Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity

Molecular & Biochemical Parasitology 156 (2007) 32 40 Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity Kota Arun Kumar a,, Celia R.S. Garcia a,b, Vandana R. Chandran c, N. Van Rooijen d, Yingyao Zhou e, Elizabeth Winzeler c,e, Victor Nussenzweig a a Michael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, New York, NY 10016, United States b Department of Physiology, Institute of Biosciences, University of Sao Paulo, Sao Paulo, Brazil c Department of Cell Biology, ICND202, The Scripps Research Institute, La Jolla, CA 92037, United States d Department of Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands e Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, United States Received 3 June 2007; received in revised form 9 July 2007; accepted 10 July 2007 Available online 13 July 2007 Abstract Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K + concentration. We report here that incubation of sporozoites to the intracellular K + concentration enhances 8 10 times the infectivity of Plasmodium berghei and 4 5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K + enhancing effect was time and concentration dependent, and was significantly decreased by K + channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K + treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K + treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants. 2007 Elsevier B.V. All rights reserved. Keywords: Potassium shifts; Plasmodium; Sporozoites; Cell invasion 1. Introduction Plasmodium sporozoites traverse the cytosol of cells prior to transformation into exoerythrocytic (EEF) stages [1]. The ability of sporozoites to traverse host cells is required for the completion of the Plasmodium life cycle. When infected mosquitoes feed on the mammalian hosts, the sporozoites are deposited in a pool of blood formed by the rupture of skin capillaries [2].As shown by real time studies, the parasites pass through skin cells prior to entering the blood or lymph [2,3]. Once in the blood Corresponding author. Tel.: +1 212 263 5346; fax: +1 212 263 8179. E-mail address: arun.kumar@med.nyu.edu (K.A. Kumar). circulation, sporozoites are retained in the liver sinusoids by the interaction of the circumsporozoite protein (CS) with heparan sulfate proteoglycans (HSPGs) [4,5]. Then the parasites glide onto the endothelium, and traverse Kupffer cells [6], to enter the liver parenchyma. Only after navigating rapidly through the cytoplasm of several hepatocytes [7], they turn on a program for a productive invasion that leads to the formation of a parasitophorous vacuole (PV) whereupon sporozoites transform into EEFs. The sporozoite activation is associated with the exocytosis of microneme products including TRAP (thrombospondin related anonymous protein), [8] an essential component of the molecular motor that drives the active invasion process [9,10]. While sporozoites enter and exit the cytoplasm of host cells, the parasites are exposed to profound changes in K + concentration. 0166-6851/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.07.004

K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 33 Here we explore the possibility that these shifts in [K + ] trigger the profound phenotypic changes that sporozoites undergo when they travel from the skin to the liver. 2. Materials and methods 2.1. Sporozoites P. berghei and P. yoelii cycles were maintained by allowing Anopheles stephensi mosquitoes to feed on SW mice (Jackson Laboratory) that had been infected with sporozoites. Midgut infection level was assessed on day 12 for P. berghei and day 8 for P. yoelii by checking for the presence of the midgut oocyst stages. The infected salivary glands were dissected on days 18 20 post blood meal for P. berghei and on days 14 16 for P. yoelli in DMEM (Gibco) containing 2 mm l-glutamine and 4.5 g/l glucose supplemented with 10% FCS (HyClone Laboratories), 3 antibiotic and antimycotic (Gibco). The glands were disrupted, and the sporozoites were isolated and counted in a hemocytometer. 2.2. Cell cultures All the cell lines were obtained from American Type Culture Collection (ATCC). HepG2-A16 cells, Hepa 1-6 cells, Hela, COS and MDCK cells were seeded at a density of 1 10 5 cells/well in eight well chamber slide (Labtek). The cells were maintained in control medium. Cells were seeded 36 h prior to the addition of sporozoites. 2.3. Media Control medium: DMEM (Gibco) containing 2 mm l- glutamine and 4.5 g/l glucose supplemented with 10% FCS (HyClone Laboratories), 1 antibiotic and antimycotic (Gibco). Intracellular medium: 5 mm NaCl, 142 mm KCl, 2 mm EGTA, 1 mm MgCl 2, 5.6 mm glucose, 25 mm HEPES-KOH, ph 7.2 [23]. The intracellular medium was prepared as 10 stock, diluted to 1 after supplementing with 10% FCS. Intracellular control medium was identical to the intracellular medium except the 142 mm KCl was substituted for 142 mm NaCl. 2.4. Infectivity assays Salivary gland sporozoites were washed and 2 10 4 sporozoites were incubated in 20 l of following media: control medium, intracellular control medium, [142 mm] K + medium containing 2 mm EGTA, [142 mm] K + medium without EGTA or control media containing 10 g/ml cytochalasin-d for 35 min at room temperature. The parasites were then added to hepatoma cells maintained in 450 l of control medium. For evaluating the in vivo infectivity, the treated sporozoites were injected intravenously into C57BL/6 mice. Two K + channel blockers, TEA and quinine were used to demonstrate the specific effect of K + on sporozoite infectivity. 