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• W2059692646 abstract "Molecular mechanisms by which signaling pathways operate in the malaria parasite and control its development are promiscuous. Recently, we reported the identification of a signaling pathway in Plasmodium falciparum, which involves activation of protein kinase B-like enzyme (PfPKB) by calcium/calmodulin (Vaid, A., and Sharma, P. (2006) J. Biol. Chem. 281, 27126–27133). Studies carried out to elucidate the function of this pathway suggested that it may be important for erythrocyte invasion. Blocking the function of the upstream activators of this pathway, calmodulin and phospholipase C, resulted in impaired invasion. To evaluate if this signaling cascade controls invasion by regulating PfPKB, inhibitors against this kinase were developed. PfPKB inhibitors dramatically reduced the ability of the parasite to invade erythrocytes. Furthermore, we demonstrate that PfPKB associates with actin-myosin motor and phosphorylates PfGAP45 (glideosome-associated protein 45), one of the important components of the motor complex, which may help explain its role in erythrocyte invasion. Molecular mechanisms by which signaling pathways operate in the malaria parasite and control its development are promiscuous. Recently, we reported the identification of a signaling pathway in Plasmodium falciparum, which involves activation of protein kinase B-like enzyme (PfPKB) by calcium/calmodulin (Vaid, A., and Sharma, P. (2006) J. Biol. Chem. 281, 27126–27133). Studies carried out to elucidate the function of this pathway suggested that it may be important for erythrocyte invasion. Blocking the function of the upstream activators of this pathway, calmodulin and phospholipase C, resulted in impaired invasion. To evaluate if this signaling cascade controls invasion by regulating PfPKB, inhibitors against this kinase were developed. PfPKB inhibitors dramatically reduced the ability of the parasite to invade erythrocytes. Furthermore, we demonstrate that PfPKB associates with actin-myosin motor and phosphorylates PfGAP45 (glideosome-associated protein 45), one of the important components of the motor complex, which may help explain its role in erythrocyte invasion. Almost 500 million cases of malaria are reported each year, and 1–3 million of these cases result in human death. Apicomplexan parasite Plasmodium, which is responsible for malaria, has a complex life cycle. It develops inside both human and insect hosts, but its development inside human erythrocytes is the major cause of malarial pathology. Upon invasion of the erythrocyte, the parasite resides inside the parasite vacuole and undergoes various developmental changes. Subsequent to nuclear division, several merozoites are generated, which upon release invade fresh erythrocytes resulting in higher parasitemia. It is clear that erythrocyte invasion is a key step for the parasite to infect the human host and therefore is a key target for intervention. Erythrocyte invasion is a multistep process wherein interaction between the merozoite and erythrocyte is followed by reorientation of the merozoite, which results in the formation of a tight junction between the merozoite apical end and the erythrocyte membrane (2Soldati D. Foth B.J. Cowman A.F. Trends Parasitol. 2004; 20: 567-574Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Although reasonable information is available about the receptor-ligand interactions involved in recognition and formation of the junction, parasite events that cause the initiation of invasion process are poorly understood. It has been reported that parasite calcium levels need to be optimal for successful invasion (3McCallum-Deighton N. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 317-323Crossref PubMed Scopus (45) Google Scholar, 4Moreno S.N. Docampo R. Curr. Opin. Microbiol. 