in A) can be amplified only from WT rate for PbKch1-null parasites was 1.6 Ϯ 2.5 ϫ 10 9 mol K /(109 parasites, not from PbKch1-null parasites. The primers used to generate cell ϫ min) (95% CI: 0–4.1 ϫ 10Ϫ9), three to four times lower fragments 3 and 4 (same as in A) target the Tg-DHFR ORF from outside the than for WT parasites. Theoretically, a lower uptake rate in recombination sites. These fragments can be amplified only from PbKch1- PbKch1-null parasites could be caused by a maturation arrest in null parasites, not from WT parasites, nor from parasites containing circular those parasites, resulting in early immature stage parasites with (nongenomic) copies of the targeting plasmid. Lanes 1–4: PCR products amplified from WT parasite DNA. The presence of fragments 1 (1,022 bp) low uptake rates only. However, successful production of and 2 (667 bp), but not 3 and 4, confirms the undisrupted genotype. Lanes PbKch1-null parasites clearly demonstrates that they developed 5–8: PCR products amplified from PbKch1-null parasite DNA. The presence as erythrocytic asexual and sexual stages (see below) similar to of fragments 3 (968 bp) and 4 (625 bp), but not 1 and 2, confirms that the those in the WT parasites. ϩ core region has been correctly replaced by the Tg-DHFR ORF, and that there The pharmacological profile of the parasites 86Rb uptake was is no contamination from WT parasite DNA. Standard (St) lanes contain further investigated with Kϩ channel blockers. Isolated parasites BstEII-digested Escherichia coli ␭-phage DNA with base-pair length of were incubated in the presence of Kϩ channel blockers before selected markers shown to the right. (C) RT-PCR with RNA from WT para- sites (lanes 1–3) and PbKch1-null parasites (lanes 4–6). Primer pairs were the same as those used above to assess the presence of the uninterrupted PbKch1 gene and the interrupted gene where the core region of PbKch1 not from WT RNA (lane 2). PCR amplification not preceded by the reverse- has been replaced by the Tg-DHFR ORF. Fragment 2 (667 bp) could be transcriptase step gave no bands from WT RNA (lane 3) or PbKch1-null RNA amplified from WT RNA (lane 1), but not from PbKch1-null RNA (lane 4). (lane 6), which confirms the absence of genomic DNA contamination in the Fragment 3 (968 bp) could be amplified from PbKch1-null RNA (lane 5), but RNA preparations. Standard (St) marker is as mentioned in B.

6400 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802384105 Ellekvist et al. Downloaded by guest on September 24, 2021 86 ϩ the addition of Rb , and parasite samples were analyzed at a 60 ϩ A fixed time point (t ϭ 10 min). Prior treatment with 10 mM Ba2 or 0.5 mM quinine resulted in Ϸ80% inhibition of uptake of 50 86 ϩ Rb in WT parasites as compared with parasites pretreated 40 with solvents only (control) (Fig. 3B). When tested in parallel, PbKch1-null parasites revealed 86Rbϩ uptake to the level de- 30 tected as residual uptake in WT parasites upon treatment with ϩ 20 Ba2 or quinine. Furthermore, this already reduced uptake of (%) Parasitaemia ϩ 86Rb in PbKch1-null parasites was insensitive to further inhi- 10 bition by Ba2ϩ and quinine. Taken together, these results ϩ 0 strongly suggest that PbKch1 is the predominant K channel in 0 2 4 6 8 10 12 14 16 18 20 the erythrocytic stages of P. berghei parasites. The residual Days post-infection uptake seen in WT parasites in the presence of Ba2ϩ or quinine, and in the PbKch1-null parasites, is most likely mediated by other B 100 membrane transporters, e.g., cotransporters, present in the plasma membrane of the parasites. In addition, another putative 75 Kϩ channel has been identified in P. falciparum (11), and this channel is highly conserved among members of the Plasmodium 50 genus (unpublished observation). It cannot, a priori, be excluded that this channel could mediate Ba2ϩ- and quinine-insensitive Survival (%) 86Rbϩ fluxes. The nonspecific Kϩ channel blocker tetraethyl- 25 ammonium (TEA; 20 mM) had a small and nonsignificant ϩ inhibitory effect on 86Rb uptake in WT parasites, suggesting 0 0 2 4 6 8 10 12 14 16 18 20 22 that PbKch1 is insensitive to TEA. In a recent study (19), it has Days post-infection been shown that exposure of P. berghei sporozoites to