2 10 4 sporozoites were incubated in 20 l of control medium that contained either 20 mm TEA or 0.5 mm quinine for 30 min at room temperature. Following this treatment, sporozoites were washed at 6800 g for 5 min at 4 C and then exposed to [142 mm] K + in the continued presence of channel blockers. Control sporozoites were only exposed to [142 mm] K +. The sporozoites were then added to HepG2 cells and infectivity was evaluated at 60 h by qrt PCR. 2.5. Hepatocyte invasion assay The double staining test to differentiate sporozoites outside versus inside host cell (hepatocyte entry) was performed as published earlier [31]. 2 10 4 P. berghei or P. yoelli sporozoites were exposed to different media as described above for 35 min at room temperature and added to confluent HepG2 or Hepa 1-6 cells maintained in 450 l control medium. After 60 min incubation at 37 C, cells were fixed with 4% paraformaldehyde, blocked with 1% BSA in PBS and stained with anti-cs 3D11 Ab (primary Ab) followed by Alexa Fluor 594 (red color) goat anti-mouse IgG (Molecular Probes) as secondary Ab. After washing with PBS cell membranes were permeabilised with 100% methanol at room temperature. The cells were again blocked and stained with anti-cs 3D11 Ab followed by Alexa Flour 488 (green color) goat anti-mouse IgG as secondary Ab. Sporozoites were counted under a fluorescent microscope. 2.6. Immunofluorescence assays Immnofluorescense assays were done on infected cell cultures after fixation with 4% paraformaldehyde (PFA, Sigma) for 20 min at room temperature. The cells were washed two times with PBS followed by permeabilization with absolute methanol for 15 min at 4 C. Non-specific blocking was performed with 1% BSA in PBS for 1 h at 37 C. Following this treatment, the cells were stained with primary antibodies 2E6 (anti-hsp antibody, 1:1000 dilution), anti-uis4 (antibody against a PV membrane associated protein, 1:1000 dilution) or mab 25.1 (anti-msp-1, 1:500 dilution). The immunoreactivity was revealed by using anti-mouse or anti-rabbit secondary antibodies conjugated to FITC and Rhodamine, respectively. The parasite and host cell nuclei were visualized by DAPI staining. 2.7. Quantitative real time RT-PCR Two million sporozoites were either exposed to [142 mm] K + medium or the control medium and total RNA was isolated using micro to midi RNA isolation kit (Invitrogen) and treated with DNase I (Invitrogen). Thirty nanograms of RNA was used as a template in first-strand cdna synthesis, using MuVL reverse transcriptase (Applied Biosystems). PCR amplification of the P. yoelli PP2C and CS gene was performed using genomic DNA extracted from mixed blood stage parasites using the DNEasy kit (Qiagen). The primers used for amplification were PyPP2C (sense, 5 -CAAAAGGAATT- ACCATACATGATTATAAGAG-3 ; antisense, 5 -AATGTTGG- TAGTTTCATTAACTTTATT-3 ) and CSP (sense, 5 -TATAA- GCTTAAAATGCAAAATAAAAGTGTCCAAGCCCAAAG- 3 ; antisense, 5 -TCTGTTGACTATATTTCGATTGTATAT-3 ). The PCR products were cloned into pcr4 TOPO (Invitrogen)

34 K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 and standard curves were determined by 10-fold dilution series (100 copies to 10 7 copies, each in duplicate) of the plasmids. Reactions were subjected to one cycle of 3 min at 95 C and 45 cycles of 30 s at 95 C, 30 s at 50 C and 30 s at 72 C. The in vitro and in vivo infectivity of Plasmodium sporozoites was assessed by real time PCR as described earlier [32,33]. The amounts of parasite-derived 18S rrna copies were determined from a standard curve, generated with known amounts of 18S rrna plasmid. 2.8. Kupffer cell depletion Kupffer cell depletion [34] was performed by intravenous injection of 200 l of Clodronate liposomes suspended in PBS per mouse into a group of 5 C57BL/6 mice for three consecutive times at 2 days intervals. Liposomes containing PBS, were used as controls for depletion experiment. The effect of K + treated sporozoite infectivity subsequent to Kupffer cell depletion, was assessed by measurement of liver stage burden by real time PCR as described above. 2.9. Calcein green assay to quantify in vitro sporozoite migration In vitro sporozoite migration was quantified by calcein green assay (Coppi and Sinnis, personnal communication). Hepa 1-6 cells were seeded in 96 well plates, and grown to confluency. Cells were labeled with 10 M calcein green (Molecular Probes) made in Hank s Balanced Salt Solution (HBSS) (Gibco) and loading was performed for 1 h in tissue culture incubator (37 C and 5% CO 2 ) followed by three gentle washes with HBSS buffer for 2 min each to remove any residual unloaded calcein green. During incubation, calcein green passively diffuses across cell membrane and generates a fluorescent product upon cleavage by non-specific cellular esterases. Efficiency of calcein green loading was confirmed by observing the cultures under a fluorescent microscope, prior to the addition of sporozoites. One million sporozoites were spun at 6800 g for 5 min at 