Toxoplasma gondii Attachment to Host Cells Is Regulated by a Calmodulin-like Domain Protein Kinase (2001)

The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion. The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion. Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Infection with this parasite is typically asymptomatic, although acute toxoplasmosis can be fatal in immunocompromised individuals and result in severe birth defects or abortion during the first trimester of pregnancy (1Frenkel J.K. Parasitol. Today.. 1988; 4: 273-278Google Scholar). Three developmental stages of Toxoplasma have been described. The highly infective sporozoite stage is shed in the feces of infected felines and can infect all warm-blooded animals, where it differentiates into the rapidly replicating tachyzoite stage. This form of the parasite rapidly spreads throughout the infected animal and eventually differentiates into the slow growing, encysted bradyzoite stage. The latter can infect another animal upon ingestion of infected tissues. The three stages are immunologically and biochemically distinct, due to the expression of many stage-specific proteins (2Bohne W. Holpert M. Gross U. Immunobiology.. 1999; 201: 54-248Google Scholar). All three developmental stages are obligate intracellular parasites, suggesting that infected host cells supply nutrients that supplementT. gondii biosynthetic deficiencies, such as its inability to make purines de novo (3Pfefferkorn E.R. Wyler D.J. Modern Parasite Biology: Cellular, Immunological and Molecular Aspects.W. H. Freeman and Co. 1990; : 26-50Google Scholar). As a result of these biosynthetic requirements, invasion of a host cell is critical for the growth and reproduction of T. gondii. The biochemical pathways involved in host cell invasion have not yet been identified, although it has been shown that cytoplasmic calcium in the parasite is essential for this process (4Carruthers V.B. Sibley L.D. Mol. Microbiol... 1999; 31: 421-428Google Scholar, 5Pezzella N. Bouchot A. Bonhomme A. Pingret L. Klein C. Burlet H. Balossier G. Bonhomme P. Pinon J.M. Eur. J. Cell Biol... 1997; 74: 92-101Google Scholar). The involvement of calcium in regulating parasite interaction with host cells was further strengthened by the observation of Sibley et al. that both the attachment of Toxoplasma tachyzoites to its host cells as well as parasite motility are sensitive to an inhibitor of calcium-dependent protein kinases, KT5926 (6Hashimoto Y. Nakayama T. Teramoto T. Kato H. Watanabe T. Kinoshita M. Tsukamoto K. Tokunaga K. Kurokawa K. Nakanishi S. Biochem. Biophys. Res. Commun... 1991; 181: 423-429Google Scholar, 7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). In eukaryotic cells, protein kinases often mediate the cellular responses to external stimuli. Three major classes of protein kinases are often involved in this: cyclic nucleotide-dependent protein kinases, calcium/calmodulin-dependent protein kinases, and calcium/phospholipid-dependent protein kinases. In a number of organisms, an unusual class of calcium-dependent protein kinases has been described. These enzymes, the calmodulin-like domain protein kinases (CDPKs),1 are activated by calcium in the absence of calmodulin or phospholipids. Initially identified, characterized, and cloned in plants (8Harmon A.C. Putnam-Evans C. Cormier M.J. Plant Physiol... 1987; 83: 830-837Google Scholar, 9Putnam-Evans C.L. Harmon A.C. Cormier M.J. Biochemistry.. 1990; 29: 2488-2495Google Scholar, 10Harper J.F. Sussman M.R. Schaller G.E. Putnam-Evans C. Charbonneau H. Harmon A.C. Science.. 1991; 252: 951-954Google Scholar), CDPKs have also been identified in algae (11McCurdy D.W. Harmon A.C. Planta ( Heidelberg ).. 1992; 188: 54-61Google Scholar, 12Yuasa T. Muto S. Arch. Biochem. Biophys... 1992; 296: 175-182Google Scholar), Paramecium tetraurelia (13Gundersen R.E. Nelson D.L. J. Biol. Chem... 1987; 262: 4602-4609Google Scholar, 14Son M. Gundersen R.E. Nelson D.L. J. Biol. Chem... 1993; 268: 5940-5948Google Scholar, 15Kim K. Messinger L.A. Nelson D.L. Eur. J. Biochem... 1998; 251: 605-612Google Scholar), and the apicomplexan parasitesPlasmodium falciparum (16Zhao Y. Kappes B. Franklin R.M. J. Biol. Chem... 1993; 268: 4347-4354Google Scholar, 17Li J.L. Baker D.A. Cox L.S. Biochim. Biophys. Acta.. 2000; 1491: 341-349Google Scholar), Eimeria maxima, and Eimeria tenella (18Dunn P.P. Bumstead J.M. Tomley F.M. Parasitology.. 1996; 113: 439-448Google Scholar). The typical CDPK domain structure consists of an N-terminal serine/threonine kinase domain homologous to that of calcium/calmodulin-dependent protein kinases, followed by a highly conserved junction domain, which joins the kinase region to a C-terminal calmodulin-like domain (10Harper J.F. Sussman M.R. Schaller G.E. Putnam-Evans C. Charbonneau H. Harmon A.C. Science.. 