intracel- lular concentrations of Kϩ enhances their infectivity by 8–10 Fig. 4. Phenotype characterization of the PbKch1-null parasite. (A) Growth times. This effect of a high Kϩ concentration is significantly kinetics of the PbKch1-null parasite. Parasitemia of mice infected with WT ϩ parasites (squares) or PbKch1-null parasites (triangles) is shown. Each point attenuated by K channel blockers, including TEA. The latter represents mean value Ϯ SEM. (B) Survival of mice infected with WT parasites observation is not consistent with the lack of TEA inhibition (stippled curve) or PbKch1-null parasites (continuous curve). Median survival found in the present study, and it cannot be excluded that the time was 15 days for mice infected with WT parasites and 21 days for mice effect of TEA reported in ref. 19 can be caused by other actions infected with PbKch1-null parasites (P Ͻ 0.001, ␹2 test). of TEA than Kϩ channel blockage.

Comparison of Blood-Stage Parasite Growth Kinetics and Virulence. mature oocysts. Because of the deficient oocyst development we Next, we investigated in vivo asexual growth kinetics of PbKch1- were not able to generate sporozoites from PbKch1-null para- null and WT parasites. Naı¨ve mice were infected with an equal sites and directly evaluate liver cell infection by PbKch1-null inoculum of WT and PbKch1-null parasites, and parasitaemia sporozoites. Others have shown that infectivity of Plasmodium was determined daily. Both groups of mice rapidly progressed in sporozoites can be attenuated by Kϩ channel blockers (19), and parasitaemia from day 4. In contrast to WT-infected mice, we suggest that this effect is most likely caused by the inhibition however, parasitaemia in PbKch1-null-infected mice leveled off of PbKch1. at a plateau 9 days after infection, before approaching the parasitaemia of WT-infected mice at day 14 (Fig. 4A). This delay Conclusion in parasitaemia was not statistically significant, but it may Data in the present article show that PbKch1 functions as a Kϩ provide an explanation for the slightly prolonged survival of channel and it mediates Kϩ transport in the intraerythrocytic PbKch1-null-infected mice compared with WT-infected mice stages of P. berghei parasites. The function of PbKch1 was not (Fig. 4B). In addition to the asexual parasite stages, normally critical for asexual replication of P. berghei. However, the appearing gametocytes were observed in the blood taken from dependency of sexual replication in mosquitoes on PbKch1 both groups of mice (data not shown). implies that PfKch1 may be used as a potential drug target in P. falciparum parasites. Evaluation of Malaria Transmission to Anopheles Mosquitoes. In our quest for functional significance of PbKch1, we extended our analysis to evaluate transmission of malaria parasites from 350 infected vertebrate hosts to anopheline mosquito vector. Adult mosquitoes were fed on PbKch1-null- or WT-infected mice, and 300 10–12 days after feeding the number of oocysts per mosquito 250 midgut was assessed (Fig. 5). Mosquitoes infected (81 of 89 dissected) with WT parasites revealed a median oocyst burden 200 of 115 per mosquito (25 and 75 percentiles, oocyst numbers 37 150 and 251). In sharp contrast, only two mosquitoes of 112 fed on PbKch1-null-infected mice had oocysts in their midguts, thus 100 50 demonstrating a 98% reduction in the infectivity of PbKch1-null Oocysts midgut per mosquito parasites during transmission through mosquitoes. Approxi- 0 mately 10 mosquitoes from each group were dissected 24 h after WT PbKch1-null the blood meal, and the presence of ookinetes was validated by Fig. 5. Transmission results of the WT and PbKch1-null parasite. Mosquitoes light microscopy (data not shown). By this semiquantitative were fed on PbKch1-null infected mice or WT-infetced mice. Of 89 mosquitoes method, no gross difference in ookinete count could be detected fed on WT-infected mice (n ϭ 3), 81 contained oocysts (115 median oocyst per between mosquitoes fed on WT- and PbKch1-null-infected mice, mosquito). Of 112 mosquitoes fed on PbKch1-null infected mice (n ϭ 3) only MEDICAL SCIENCES thus pointing to a compromised maturation of ookinetes into two contained oocysts (1 and 22 oocysts, respectively).