4 C and the sporozoite pellet was gently resuspended in 10 15 l of either control medium, intracellular control medium, media containing [142 mm] K + or control medium containing 10 g/ml of cytochalasin-d and incubated for 35 min at room temperature. Following incubation, the sporozoites were added to the wells and immediately spun for 5 min at 300 g at 4 C. The plates were left at 37 C for 1 h. Following this incubation, 100 l of supernatant from each well were transferred to a ThermoElectron microfluor 96-well black plate (Fisher). Controls included media obtained from wells treated with uninfected salivary gland extracts or media from labeled cells not infected with sporozoites served as a reference for background fluorescence. The plates were read in a Fluoroscan II (labsystems) that was set at an excitation wavelength of 494 and emission wavelength of 517 nm. The assay was performed twice with identical results. 2.10. Array design In order to monitor transcription in P. yoelli a full genome oligonucleotide array that contains approximately 500,000 single-stranded 25 base probes to the P. falciparum and P. yoelli genomes was used [35]. The sequences in this microarray were derived from the P. falciparum and P. yoelli sequence data released in July 2001 and carries 124,957 probes from P. yoelli contigs. A further 6000 control sequences corresponding to human and mouse genes that are reported to be highly expressed in blood cells, 3602 traditional Affymetrix controls and 2397 background probes were selected. These controls allow us to unambiguously assure expression patterns are from the parasite in the analysis of human samples [36]. Following release of the P. yoelli genome sequence and annotations in October 2002 probe nucleotide sequences were mapped to the sense nucleotide sequence of predicted coding regions (introns removed) using BLASTN. Gene expression levels were determined using the MOID algorithm [35]. For very large genes with more than 20 probes only the 20 probes closest to the 3 end of the gene were used. To identify genes that were changing in the treated and untreated samples statistical tests were performed. First expression levels were determined using the MOID algorithm: using criteria of LopP < 0.5 and E > 25 transcripts for 2683 genes were detected above background with confidence in untreated sporozoites. To identify genes that were potentially changing a sensitive, two-way ANOVA test was applied [37]. This test calculates the probability that two or more data sets are similar examining the intensity levels for the different probes for a gene across conditions. Because the microarray has not been previously evaluated for use with P. yoelli sporozoites, we compared expression levels to those observed in previous analyses of the P. yoelli sporozoite expression obtained through sequencing of cdna libraries or comparison with the P. falciparum. As expected the most abundant transcripts map to genes that are largely known to be highly expressed in sporozoites (Supplementary information Table 2). While a number of the most highly expressed genes such as PY04653 do not have a recognizable ortholog in P. falciparum, the second most highly expressed gene was the circumsporozite protein (2nd). Also ranking highly were the sporozoite surface protein (5th), pbs36 (14th), and the translation initiation factor, SUI1 (8th). PY00204 (uis4), which when disrupted causes liver stage developmental arrest [12] is highly expressed in P. falciparum (3rd) and in P. yoelli (1st). UIS3 (PY03011), which was abundantly expressed in a P. yoelli cdna sporozoite library, ranked 3rd [38]. Given that completely different oligonucleotide probes and experimental material were used in the P. falciparum and P. yoelli experiments the degree of concordance was striking with CSP ranked first out of 5000 genes in P. falciparum and 2nd in P. yoelli., SSP2 ranked 2nd in P. falciparum and 6th in P. yoelli, the heat shock 70 protein 4th in P. falciparum and 4th in P. yoelli and SUI1 ranked 14th in P. falciparum and 8th in P. yoelli confirming that expression analysis, even when amplification of samples is used, is a very robust and sensitive method for studying changes in parasite physiology as well as for detecting relative transcript levels. Almost all genes ranked in the top 10% in P. falciparum although there were striking exceptions, such PY3963, which is not expressed at detectable levels in P. falciparum but is highly expressed in the rodent parasite. This