1991; 252: 951-954Google Scholar). This calmodulin-like domain, which is 30–40% homologous to calmodulin, imparts calcium sensitivity to the CDPKs (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar, 20Zhao Y. Pokutta S. Maurer P. Lindt M. Franklin R.M. Kappes B. Biochemistry.. 1994; 33: 3714-3721Google Scholar). Activation of CDPKs has been shown to be dependent on calcium and independent of calmodulin (8Harmon A.C. Putnam-Evans C. Cormier M.J. Plant Physiol... 1987; 83: 830-837Google Scholar). The elucidation of the function of specific CDPKs has been complicated, in part, by the fact that often multiple isoforms are expressed at the same time (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar). Three soybean isoforms were found to differ in sensitivity to calcium and substrate specificity (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar), suggesting that they are involved in the regulation of different phenomena. In plants, CDPKs have been found to regulate Ca2+-pumps (21Hwang I. Sze H. Harper J.F. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 6224-6229Google Scholar), K+-transport (22Li J. Lee Y.R. Assmann S.M. Plant Physiol... 1998; 116: 785-795Google Scholar), and a Cl− channel (23Pei Z.M. Ward J.M. Harper J.F. Schroeder J.I. EMBO J... 1996; 15: 6564-6574Google Scholar) and have also been implicated in the response to environmental stress and infections (24Romeis T. Piedras P. Jones J.D. Plant Cell.. 2000; 12: 803-816Google Scholar, 25Saijo Y. Hata S. Kyozuka J. Shimamoto K. Izui K. Plant J... 2000; 23: 319-327Google Scholar). The role of CDPKs in protists and apicomplexan parasites is unclear at this time. In light of the importance of calcium-dependent regulation in host cell invasion by Toxoplasma tachyzoites and the suggestion that this might be affected in the of KT5926, an of calcium-dependent protein kinase in tachyzoites and their sensitivity to the of two TgCDPK1 and in T. gondii. only TgCDPK1 is expressed in the tachyzoite stage of the This to be the of KT5926 in tachyzoites and is to a role in their invasion of host cells. T. was S. of and was by in or in the of as has been J.F. J. Cell Biol... 1994; Scholar). were the or the by and a These were as by et al. J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). attachment and invasion parasites were to of cells at a of in in the of concentrations of KT5926 or and invasion were to for at All were on The were three in to parasites were detected by with an in a the were as were in in and for in and intracellular parasites were detected the J. to the parasite were and in the and and to and the number of parasites, parasites, and host cell were in at All were in and invasion were expressed as the number of host cell were and as but essential In a of parasites were with of was and to a of KT5926 was a at the parasites were by and in of and of were by All were in All were on or at as were to a of in and at were by two by or by the of to a of were by for at extracts were by at for and proteins were by of a parasite by was a in The was with of and protein was with a of in was for the of protein kinase activity as The three the peak of KT5926-sensitive protein kinase activity were and was to a of This was a in The was with of and proteins were with a of to in and protein kinase activity in was as were in a of and The was in and during and were calcium-dependent activity of the the were in the of or the of calcium was by a of a of to A. 1988; Scholar). KT5926, and were in the kinase at the the concentrations of and were to I. for for were at for to an and by on a were and with for in of followed by in were and was a of was with and a of was in All other were in are of in a was with and a of was in All other were in are of for and were by to and of parasite in a of and was for at or and of were by the of of the first were and was for at followed by at and a in the were the were and was to was parasites with the the suggested by the was and or to the The was for the of protein and two conserved in a of serine/threonine protein and typical of of and of and The were of at at and at followed by a at of the were and cloned into to the were identified, and the were with and were by in of of and of and of and the TgCDPK1 was for the TgCDPK2 the was The were of at at and at Toxoplasma in and was with or were to in to the The were by and were the the on the The was the the and in the D.L. 1998; Scholar). In the during the process were not as all were and the were not single was identified in the