2003; 6: 359-364Crossref PubMed Scopus (127) Google Scholar). Therefore, it is reasonable to speculate that calcium-dependent signaling pathways may be critical in controlling signaling events. Sequencing of the Plasmodium genome and subsequent in silico work has indicated the presence of several putative signaling proteins in the parasite (5Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. Craig A. Kyes S. Chan M.S. Nene V. Shallom S.J. Suh B. Peterson J. Angiuoli S. Pertea M. Allen J. Selengut J. Haft D. Mather M.W. Vaidya A.B. Martin D.M. Fairlamb A.H. Fraunholz M.J. Roos D.S. Ralph S.A. McFadden G.I. Cummings L.M. Subramanian G.M. Mungall C. Venter J.C. Carucci D.J. Hoffman S.L. Newbold C. Davis R.W. Fraser C.M. Barrell B. Nature. 2002; 419: 498-511Crossref PubMed Scopus (3443) Google Scholar, 6Ward P. Equinet L. Packer J. Doerig C. BMC Genomics. 2004; 5: 79Crossref PubMed Scopus (403) Google Scholar, 7Aravind L. Iyer L.M. Wellems T.E. Miller L.H. Cell. 2003; 115: 771-785Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 8Anamika Srinivasan N. Krupa A. Proteins. 2005; 58: 180-189Crossref PubMed Scopus (141) Google Scholar). However, the information about parasite signaling networks is very limited. Mammalian protein kinase B (PKB) 3The abbreviations used are:PKBmammalian protein kinase BCaMcalmodulinCBDcalmodulin binding domainPfGAP45P. falciparum homologue of Glideosome-Associated Protein 45NTRN-terminal regionPfPKBprotein kinase B-like enzyme in P. falciparumΔPfPKBdeletion of PfPKB lacking the NTR that is catalytically activePLCphospholipase CIPimmunoprecipitateIMCinner membrane complexscrscrambledFITCfluorescein isothiocyanatePfMTIPP. falciparum homologue of myosin tail interacting protein.3The abbreviations used are:PKBmammalian protein kinase BCaMcalmodulinCBDcalmodulin binding domainPfGAP45P. falciparum homologue of Glideosome-Associated Protein 45NTRN-terminal regionPfPKBprotein kinase B-like enzyme in P. falciparumΔPfPKBdeletion of PfPKB lacking the NTR that is catalytically activePLCphospholipase CIPimmunoprecipitateIMCinner membrane complexscrscrambledFITCfluorescein isothiocyanatePfMTIPP. falciparum homologue of myosin tail interacting protein. is regulated by phosphoinositides as they regulate its cellular localization and activation by interacting with a pleckstrin homology domain present at its N terminus. PKB is activated upon phosphorylation by PDK1 (9Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar). In contrast, PfPKB, which is a PKB-like enzyme in P. falciparum, is regulated via a PDK1 and Phosphatidylinositol 3-kinase-independent mechanism (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Recently, we described a signaling pathway in Plasmodium falciparum, which involved the activation of PfPKB by calcium/calmodulin (CaM). Calmodulin interacts with the N-terminal region (NTR) of PfPKB and promotes its autophosphorylation-dependent activation. The intracellular calcium necessary for the activation of this pathway was mobilized from intra-parasitic stores by phospholipase C (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Despite this information, the function of this pathway had remained unexplored. In this study, we demonstrate that CaM and intracellular calcium are important for invasion, and phospholipase C was found to play an important role in this process as it mediated intracellular calcium release needed for invasion. Because inhibition of PfPKB impaired invasion, we conclude that this calcium/calmodulin signaling pathway is crucial for erythrocyte invasion. In addition, we provide evidence that PfPKB interacts with actin-myosin motor complex and phosphorylates PfGAP45 (glideosome-associated protein 45). mammalian protein kinase B calmodulin calmodulin binding domain P. falciparum homologue of Glideosome-Associated Protein 45 N-terminal region protein kinase B-like enzyme in P. falciparum deletion of PfPKB lacking the NTR that is catalytically active phospholipase C immunoprecipitate inner membrane complex scrambled fluorescein isothiocyanate P. falciparum homologue of myosin tail interacting protein. mammalian protein kinase B calmodulin calmodulin binding domain P. falciparum homologue of Glideosome-Associated Protein 45 