K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 35 Fig. 1. Exposure of Plasmodium sporozoites to [142 mm] K + enhances infectivity of P. berghei and P. yoelli sporozoites. (a) P. berghei or (b) P. yoelli sporozoites (2 10 4 ) were incubated in the following media for 35 min at room temperature: control medium, intracellular control medium, intracellular medium [142 mm] K + containing 2 mm EGTA, intracellular medium without EGTA, and control medium containing 10 g/ml cytochalasin-d. All the treated sporozoites were added to either HepG2 or Hepa 1-6 cultures and fixed 1 h post-infection. The parasites inside and outside the HepG2 cells were quantified by the hepatocyte invasion assay. The results are representative of sporozoite numbers from 25 random fields counted under 40 magnification. may represent a difference in the physiology of the two species, although it could be due to a problem with the gene models in one of the species. The amount of similarity between treated and untreated samples was surprising given the large changes in expression programs that are observed between different life cycle stages. 2.11. crna preparation for hybridization in arrays Two million sporozoites were either treated with control media or medium containing [142 mm] K + for 35 min. Total RNA was isolated from both groups, using a micro to midi total RNA isolation kit (Invitrogen). Sporozoite RNA (initial concentration of 100 ng) was subjected to double amplification step, using a modified Eberwine protocol, to obtain 8 g ofrna. After the first cdna synthesis, using an oligodt primer containing a phage T7 promoter at its 5 end which was utilized to prime the cdna synthesis reaction, we used T7-in vitro transcription (Ambion MEGAscript Kit) following the manufacturer s protocol. The labeled crna was then fragmented, hybridized to the array, and stained with a streptavidin phycoerythrin conjugate. Hybridizations were carried out with 15 g of fragmented crna, at 45 C for 16 h, then the hybridization solution was removed and the arrays were stained and washed following the Affymetrix protocols. Arrays were scanned with an emission wavelength of 560 nm at 3 m resolution using a confocal scanner (Affymetrix), and the signal intensity for each sequence feature on the array was determined using the 70th percentile method in Microarray Suite 5 (Affymetrix). 3. Results and discussion P. berghei sporozoites were incubated for 35 min at room temperature in medium containing [142 mm] K + ( intracellular medium), or with the same medium but containing instead [142 mm] Na + ( intracellular control medium), or with control medium (DMEM containing 4.5 g/l glucose supplemented with 10% FCS). Following incubation in different media, the sporozoites were added to hepatoma cultures maintained in control medium. In this paper the sporozoites exposed to [142 mm] K + are named treated. In multiple experiments, the invasion of HepG2 cells by treated P. berghei sporozoites increased between 8 10 times, as measured by the number of sporozoites internalized at the end of 1 h incubation (Fig. 1a). This enhancement in invasion led to a similar increase in the numbers of EEFs (Fig. 2a) as determined by fluorescent microscopy after staining for two EEF antigens, HSP70 [11] and UIS4 [12] (Fig. 2c). No intracellular parasites were detected if the incubation medium contained cytochalasin-d. In addition, we measured Plasmodium 18S rrna copy numbers from HepG2 cultures exposed to treated and control sporozoites. The results also showed a 10- fold increase in infectivity of the treated parasites (Fig. 2b). Since the 18S rrna copy numbers reflect the EEF numbers in the entire HepG2 cell monolayer, it was the method of choice for measuring infectivity of sporozoites. Parallel studies were performed with P. yoelli that showed four- to five-fold increase in infectivity following exposure to [142 mm] K + (Fig. 1b). The enhanced infectivity was dependent on the time of treatment, and on the concentration of K + in the medium. Infectivity increased substantially between 15 and 35 min of incubation (Fig. 3a), but not thereafter. A suboptimal enhancement of infectivity was noted at [70 mm] K +, but lower concentrations were inactive (Fig. 3b). We also studied the effect on enhancement of two K + channel blockers, TEA (tetraethyl ammonium chloride) and quinine, known to inhibit the K + channels in Paramecium [13]. In three independent experiments, sporozoite activation was inhibited 50 60% (P < 0.05) by 20 mm TEA or by 0.5 mm quinine in both P. berghei and yoelli (Fig. 4). The inhibitors were incubated with sporozoites in a volume of 20 l. This was added to Hepatoma cultures and the total volume was increased to 500 l of culture medium resulting in final concentration of 0.8 mm TEA and 0.02 mm quinine. As a control we exposed

36 K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 Fig. 2. [142 mm] K + treated sporozoites show enhanced numbers of EEFs in hepatoma cultures. HepG2 cultures were infected with 2 10 4 P. berghei sporozoites treated either with intracellular control medium, [142 mm] K + or with 10 g/ml cytochalasin-d in control medium. Infectivity was quantified at 60 h post infection by (a) microscopically or by (b) quantitative RT-PCR. The two panels (c) show HSP staining of EEFs (20 magnification) derived from sporozoites exposed to intracellular control medium (top) or [142 mm] K + (bottom). The insets A, B in the right side show parasites stained for expression of HSP (mab 2E6, EEF marker) and UIS4 (rabbit polyclonal, marker for PV), respectively. Inset C shows a merge between HSP and UIS4 staining for each of these panels. Magnification of inserts in A, B and C are 60. HepG2 cell or Hepa 1-6 cells to the above concentrations of the inhibitors. Treated and untreated cells were equally susceptible to infection by sporozoites (not shown). Remarkably, the treated P. berghei sporozoites showed enhanced numbers of EEFs in non-permissive cell lines like MDCK, COS and HeLa (Fig. 5a). At 60 h