and which is shown the were The for the are as at the the the to the was identified in a of The of TgCDPK1 and were the and for and for TgCDPK2 with as by the were or and cloned into the expression with and in the and These the of the protein kinases at their to a and the expression of these were into were in of and at was was to a of and growth was for an at were in in and of The was with of and of The proteins were a in proteins were and as The of TgCDPK1 was the and and as The was by with the and by The expression was with the and and by The was into in a that only TgCDPK1 of the was to of and at an of was was to a of and the was for an at All were at The was in of and was to a of and a the was on the was in and at of the was and was to the was by at for was to the to the was by at for The was in of and two of of The was by and to a with The was with of this and proteins were with a of in were for kinase activity as described. The the peak of kinase activity were and in interaction was as the The peak of kinase activity were and was to a of This was to a with and The was with of followed by of and The was with and were for kinase activity as described. of the protein was by were by on or proteins were to for at The was in and or TgCDPK1 and TgCDPK2 was at a in the were detected and the concentrations were by the of Biochem... Scholar) as a was by et al. J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar) that the protein kinase inhibitor KT5926 the of T. gondii to to their host cell and to these as can be in and that the of KT5926 attachment is KT5926 is a inhibitor of and light kinases in animal cells. the to which KT5926 blocks protein phosphorylation in T. tachyzoites were with in the or absence of As a also an in phosphorylation in the of a inhibitor of serine/threonine protein kinases. the the of the inhibitor on of in proteins was by As the of blocks of into proteins not The of KT5926 to parasites, as in blocks the of in three major and These that the of KT5926 on protein phosphorylation gondii is and might be due to an of or only a protein kinases. the of KT5926 for parasite attachment is only the in phosphorylation in was in the of inhibitor to its on protein of KT5926 on protein phosphorylation was at a of the of KT5926 on protein kinase activity in Toxoplasma tachyzoites parasite extracts for the of protein kinase As can be in protein kinase activity is detected in as KT5926 is to be specific for calcium-dependent protein kinase (6Hashimoto Y. Nakayama T. Teramoto T. Kato H. Watanabe T. Kinoshita M. Tsukamoto K. Tokunaga K. Kurokawa K. Nakanishi S. Biochem. Biophys. Res. Commun... 1991; 181: 423-429Google Scholar), the or absence of calcium protein kinase activity in Toxoplasma In the of protein kinase activity in parasite extracts was with in the of These that or calcium-dependent protein kinases. The calcium-dependent in protein kinase activity was in the of KT5926, the that this inhibitor or calcium-dependent protein kinases in The of Toxoplasma calmodulin, a calmodulin or no on the KT5926-sensitive protein kinase activity. These that Toxoplasma not major protein kinase or protein kinase and that the of KT5926 be a different class of calcium-dependent protein the calcium-dependent protein kinase activity in Toxoplasma extracts parasite extracts by and for calcium-dependent and KT5926-sensitive protein kinase activity and the of KT5926 on that activity. As can be in extracts of Toxoplasma a number of protein kinase that can but only a single peak of calcium-dependent protein kinase activity was and this activity was effectively inhibited in the of of this activity by interaction only a single peak of KT5926-sensitive protein kinase activity As the of Toxoplasma calmodulin calmodulin affected of the protein kinase detected in not these tachyzoites a single KT5926-sensitive protein kinase activity that is calcium-dependent but not require calmodulin. for the KT5926-sensitive protein kinase activity in that were known to of the domain of a of serine/threonine protein kinases. The only for calcium-dependent protein kinases identified in an of protein kinase were two members of the of for protein kinases or protein kinase were were a both and by the of both are highly homologous to known these are to a TgCDPK1 and of Toxoplasma did not not The of TgCDPK1 a protein with a of TgCDPK2 is to be a protein with a of The of both proteins the typically in an N-terminal domain, a domain, and a C-terminal domain The two CDPK isoforms are identical to TgCDPK1 is homologous to the CDPKs in the apicomplexan parasites and tenella and TgCDPK2 is homologous to and falciparum and and soybean the of the protein but are in the in TgCDPK1 and in where they to found in protein kinases T. Science.. 