Ellekvist et al. PNAS ͉ April 29, 2008 ͉ vol. 105 ͉ no. 17 ͉ 6401 Downloaded by guest on September 24, 2021 Materials and Methods plasma membrane intact (18, 20). Saponin-treated infected red blood cells are Identification and Cloning of PbKch1. The P. berghei orthologue of PfKch1 referred to as isolated parasites hereafter. Infected red blood cells were treated with 0.05% saponin in RPMI medium (GenBank accession no. NP࿝701625), PbKch1 (GenBank accession no. 1640 and immediately spun at 2,000 ϫ g for 5 min. The remaining parasite XP࿝676033), was identified by BLAST search of the P. berghei genome data- pellet was washed twice at room temperature in RPMI medium 1640, thereby base (www.sanger.ac.uk). The alignment was made with the Dialign algo- removing hemoglobin and most of the red cell ghosts. Parasites were resus- rithm (http://bioweb.pasteur.fr/seqanal/interfaces/dialign2-simple.html). pended in RPMI medium1640 and placed in a water bath at 37°C. Because of the insolubility of BaSO4, Hepes-buffered saline (125 mM NaCl, 25 mM Hepes, Targeted Disruption of the PbKch1 Gene. Plasmid pB3DPbKch1 was constructed 10 mM glucose, 5.4 mM KCl, 0.4 mM MgCl2, pH 7.4) was used in the pharma- by cloning fragments from the 5Ј and 3Ј flanking regions of the deduced six cological studies, which included the presence of Ba2ϩ. Cell number was transmembrane domains-encoding part of the PbKch1 gene. The 5Ј fragment counted in a Neubauer chamber (cell concentration 0.7–4.5 ϫ 108 cells per ml). was PCR-amplified from genomic DNA of P. berghei by using the forward Microscopy revealed free parasites of all stages, and, only occasionally, eryth- primer B1.L.1548–66 (5Ј-GGTACCGTAAGAAAGGCAATCAACC-3Ј) and the re- rocyte ghosts. Trace amounts of 86RbCl was added (0.8–2 ϫ 105 Bq/ml, specific verse primer B1.R.2032–11 (5Ј-AAGCTTGTTATCTGTTTTTCTTTTATCG-3Ј). The activity 2.497 ϫ 1015 Bq/mol), and at various time points (0–30 min), 200-␮l 3Ј fragment was PCR-amplified from genomic DNA of P. berghei by using the samples were spun (16,000 ϫ g for 20 s) through 0.25-ml oil cushions, consist- forward primer B1.L.3149–70 (GGATCCGAATCCATATTTTTATTTACCC-3Ј) and ing of a 5:4 mixture of dibutyl/dioctyl phthalate, overlaid with 0.8 ml of the reverse primer B1.R.3602–3585 (GCGGCCGCTTGATCATCCTTTTCCC-3Ј). isotonic saline. The cell pellets were lysed with 0.1% Triton X-100, and the The fragments contained sites for KpnI and HindIII at the 5Ј and 3Ј ends of the protein was precipitated with 5% trichloroacetic acid. After centrifugation ϫ 5Ј flanking fragment and sites for BamHI and NotI at the 5Ј and 3Ј ends of the (10,000 g for 10 min), supernatants were transferred to plastic vials and ␤ ϩ 3Ј flanking fragment. These sites were used for cloning of the fragments into counted in a -scintillation counter. K -uptake rates were calculated from 86 ϩ the pB3D vector (kindly provided by Andrew Waters, University of Glasgow, Rb uptake by using the specific activity of the isotope, the calculated ratio [86Rbϩ]/[Kϩ] in the uptake buffer (Ϸ8.3 ϫ 10Ϫ4), and the counting efficiency of Scotland) on either side of the Toxoplasma