post-infection, the EEFs stained positive for UIS4, a marker for PV and also for MSP-1, a blood stage antigen (Fig. 5b). The mature EEFs contained merozoites that were infective for young rats (not shown). We are aware of only a single publication that documents the infection of HeLa cells by P. berghei sporozoites [14]. Next, we evaluated the cell traversal activity of treated P. yoelii sporozoites. We found that treated sporozoites migrated significantly less as compared to non-treated or those exposed to control intracellular medium (Fig. 6a). In parallel with the diminished ability to cross cells, the treated sporozoites were less infective to mice (Fig. 6b). Prior Kupffer cell depletion of the mice, however, enhanced several fold the infectivity of K + treated sporozoites. Nevertheless the infectivity of treated sporozoites was significantly lower than that of normal sporozoites injected into Kupffer cell depleted mice, consistent with the earlier published data [15]. Fig. 3. The effect of [142 mm] K + on sporozoite infectivity is both time and concentration dependent. (a) 2 10 4 sporozoites were treated with intracellular control medium or [142 mm] K + medium for indicated times and added to the HepG2 cultures. The infectivity was measured by quantitative real time PCR at end of 60 h. (b) 2 10 4 P. berghei sporozoites were treated with indicated concentrations of K + for 35 min and subsequently added to the HepG2 cultures. C: non-treated sporozoites.

K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 37 Fig. 4. Effect of K + channel blockers on sporozoite infectivity. P. berghei or P. yoelli sporozoites (2 10 4 ) were pre-treated for 30 min in either 20 mm TEA or 0.5 mm quinine made in control medium. Following incubation the sporozoites were washed and additionally incubated with [142 mm] K + for 30 min in the continued presence or absence of indicated concentrations of channel blockers before addition to hepatoma cultures. Infectivity was quantified by real time PCR and represented as percentage decrease relative to [142 mm] K + treated sporozoites (*P < 0.05). We also tested the effect of [142 mm] K + on P. berghei SPECT mutants that lack cell traversal activity but have normal infectivity in vitro [16]. We found that the infectivity of SPECT ( ) mutants and weight parasites was equally enhanced after incubation with [142 mm] K + (Fig. 6c), indicating the effect of K + is independent of SPECT. To study the possibility that treatment with [142 mm] K + changes the parasite s transcriptional program, we hybridized crna, derived from total RNA of treated and untreated parasites, to high density oligonucleotide array that contain one or more 25mer probes to 5520 different P. yoelli predicted genes. We found that 551 genes were differentially expressed using a threshold of P < 0.05 (Fig. 7a). Although there is an increase in expression levels in the K + treated samples, the fold changes were not large (1.2-fold) even though the changes were regarded as significant (P < 0.0002 rank 16). Only seven genes out of 551 that were differentially expressed between treated and nontreated sporozoites changed by more than two-fold (threshold of P < 0.05) (Supplementary information Table 1). The gene showing the highest degree of up-regulation (2.8- fold) in treated samples (at P <.05) was the P. yoelii homolog of protein phosphatase 2C (PY01654, PP2C). These results were confirmed by the comparing the expression of PP2C levels from cdna samples of non-treated and treated sporozoites by absolute qrt-pcr. We found a 1.6-fold increase (P < 0.05) in the copy number of PP2C transcripts in treated sporozoites (Fig. 7b) while there was no change in expression of CS that was amplified in parallel as a normalization control (Fig. 7c). Neverthless, correlations between treated and untreated parasites were generally quite high (r = 0.98 and 0.985). Plasmodium sporozoites have to surmount several obstacles to continue the life cycle in the mammalian host. While the infective stages of other protozoan parasites encounter the target cells in proximity to the site of entry, malaria sporozoites develop fully only in hepatocytes, very far from where they are deposited by Anopheles mosquitoes. This long trajectory, from the skin to the liver, has to be accomplished without substantial losses because very few sporozoites are injected into the skin during the mosquito bite. In its route to the liver, the sporozoites go through the wall of capillaries to reach the blood circulation, and traverse Kupffer cells of the liver sinusoids and the space of Disse to enter the parenchyma [7]. The passage through cells is required for the transformation of sporozoites into EEFs [17]. Even if the sporozoites are Fig. 5. Sporozoites treated with [142 mm] K + show higher number of EEFs in non-permissive cell lines. (a) P. berghei sporozoites (2 10 4 ) were treated with intracellular control medium or with [142 mm] K + and added to the indicated cell lines. The infectivity was quantified by microscopy. The results are representative of EEF numbers from 25 random fields counted under 20 magnification. (b) Cultures were fixed at the end of 60 h and stained for expression of MSP-1, UIS4 and DAPI and viewed under 40 magnification. Scale bar is 10 m.