1988; Scholar) are in both TgCDPK1 and a TgCDPK1 and as well as an in TgCDPK1 and in involved in also have a number of found in serine/threonine protein TgCDPK1 and in The junction of both in in are homologous to the junction and soybean The calmodulin-like C-terminal of TgCDPK1 and all known The with the calmodulin-like of other CDPKs to The of this domain and animal calmodulin is to the and not The of the Toxoplasma CDPKs to known CDPKs is shown in B. The CDPKs plants and Paramecium form two that are other and the CDPKs The latter form two the Eimeria and P. falciparum the TgCDPK2 and P. falciparum and to both TgCDPK1 and were proteins and were to their expression in the The are highly specific for of the and no is As can be in TgCDPK1 can be detected in parasite extracts as a protein with an of to the of The to TgCDPK2 to protein in tachyzoite although it with protein the that the TgCDPK2 might be of a TgCDPK2 expression in tachyzoites As can be in the TgCDPK2 can be detected by suggesting that the TgCDPK2 is but that expression of the protein be at the the TgCDPK2 protein not to be expressed in Toxoplasma tachyzoites, it was not TgCDPK1 calcium-dependent protein kinase activity. of TgCDPK1 by in protein kinase were at different concentrations of calcium of TgCDPK1 at and activity was at calcium concentrations and of activity was at calcium all were in the of Activation of TgCDPK1 was dependent on the of The of to in protein kinase to the activity of TgCDPK1 that with calcium not The of Toxoplasma calmodulin also to TgCDPK1 activity not The of of TgCDPK1 are shown in protein and in the of protein kinases were as TgCDPK1 and were which has been as a substrate for was a substrate for TgCDPK1 in the and of a number of it has been that in different of the substrate the of A.C. Yoo C. Biochemistry.. 1994; 33: Scholar). found that the and the of and and for the and found the to be and the to be with to the activity of TgCDPK1 the of a of on the of serine/threonine kinases no on the activity of an inhibitor of protein kinase an inhibitor of an in of for The of and calmodulin was not The inhibitor of TgCDPK1 was KT5926, a as an inhibitor of protein kinase and light kinase that are inhibited of and (6Hashimoto Y. Nakayama T. Teramoto T. Kato H. Watanabe T. Kinoshita M. Tsukamoto K. Tokunaga K. Kurokawa K. Nakanishi S. Biochem. Biophys. Res. Commun... 1991; 181: 423-429Google Scholar). As can be in TgCDPK1 is sensitive to KT5926 with of protein kinase activity at and at TgCDPK1 protein kinase activity and Toxoplasma attachment to host cells a identical sensitivity to KT5926 suggesting that TgCDPK1 is the of KT5926 in T. gondii and is for the attachment of the parasite host cells. the TgCDPK1 and the major KT5926-sensitive protein kinase activity detected during of Toxoplasma tachyzoite all by As can be in TgCDPK1 with the KT5926-sensitive protein suggesting that they are for in protein phosphorylation were and by and first identified proteins T. gondii that were phosphorylated as a result of calcium-dependent protein kinase this protein kinase parasite were in the and absence of and the were and Three major of calcium-dependent protein kinase activity were identified as well as the for gondii was at for to protein kinases. As is this is at the protein kinases. The of TgCDPK1 protein kinase in the calcium-dependent phosphorylation of a number of The three major TgCDPK1 identical to the major of calcium-dependent protein kinase activity in parasite with to and they to be identical to the three KT5926-sensitive phosphoproteins identified tachyzoites in vivo, In to the major also a number of for these are in they proteins that were during the of TgCDPK1 phosphorylation in the TgCDPK1 is a major protein kinase activity in parasite extracts of and that this only a of the protein kinase activity found in Toxoplasma This that other protein kinase are during and of extracts or that they are in the to In the or of or protein during the and of the extracts also result in an in its activity. calcium have been implicated in the regulation of host cell invasion by the apicomplexan Eur. J. Cell Biol... Scholar), P. falciparum M. J. Parasitol. 1996; Scholar), gondii tachyzoites (4Carruthers V.B. Sibley L.D. Mol. Microbiol... 1999; 31: 421-428Google Scholar, 7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar, Mol. Biochem. 