N-terminal region protein kinase B-like enzyme in P. falciparum deletion of PfPKB lacking the NTR that is catalytically active phospholipase C immunoprecipitate inner membrane complex scrambled fluorescein isothiocyanate P. falciparum homologue of myosin tail interacting protein. Reagents—PfPKB-pGEX4T1 plasmid used for protein expression and anti-PfPKB rabbit antisera used in these studies have been described earlier (10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The following peptides: cross-tide (GRPRTSSFAEG), CBD peptide (IGKKRLRNSMSLSYERKKRIR), scr-peptide (MKLSGKRYRNSRLKEIRSRIK), and CBD-(1–15) (IGKKRLRNSMSLSYE) were custom-synthesized by Peptron, South Korea. Scr peptides have similar amino acid composition as CBD, but their arrangement has been scrambled. U73322, U73122, and W7 were purchased from Calbiochem. A443654 and A739985.3 were provided by Dr. Vincent Giranda, Abbott. Cell Culture, Synchronization, Preparation of Free Merozoites, Inhibitor Treatment, and Invasion Assays—P. falciparum strain 3D7 was cultured at 37 °C in RPMI 1640 medium using either 10% AB+ human serum (11Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6161) Google Scholar) or 0.5% Albumax II (Invitrogen) (complete medium). Cultures were gassed with 7% CO2, 5% O2, and 88% N2, and synchronization of the parasites in culture was achieved by sorbitol treatment (12Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2832) Google Scholar). RBC-free merozoites were prepared by using a previously published method with slight modifications (13Blackman M.J. Scott-Finnigan T.J. Shai S. Holder A.A. J. Exp. Med. 1994; 180: 389-393Crossref PubMed Scopus (226) Google Scholar, 14Mohrle J.J. Zhao Y. Wernli B. Franklin R.M. Kappes B. Biochem. J. 1997; 328: 677-687Crossref PubMed Scopus (48) Google Scholar). Briefly, sorbitol synchronized parasites at ring stages were collected and washed with a buffer containing 10 mm Tris, pH 7.6, 150 mm NaCl, 10 mm glucose, 1 mm CaCl2 and incubated at 10% hematocrit in the same buffer with 1 mg/ml trypsin for 60 min at 37 °C. After washing with complete medium, parasites were plated at 6–8% parasitemia and 5% hematocrit in a T-175 cm2 culture flask containing 35 ml of complete medium. When parasites were predominantly in mid-schizont stage, 10–15 ml of fresh complete medium was added to the cultures. After 1–2 h, parasite cultures were centrifuged at 500 × g for 5 min to pellet parasitized and uninfected erythrocytes. Culture supernatant containing merozoites was centrifuged at 3300 × g to obtain the pellet of erythrocyte-free merozoites. Merozoites were resuspended immediately in complete medium and were incubated with fresh erythrocytes for invasion. Smears were stained with Giemsa and examined microscopically and were typically found free of either erythrocytes or intracellular parasitic stages. To determine the effect of inhibitors on PfPKB activity, inhibitors were added to either schizonts or merozoites in culture. Subsequently, immunoprecipitation was performed, and the IP was used for kinase assays (described below). For typical invasion assays, parasite cultures were synchronized using sorbitol as described above, and peptides and pharmacological inhibitors were added to mid-late schizonts (∼3% parasitemia) and 5% hematocrit, which was maintained using fresh erythrocytes. The formation of rings was analyzed periodically after every 2 h. For experiments with free merozoites, 1–5 × 109 merozoites were incubated with peptides or inhibitors for ∼10 min in complete medium prior to the addition of fresh erythrocytes at 5% hematocrit in culture medium. New ring stage infection was scored after 3 h by microscopic examination of Giemsa-stained blood smears. At least 1000 erythrocytes from several different fields were counted for each experiment. Recombinant Protein Expression and Generation of Antisera—For expression of ΔPfPKB as a GST fusion protein, the pGEX4T1-ΔPfPKB plasmid construct was used, as described previously (10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). For expression of PfGAP45 and PfMTIP, corresponding cDNAs were amplified and cloned in PET28a and PQE30-UA vectors, respectively, and expressed as His6-tagged proteins in Bl21(DE3)-RIL Escherichia coli strain and purified using nickel-nitrilotriacetic acid affinity chromatography. These recombinant proteins were used to raise antisera in rabbits or mice using standard procedures. PfMTIP was also expressed as a GST fusion protein. For this purpose, PfMTIP was cloned in pGEX4T3 vector and expressed in BL21-RIL strain as described previously (15Jones M.L. Kitson E.L. Rayner J.C. Mol. Biochem. Parasitol. 