38 K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 Fig. 6. Sporozoites exposed to [142 mm] K + demonstrate reduced migration in vitro and in vivo. (a) Calcein green assay to measure in vitro migration of sporozoites. Calcein green was loaded into Hepa 1-6 cells for 1 h at 37 C. Inset within the figure shows the efficiency of loading. Following labeling, 1 10 6 sporozoites treated either with control medium, control intracellular medium, [142 mm] K +,or10 g/ml cytochalasin-d and were added to the cells. Controls that included media obtained from wells treated with uninfected salivary gland extracts or media from labeled cells not infected with sporozoites served as a reference for background fluorescence. Following incubation for 1 h at 37 C, supernatants were gently recovered from sample wells and read on spectrofluorometer set at excitation wavelength of 494 nm and emission wavelength of 517 nm. (b) P. yoelli sporozoites treated with [142 mm] K + are less infective to mice, but infectivity is partially restored by depletion of Kupffer cells. 2 10 4 P. yoelli sporozoites, non-treated or exposed to [142 mm] K + were injected into intact C57BL/6 mice ( ), or into Kupffer cell-depleted C57BL/6 mice (+). A control sample of 2 10 4 P. yoelli sporozoites were also injected into mice treated with PBS liposomes. The infectivity was assessed at the end of 48 h by quantitative RT-PCR measurement of parasite specific 18S rrna copy number. (c) SPECT mutants demonstrate infectivity identical to Wild Type (WT) parasites following exposure to [142 mm] K +.WTP. berghei parasites or SPECT mutants were either exposed to control intracellular medium or to 142 mm K + and added to HepG2 cultures. Infectivity was quantified by measuring the transcript abundance of P. berghei 18S rrna by real time PCR. deposited directly on top of hepatocyte monolayers they cross several cells prior to the productive invasion that requires the engagement of a parasite actin/myosin motor [1]. This motor drives the moving junction between the parasite and hepatocyte membranes leading to sporozoite invasion of the hepatocyte. An essential component of the motor is TRAP that connects the motor to the target cell surface receptors [10]. This active invasion process leads to the formation of a PV in which the Fig. 7. (a) Microarray analysis of the sporozoite transcriptome following exposure to [142 mm] K +. Two million P. yoelli sporozoites were either treated with control medium or medium containing [142 mm] K + for 35 min and RNA was isolated immediately from the respective samples. The RNA samples were amplified prior to cdna synthesis. Transcript abundance was determined using custom microarray for asexual stages vs. sporozoites (black), and sporozoites incubated in normal medium vs. [142 mm] K + (red). Quantitative RT-PCR analysis of (b) PP2C and (c) CS levels in sporozoites incubated in control medium (C) vs. [142 mm] K +. cdna was generated from two million sporozoites that were either treated with control media or media containing [142 mm] K +. Gene specific standards were generated for both PP2C and CS and used to quantify by absolute RT-PCR method, the transcript levels of the respective genes from parasites incubated in control medium and from those treated with [142 mm] K + (*P < 0.05).

K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 39 parasites round up and transform into EEFs. In contrast, the rupture of membranes during passage through cells is accomplished through the secretion of perforin-like molecules [18,19] and a lipoprotein lipase [20]. Thus two distinct and sequential invasion programs are utilized during sporozoite infection, but only salivary gland sporozoites are primed to accomplish cell passage. What are the signals required to repress passage through cells, and activate the productive invasion machinery? A prior study showed that the treatment of sporozoites with ionomycin, a Ca 2+ ionophore that stimulates the secretion of apical organelles containing TRAP, led to an increase of infectivity and inhibition of passage through cells [8]. The same effect was obtained by incubation of sporozoites with cell extracts, but the active component in the extracts was not identified. The present findings suggest that one of them might be an increased K + concentration. When sporozoites traverse cells in vivo or in vitro, they are exposed rapidly and successively to large differences in concentrations of K +, from [5 mm] outside cells, to [142 mm] in the cytoplasm. In our in vitro experiments, sporozoites were also subjected to a shift in [K + ]. Following incubation with [142 mm] K +, they were added to HepG2 cells maintained in the control medium. We show that the enhancing effect of [142 mm] K + is time dependent; the increase in infectivity reaches a maximum only after 35 min of incubation. This is approximately the time that sporozoites