2000; Scholar), suggesting that this process is by a in apicomplexan the specific calcium-dependent involved has not been identified inhibitor have implicated calmodulin and a light activity N. Bouchot A. Bonhomme A. Pingret L. Klein C. Burlet H. Balossier G. Bonhomme P. Pinon J.M. Eur. J. Cell Biol... 1997; 74: 92-101Google Scholar, 7Dobrowolski J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). the of calcium-dependent attachment to host cells as a of the CDPK members of this TgCDPK1 and were identified in T. gondii. TgCDPK1 protein is expressed in tachyzoites, TgCDPK2 protein is not in this stage of the Toxoplasma The of these protein kinases are highly homologous to of CDPKs in plants (8Harmon A.C. Putnam-Evans C. Cormier M.J. Plant Physiol... 1987; 83: 830-837Google Scholar, 9Putnam-Evans C.L. Harmon A.C. Cormier M.J. Biochemistry.. 1990; 29: 2488-2495Google Scholar, 10Harper J.F. Sussman M.R. Schaller G.E. Putnam-Evans C. Charbonneau H. Harmon A.C. Science.. 1991; 252: 951-954Google Scholar) and in the apicomplexan parasites P. falciparum (16Zhao Y. Kappes B. Franklin R.M. J. Biol. Chem... 1993; 268: 4347-4354Google Scholar, 17Li J.L. Baker D.A. Cox L.S. Biochim. Biophys. Acta.. 2000; 1491: 341-349Google Scholar) and tenella (18Dunn P.P. Bumstead J.M. Tomley F.M. Parasitology.. 1996; 113: 439-448Google Scholar). these enzymes, CDPKs are of three an N-terminal domain, a junction domain, and a C-terminal calmodulin-like domain with of TgCDPK1 in to the Toxoplasma also with other CDPKs in that its activity is dependent on the of calcium yet is to calmodulin the of the of TgCDPK1 are different other CDPKs with to the for and C.L. Harmon A.C. Cormier M.J. Biochemistry.. 1990; 29: 2488-2495Google Scholar, J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar). These the different substrate of these in vivo. of cellular CDPKs in their by calcium (19Lee J.Y. Yoo B.C. Harmon A.C. Biochemistry.. 1998; 37: 6801-6809Google Scholar). the activity of TgCDPK1 is at a often in activated cells, the activity of the P. falciparum is at calcium Y. Pokutta S. Maurer P. Lindt M. Franklin R.M. Kappes B. Biochemistry.. 1994; 33: 3714-3721Google Scholar). This the for different CDPKs to be activated at different of cellular although it also the absence of essential in the kinase for P. falciparum function for TgCDPK1 in T. gondii is suggested by its sensitivity to KT5926, an inhibitor of light kinases in animal cells. This was shown to be an inhibitor of Toxoplasma motility as well as its attachment to host cells. The of KT5926 for in kinase activity of TgCDPK1 and parasite attachment to host cells is In in that the of KT5926 on protein phosphorylation in Toxoplasma is it blocks the phosphorylation of only three These three proteins with to their and the three major of TgCDPK1 identified in Toxoplasma extracts in during of Toxoplasma a single peak of KT5926-sensitive protein kinase activity was which cofractionated with these that TgCDPK1 is the for KT5926 and is to be involved in parasite motility and attachment to host cells. The of TgCDPK1 are by the phosphorylation of the three detected in Toxoplasma The of these and the in which their phosphorylation to parasite motility and host attachment is unclear at this time. is suggested by the observation that of Toxoplasma with KT5926 the of the other proteins as the J.M. Carruthers V.B. Sibley L.D. Mol. Microbiol... 1997; 26: 163-173Google Scholar). is that phosphorylation of or of the TgCDPK1 is to an of such as of to their of or the of with the parasite the of and other by Toxoplasma to be an essential for parasite it is that all three TgCDPK1 are involved in is that and parasite motility are by TgCDPK1 phosphorylation of different of the TgCDPK1 shed light on these The role of TgCDPK2 is unclear at this time. its is detected in the tachyzoite stage of the Toxoplasma the protein is number of have been where the of are in both tachyzoites and but the proteins are only in the tachyzoites S. ( ).. 1997; Scholar) or the Mol. Biol... 1998; Scholar). the that TgCDPK2 protein be expressed in or The observation falciparum is only expressed in the stage of P. falciparum J.L. Baker D.A. Cox L.S. Biochim. Biophys. Acta.. 2000; 1491: 341-349Google Scholar) this The role of CDPKs in other apicomplexan parasites is unclear at this time. that all apicomplexan parasites are obligate intracellular parasites and that cytoplasmic calcium to be essential for host cell they are to calcium-dependent to host cell attachment and invasion. is that or of the CDPKs identified in other apicomplexan parasites a role to that of TgCDPK1 in Toxoplasma gondii. this apicomplexan CDPKs in of of the essential in parasite the to animal host cells. no members of the CDPK have been in the animal of apicomplexan parasites, these to be for the of for and and and Carruthers for also for the to are to for the of the CDPK calmodulin-like domain protein kinase T. gondii and