2006; 147: 74-84Crossref PubMed Scopus (66) Google Scholar). Recombinant proteins were quantified and normalized by performing densitometry of SDS-polyacrylamide gels. Metabolic Labeling of Parasites—Tightly synchronized parasite cultures (∼6–8% parasitemia) were washed at least three times either with phosphate or methionine-deficient RPMI 1640 medium (HyClone). Subsequently, parasites were cultured in media supplemented with 10% human serum. CBD peptide or pharmacological inhibitors were added to schizonts, which corresponded to ∼42–44-h post-invasion parasites. Inorganic 32P radionuclide in the form of [32P]orthophosphoric acid (2 mCi/ml) (16Mamoun C.B. Goldberg D.E. Mol. Microbiol. 2001; 39: 973-981Crossref PubMed Scopus (45) Google Scholar) or [35S]Met/Cys (0.3 mCi/ml) (17Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 18Rees-Channer R.R. Martin S.R. Green J.L. Bowyer P.W. Grainger M. Molloy J.E. Holder A.A. Mol. Biochem. Parasitol. 2006; 149: 113-116Crossref PubMed Scopus (86) Google Scholar) was added to cultures and incubated for 2 and 4 h, respectively. Subsequently, parasite lysates were prepared, and immunoprecipitation was performed as described below. The IP was resuspended in SDS-PAGE loading buffer and electrophoresed, and labeled proteins were detected using a Fuji FLA5000 scanner. Western Blotting and Immunoprecipitation—Parasites were released from infected erythrocytes by 0.05% (w/v) saponin treatment. Cell-free protein extracts were prepared by homogenizing parasite pellets in a buffer containing 10 mm Tris, pH 7.5, 100 mm NaCl, 5 mm EDTA, 1% Triton X-100, 20 μm sodium fluoride, 20 μm β-glycerophosphate, 100 μm sodium orthovanadate, and 1× Complete Protease inhibitor mixture (Roche Applied Science). Particulate material was removed by centrifugation at 14,000 × g for 30 min. PfPKB, PfGAP45, and PfMTIP were immunoprecipitated from the schizont or merozoite lysates using appropriate antisera as described previously (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For co-immunoprecipitation experiments, IP was electrophoresed on SDS-PAGE followed by Western blotting (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Immunofluorescence and FITC-Peptide Localization—Immunofluorescence was performed on thin blood smears of parasite cultures. Thin blood smears were first fixed with cold methanol and blocked with 3% bovine serum albumin (prepared in phosphate-buffered saline) before incubation with primary antibody. After washing with blocking buffer, samples were incubated with secondary antibodies labeled with FITC/Texas red or AlexaFluor488/594. For experiments performed with FITC-labeled peptides, CBD peptides were added to schizont stage parasite cultures. After 2 h, thin smears were processed for immunofluorescence for MSP-1 as described above. Slides were viewed using a Olympus fluorescence microscope. For indirect immunofluorescence assays described in Fig. 6A, confocal microscopy was performed using a Olympus FV1000 confocal microscope. The single z-stack shown was processed using Olympus Fluoview 1.6 software. Images were processed using Image ProPlus or Adobe Photoshop software. Assay of Kinase Activity—Catalytic activity of GST-ΔPfPKB or 10 μl of immunoprecipitated PfPKB were assayed as described previously (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) in a buffer containing 50 mm Tris, pH 7.5, 10 mm magnesium chloride, 1 mm dithiothreitol, and 100 μm [γ-32P]ATP (6000 Ci/mmol)) using a small peptide substrate “crosstide” (100 μm, unless otherwise indicated) or other recombinant proteins like PfGAP45. Reactions were stopped by spotting the assay mixture on P11 phosphocellulose paper, and phosphate incorporation was measured by scintillation counting of the P11 paper. When recombinant proteins were used as substrate, reactions were stopped by boiling the mixtures in SDS-PAGE loading buffer (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). After electrophoresis, phosphate incorporation in substrate proteins was visualized by using a Fuji FLA5000 scanner. For PfPKB inhibition assays, pharmacological or peptide inhibitors were preincubated with ΔPfPKB 15 min prior to the addition of phosphoacceptor substrate and ATP. Phospholipase C and Calmodulin Inhibitors Block Erythrocyte Invasion—PfPKB is regulated by CaM by promoting its autophosphorylation in a calcium-dependent manner in vitro and in parasites. The calcium necessary for PfPKB activation by CaM is dependent on the activation of phospholipase C (PLC) (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Therefore, the PfPKB pathway is regulated by CaM and phospholipase C-mediated calcium release (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Specific inhibitors of both CaM (19Matsumoto Y. Perry G. Scheibel L.W. Aikawa M. Eur. J. Cell Biol. 1987; 45: 36-43PubMed Google Scholar) and PLC (20Gazarini M.L. Thomas A.P. Pozzan T. Garcia C.R. J. Cell Biol. 2003; 161: 103-110Crossref PubMed Scopus (121) Google Scholar, 21Hotta C.T. Markus R.P. Garcia C.R. Braz. J. Med. Biol. Res. 2003; 36: 1583-1587Crossref PubMed Scopus (40) Google Scholar) have been used successfully to block the activity of these proteins in Plasmodium. In this study, we used these inhibitors to evaluate the function of CaM and PLC in invasion. When the CaM inhibitor W7 was added to schizonts, it blocked the formation of rings suggesting that CaM plays a role in invasion (Fig. 1A), which is consistent with results published previously (19Matsumoto Y. Perry G. Scheibel L.W. Aikawa M. Eur. J. Cell Biol. 1987; 45: 36-43PubMed Google Scholar). The role of PLC was determined by using its inhibitor U73122 and an inactive analogue of this compound U73322, which was used as a negative control. In comparison with U73322 or Me2SO treatment of schizonts, addition of U73122 inhibited new ring formation significantly suggesting that phospholipase C may be important for invasion. To explore if PLC controls invasion by regulating calcium release, U73122 was used in combination with ionomycin, a calcium ionophore. In this case, inhibition of invasion was significantly protected suggesting that PLC may control this process by regulating intracellular calcium release (Fig. 1B). Similar results were obtained when free merozoites were used for invasion assays (data not shown). Previously, we have demonstrated that CaM and PLC inhibitors block PfPKB activity. When U73122 was used in the presence of ionomycin, PfPKB activity was restored suggesting that PLC-mediated calcium release is critical for PfPKB to function (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Therefore, it was reasonable to speculate that the modulation of PfPKB activity caused because of the inhibition of CaM and PLC may contribute to impaired invasion. Calmodulin-dependent Activation of PfPKB May Be Important for Erythrocyte Invasion; Design and Use of Peptide Inhibitors of PfPKB—CaM and PLC appear to play a role in invasion (Fig. 1), and because they act as upstream regulators of PfPKB (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), the contribution of PfPKB in this process was worth investigating. Two different strategies were used to inhibit PfPKB as follows: 1) a pharmacological inhibitor competitive against ATP, and 2) peptide inhibitors, which target either PfPKB catalytic cleft and/or compete with CaM for PfPKB. Previous biochemical studies (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) had suggested that a calmodulin binding domain (CBD) exists