need to reach and infect the liver cells when injected into the skin [21]. Although during their trajectory from the skin to hepatocytes the parasite traverses several cells, our findings suggest that, given sufficient time of incubation with [142 mm] K + is sufficient to activate the program for productive invasion. Pre-treatment with [142 mm] K + decreased substantially the sporozoite infectivity when injected intravenously into mice. Since the treatment reduces in vitro cell crossing activity, it is possible that the injected parasites were unable to cross the wall of the liver sinusoids, and/or invaded and partially developed in non-permissive cells, prior to entering hepatocytes. This hypothesis is supported by the observation that the prior depletion of Kupffer cells, enhanced the infectivity of the treated sporozoites. Similarly to SPECT mutants, pretreatment with [142 mm] K + reduces cell traversal. Thus we considered the possibility that K + and SPECT signaling pathways intersect, and that SPECT signaling might be down stream of K + signaling. However, the infectivity of SPECT mutants and wt sporozoites are equally enhanced by exposure to [142 mm] K +, indicating that the K + mediated effects are independent of SPECT. [K + ] shifts have also a striking effect on the life cycle of Toxoplasma gondii. Treatment of tachyzoites with a high K + concentration immobilizes the parasites [22]. At the end of its intracellular replicative cycle, when the host cell membrane is ruptured, there is an abrupt decrease in [K + ] and Toxoplasma tacyzoites move out, ready to invade other host cells [23]. However, if the extracellular [K + ] is maintained at [142 mm] the parasites do not egress, indicating that it is the [K + ] shift that initiates it. It appears that the decrease in [K + ] is followed by the activation of a parasite phospholipase C and an increase of cytoplasmic Ca 2+ [23]. There are other examples where passage through cells has been documented in apicomplexan parasites. During the Plasmodium development in Anopheles, the ookinetes migrate through several cells of the mosquito midgut prior to their transformation into oocysts [24,25]. In Eimeria, the tachyzoites enter and exit cells and only then continue the cycle inside a PV [26,27]. A possible role of [K + ] shifts in these models has not been investigated. What are the mechanisms that govern the K + mediated changes in sporozoite properties? As documented in our microarray results, large transcriptional changes have been excluded by comparing hybridization arrays using a sensitive methodology. While one might dismiss the small number of observed changes as being in the realm of chance, our ability to reproducibly confirm the changes using independent methods and samples suggests that the observed low level changes are indeed real. However, it is clear that the types of transcriptional changes that are occurring are on a different scale to those which occur as the parasite goes through its lifecycle. The highest degree of up-regulation was in a putative PP2C phosphatase (PY01654). The corresponding gene product in Arabidopsis thaliana de-phospholylates the K + channel AKT3 in response to Ca 2+ signaling [28]. However, PP2C is already expressed in untreated sporozoites, and it is unlikely that a 2.8- fold increase leads to a new signaling cascade. We conclude that the major phenotypic changes that we have documented are likely to be regulated post transcriptionally, for example by phosphorylation and dephosphorylation or proteolysis of signaling proteins. Pre-incubation of the sporozoites with the classical K + channel blockers inhibitors, TEA and quinine, inhibits significantly their activation by [142 mm] K +, suggesting that the parasites bear K + channels. Two Plasmodium proteins have the signature sequences of K + channels. One of them (PF14 0043), that is expressed in sporozoites but not in blood stages, is an orthologue of a bi-functional adenyl cyclase/[k + ] channel of Paramecium tetraurelia [29]. The Paramecium bifunctional adenyl cyclase/[k + ] channel has been well characterized, its N-terminal is the ion channel domain, and the C-terminal is the adenyl cyclase and it functions as an ion channel when incorporated into lipid membranes [13]. The other Plasmodium putative ion channel, Pfkch1 (PFL1315w) is expressed in all erythrocytic stages of P. falciparum [30], and in sporozoites but its function has not yet been formally demonstrated. Further studies are required to verify whether one of these K + channels mediates the ion transport that lead to the profound changes in the biology of the infective stage of malaria parasites. Acknowledgements The authors thank Dr. Ruth Nussenzweig for valuable comments on the manuscript. We also thank Dr. Photini Sinnis and Dr. Alida Coppi for sharing the protocol and assistance with performing calcein green migration assay.