gondii dihydrofolate reductase the ␤-scintillation counter (0.30 cpm/dpm). (Tg-DHFR) cassette. Before transfection, pB3DPbKch1 was linearized with three restriction enzymes (KpnI, NotI, and ScaI). Schizont-stage parasites of P. Asexual Growth Kinetics and Virulence of PbKch1-Null Parasites. To detect berghei 2.34 ANKA strain were transfected by electroporation with Ϸ25 ␮gof differences in parasite growth during blood-stage development between the linearized targeting plasmid by using the Amaxa Nucleofactor device, pro- PbKch1-null parasites and WT parasites, 4-week-old female Swiss–Webster gram T-001 (13). Transfected parasites were immediately injected into female mice were infected i.p. with either 105 PbKch1-null parasites or WT parasites. Swiss–Webster mice (4 weeks old), and the mice were treated with py- Parasite growth, monitored by Giemsa-stained blood smears, and survival of rimethamine in the drinking water (70 mg/liter) starting 24 h after transfec- the mice, was assessed daily. tion. Pyrimethamine-resistant parasite populations were detected 7 days after infection in mice. Integration of the plasmid into the PbKch1 locus was Mosquito Transmission Differences. Starved adult (5–6 days old) A. stephensi confirmed by PCR analysis. Parasite clones were obtained by the method of mosquitoes were allowed to feed on mice infected for 4 days with equal limiting dilution. Expression of the uninterrupted PbKch1 gene in WT para- numbers of WT or PbKch1-null parasites (three mice in each group). Blood-fed sites and lack of expression in PbKch1-null parasites was assessed by RT-PCR mosquitoes were then maintained at 20°C in insectary chambers at 70–80% using poly(A)-selected, DNase-treated RNA from WT parasites and PbKch1- relative humidity. Ten to 12 days after blood feeding, mosquito midguts were null parasites. As a control for genomic DNA contamination in the RNA dissected and oocyst numbers were enumerated after staining with 0.1% preparations, PCR amplification without the reverse-transcriptase step was mercurochrome. performed in parallel. ACKNOWLEDGMENTS. Research in P.E.’s and D.A.K.’s laboratories is sup- 86Rb؉ Transport in PbKch1-Null P. berghei Parasites. The alkali ion 86Rbϩ was ported by grants from Danish International Development Assistance/Danish used as a tracer for Kϩ in an uptake protocol. To detect differences in 86Rbϩ Ministry of Foreign Affairs, the A. P. Møller Foundation, the Danish Medical transport between the PbKch1-null parasites and WT parasites, parasites were School Foundation, ‘‘Apotekerfonden,’’ ‘‘Fonden af 17-12-1981,’’ the Novo Nordic Foundation, the Lundbeck Foundation, and the Danish Research Coun- isolated from their red blood cells by saponin treatment. Saponin is a plant cil for Natural Sciences. Research in N.K.’s laboratory is supported by National detergent that permeabilizes the cholesterol-containing red blood cell mem- Institutes of Health Grant AI46760 and the Johns Hopkins Malaria Research brane and parasitophorous vacuole membrane, but leaves the parasite’s Institute.

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