in the NTR of PfPKB (Fig. 2A). In addition, a pseudo-substrate RXRXS motif is also embedded in the CBD, which probably holds PfPKB in an inactive state by interacting with its catalytic site. This was confirmed when a 21-amino acid peptide corresponding to the CBD motif could interact with CaM and prevent the activation of PfPKB in vitro (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). This peptide can also compete directly with the catalytic site of PfPKB (Fig. 2B) as it inhibited the activity of ΔPfPKB, a variant of PfPKB that lacks the CBD and the NTR of PfPKB and therefore is active independent of Ca2+/CaM (1Vaid A. Sharma P. J. Biol. Chem. 2006; 281: 27126-27133Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This “two-pronged” ability of the CBD peptide to inhibit PfPKB activity made it a putative tool for studying PfPKB function. Because this peptide may alter the function of other CaM-binding proteins, it was also important to generate a version of this peptide that could inhibit PfPKB exclusively by interacting with its active site without interacting with CaM. For this purpose, a truncated peptide, which lacked the last six amino acids of CBD, was synthesized. This peptide retained the pseudosubstrate RXRXS motif (Fig. 2A). Even though the CBD-(1–15) peptide failed to interact with CaM (supplemental Fig. IA), it effectively inhibited the activity of ΔPfPKB (Fig. 2B). Therefore, CBD-(1–15) does not interact with CaM and works exclusively as a pseudosubstrate inhibitor of PfPKB, which interacts with the catalytic domain. These peptide inhibitors were added to parasites followed by immunoprecipitation of PfPKB, and the kinase activity associated with PfPKB-IP was assayed. Both CBD and CBD-(1–15) peptides inhibited PfPKB activity, and the control scrambled version of peptides were ineffective (Fig. 2C). To demonstrate if these peptides enter the parasite, FITC-labeled CBD-(1–15) (Fig. 2D) and CBD (supplemental Fig. IB) were added to parasite cultures. The peptide-associated fluorescence was observed inside most schizonts/merozoites, which indicated that these peptides might work intracellularly. Like PfPKB, peptides also concentrated at the apical end of the parasite as observed for PfPKB (supplemental Fig. IB), and peptide associated fluorescence was largely absent from uninfected erythrocytes (data not shown). In contrast, FITC-labeled scr-CBD peptide, which does not inhibit and interact with PfPKB (Fig. 2B, supplemental Fig. ID), was not observed inside the parasite. These peptides were subsequently used to probe PfPKB function. When CBD and CBD-(1–15) peptide were added to segmenter/mature schizonts, a marked decrease in the formation of fresh rings was observed (Fig. 3A, left panel). Because there was no significant change in the number of schizonts (Fig. 3A, right panel) or their morphology, it is reasonable to infer that the decreased ring formation may be due to altered invasion and not due to defects on egress and maturation. Furthermore, invasion assays were also performed with free merozoites isolated from P. falciparum cultures. These merozoites when added to erythrocytes in culture resulted in the formation of rings, which was indicative of successful invasion (supplemental Fig. II). Although scr-CBD peptide-treated merozoites efficiently invaded erythrocytes, both CBD and CBD-(1–15) caused a significant reduction in the formation of rings indicating that invasion was significantly impaired (Fig. 3B). To rule out the effect of these peptides on other parasitic stages, changes in response to these peptides on the entire life cycle were monitored. The growth of parasites was normal until ∼46 h, and there were no significant changes in morphology of intraerythrocytic stages. Subsequent formation of rings in control cultures was indicative of the initiation of the next cycle. The peptide inhibitor-treated cultures had significantly less number of rings (supplemental Fig. IIIA), which was most likely due to impaired invasion as observed in above-described experiments (Fig. 3). A443654 Is an Effective Inhibitor of PfPKB Which Blocks Invasion—Until recently, there were no commercial inhibitors available for PKB-like enzymes from any organism. A443654 was developed as a specific inhibitor of mammalian PKB/AKT enzymes, which is competitive against ATP (22Luo Y. Shoemaker A.R. Liu X. Woods K.W. Thomas S.A. De Jong R. Han E.K. Li T. Stoll V.S. Powlas J.A. Oleksijew A. Mitten M.J. Shi Y. Guan R. McGonigal T.P. Klinghofer V. Johnson E.F. Leverson J.D. Bouska J.J. Mamo M. Smith R.A. Gramling-Evans E.E. Zinker B.A. Mika A.K. Nguyen P.T. Oltersdorf T. Rosenberg S.H. Li Q. Giranda V.L. Mol. Cancer Ther. 2005; 4: 977-986Crossref PubMed Scopus (214) Google Scholar). Given the sequence the high similarity between PfPKB and PKB/AKT in the catalytic domain (10Kumar A. Vaid A. Syin C. Sharma P. J. Biol. Chem. 2004; 279: 24255-24264Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), A443654 was chosen as a possible candidate to inhibit PfPKB. A443654 successfully inhibited the activity of recombinant PfPKB in vitro with reasonable IC50 values (∼200 nm) (Fig. 4A). Importantly, A443654 treatment of schizonts resulted in a significant reduction of parasite PfPKB activity (Fig. 4B). To explore the function of PfPKB, A443654 was added to the ring stage parasites, and their growth was monitored at regular intervals. There was no change in parasitemia until ∼46 h, and at this point, a sudden decrease in rings formed in the next round of the life cycle (supplemental Fig. IIIB) was observed, which could be a result of impaired invasion. To explore this more specifically, invasion assays were performed using schizonts as well as free merozoites as described above for peptide inhibitors (Fig. 3). When schizonts were incubated with the inhibitor, no significant change in their number or morphology was observed. However, the number of rings formed subsequent to invasion was effectively reduced, IC50 ∼ 250 nm (Fig. 5A). Similar impairment of invasion was observed when free merozoites were used for assays indicating that A443654 blocks invasion (Fig. 5B). The inactive analogue of A443654 failed to cause any significant changes to PfPKB activity or invasion of the parasites in control experiments (supplemental Fig. IV). These data corroborate well with results obtained with peptide inhibitors and collectively highlight the importance of PfPKB activity in invasion of erythrocytes.FIGURE 5PfPKB inhibitor, A443654, blocks invasion. A, schizont-infected erythrocytes were incubated with Me2SO (DMSO) or different concentrations of A443654, and the number of schizonts (right panel) and rings (left panel) was individually scored at the indicated times post-addition of the inhibitor. Although there was a significant decrease in the number of rings formed, schizont number remained almost unaltered upon inhibitor treatment at any time point. No schizonts were observed after the 8-h time point (right panel). B, free merozoites isolated from P. falciparum cultures were incubated either with Me2SO (DMSO) or 0.5 μm A443654 as described under “Experimental Procedures.” Thin blood smears were prepared after 3 h, and the number of ring-infected parasites was counted. Representations of more than three independent experiments are shown in the figure. Error bars represent S.E. between the replicates of the same experiment. Right panel shows a representative field from cultures of inhibitor or Me2SO-treated merozoites post-invasion.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PfPKB Interacts with PfGAP45 and Actin-Myosin Motor Complex—It has been demons" @default.
• W2059692646 created "2016-06-24" @default.
• W2059692646 creator A5005231542 @default.
• W2059692646 creator A5023002699 @default.
• W2059692646 creator A5041282587 @default.
• W2059692646 date "2008-02-01" @default.
• W2059692646 modified "2023-10-16" @default.
• W2059692646 title "Role of Ca2+/Calmodulin-PfPKB Signaling Pathway in Erythrocyte Invasion by Plasmodium falciparum" @default.
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