40 K.A. Kumar et al. / Molecular & Biochemical Parasitology 156 (2007) 32 40 Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2007.07.004. References [1] Mota MM, Pradel G, Vanderberg JP, et al. Migration of Plasmodium sporozoites through cells before infection. Science 2001;291(5501):141 4. [2] Vanderberg JP, Frevert U. Intravital microscopy demonstrating antibodymediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol 2004;34(9):991 6. [3] Amino R, Thiberge S, Martin B, et al. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med 2006;12(2):220 4. [4] Pinzon-Ortiz C, Friedman J, Esko J, Sinnis P. The binding of the circumsporozoite protein to cell surface heparan sulfate proteoglycans is required for Plasmodium sporozoite attachment to target cells. J Biol Chem 2001;276(29):26784 91. [5] Frevert U, Sinnis P, Cerami C, et al. Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J Exp Med 1993;177(5):1287 98. [6] Pradel G, Frevert U. Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion. Hepatology 2001;33(5):1154 65. [7] Frevert U, Engelmann S, Zougbede S, et al. Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol 2005;3(6):e192. [8] Mota MM, Hafalla JC, Rodriguez A. Migration through host cells activates Plasmodium sporozoites for infection. Nat Med 2002;8(11):1318 22. [9] Menard R. Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite. Cell Microbiol 2001;3(2):63 73. [10] Sultan AA, Thathy V, Frevert U, et al. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 1997;90(3): 511 22. [11] Tsuji M, Mattei D, Nussenzweig RS, Eichinger D, Zavala F. Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites. Parasitol Res 1994;80(1):16 21. [12] Mueller AK, Camargo N, Kaiser K, et al. Plasmodium liver stage developmental arrest by depletion of a protein at the parasite host interface. Proc Natl Acad Sci USA 2005;102(8):3022 7. [13] Schultz JE, Klumpp S, Benz R, Schurhoff-Goeters WJ, Schmid A. Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance. Science 1992;255(5044):600 3. [14] Calvo-Calle JM, Moreno A, Eling WM, Nardin EH. In vitro development of infectious liver stages of P. yoelii and P. berghei malaria in human cell lines. Exp Parasitol 1994;79(3):362 73. [15] Baer K, Roosevelt M, Clarkson Jr AB, et al. Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver. Cell Microbiol 2007;9(2):397 412. [16] Ishino T, Yano K, Chinzei Y, Yuda M. Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol 2004;2(1):E4. [17] Mota MM, Rodriguez A. Migration through host cells by apicomplexan parasites. Microbes Infect 2001;3(13):1123 8. [18] Ishino T, Chinzei Y, Yuda M. A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell Microbiol 2005;7(2):199 208. [19] Kaiser K, Camargo N, Coppens I. A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Mol Biochem Parasitol 2004;133(1):15 26. [20] Bhanot P, Schauer K, Coppens I, Nussenzweig V. A surface phospholipase is involved in the migration of Plasmodium sporozoites through cells. J Biol Chem 2005;280(8):6752 60. [21] Sidjanski S, Vanderberg JP. Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood. Am J Trop Med Hyg 1997;57(4):426 9. [22] Kafsack BF, Beckers C, Carruthers VB. Synchronous invasion of host cells by Toxoplasma gondii. Mol Biochem Parasitol 2004;136(2):309 11. [23] Moudy R, Manning TJ, Beckers CJ. The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii. J Biol Chem 2001;276(44):41492 501. [24] Shahabuddin M. Do Plasmodium ookinetes invade a specific cell type in the mosquito midgut? Trends Parasitol 2002;18(4):157 61. [25] Zieler H, Dvorak JA. Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells. Proc Natl Acad Sci USA 2000;97(21):11516 21. [26] Fayer R, Hammond DM. Development of first-generation schizonts of Eimeria bovis in cultured bovine cells. J Protozool 1967;14(4):764 72. [27] Roberts WL, Speer CA, Hammond DM. Penetration of Eimeria larimerensis sporozoites into cultured cells as observed with the light and electron microscopes. J Parasitol 1971;57(3):615 25. [28] Vranova E, Tahtiharju S, Sriprang R, et al. The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C. J Exp Bot 2001;52(354):181 2. [29] Weber JH, Vishnyakov A, Hambach K, et al. Adenylyl cyclases from Plasmodium, Paramecium and Tetrahymena are novel ion channel/enzyme fusion proteins. Cell Signal 2004;16(1):115 25. [30] Ellekvist P, Ricke CH, Litman T, et al. Molecular cloning of a K(+) channel from the malaria parasite Plasmodium falciparum. Biochem Biophys Res Commun 2004;318(2):477 84. [31] Renia L, Miltgen F, Charoenvit Y, et al. Malaria sporozoite penetration. A new approach by double staining. J Immunol Methods 1988;112(2):201 5. [32] Kumar KA, Oliveira GA, Edelman R, Nardin E, Nussenzweig V. Quantitative Plasmodium sporozoite neutralization assay (TSNA). J Immunol Methods 2004;292(1/2):157 64. [33] Bruna-Romero O, Hafalla JC, Gonzalez-Aseguinolaza G, et al. Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR. Int J Parasitol 2001;31(13):1499 502. [34] Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994;174(1/2):83 93. [35] Le Roch KG, Zhou Y, Blair PL, et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 2003;301(5639):1503 8. [36] Daily JP, Le Roch KG, Sarr O, et al. In vivo transcriptome of Plasmodium falciparum reveals overexpression of transcripts that encode surface proteins. J Infect Dis 2005;191(7):1196 203. [37] Barrera L, Benner C, Tao YC, Winzeler E, Zhou Y. Leveraging two-way probe-level block design for identifying differential gene expression with high-density oligonucleotide arrays. BMC Bioinformatics 2004;5:42. [38] Kappe SH, Gardner MJ, Brown SM, et al. Exploring the transcriptome of the malaria sporozoite stage. Proc Natl Acad Sci USA 2001;98(17):9895 900.