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

Abstract

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-dependentbutdidnotrequirecalmodulin.Inasearchforcalciumdependent 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 Toxoplasma tachyzoites 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 regulating Toxoplasma motility and host cell invasion.

A novelToxoplasma gondiicalcium-dependent protein kinase

“…Our results demonstrate that as for TgCDPK1 described by Kieschnick et al, 2001, TgCDPK4 can phosphorylate both MBP and histone H1 in vitro and that incubation with calcium ions induced an increase in its kinase activity, demonstrating Ca 2+ -dependence. On the contrary, the TgCDPK3 recently described by Donald et al, 2006 was insensitive to EGTA and not dependent on exogenous calcium.…”

“…The predicted amino-acid sequence of TgCDPK4 shows similarity to CDPKs of plants and apicomplexan parasites. Among the 20 CDPK or CDPK-related kinases identified in T. gondii, only TgCDPK1 (Kieschnick et al, 2001) and TgCDPK3 proteins (Donald et al, 2006) were detected in tachyzoïtes. Because TgCDPK4 shares low identities with other TgCDPKs isoforms, we can assume that anti-TgCDPK4 antibodies are specific to TgCDPK4 and the cross reactivity with other TgCDPKs isoforms seems unlikely.…”

“…T. gondii contains more than 20 CDPK or CDPK-related kinases (Nagamune & Sibley, 2006). In the present, two related T. gondii CDPK isoforms have been recently identified, where TgCDPK1 seems to play a central role in regulating parasite motility and host cell invasion (Kieschnick et al, 2001). Indeed, TgCDPK1 was found to be inhibited by KT5926, a selective inhibitor of calmodulin-dependent and myosin light chain kinase, which is known to block the mobility of T. gondii (Dobrowolski et al, 1997).…”

N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii

“…The isolated parasites were also tested for their ability to perform gliding motility and to attach to and invade HFF cells as described before [22].…”

Molecular and functional aspects of parasite invasion

“…The latter source of Ca 2C is also likely to contribute to the loading of parasite intracellular Ca 2C stores, a prerequisite for the use of a Ca 2C -based signaling mechanism [35]. The signaling cascade that operates downstream of Ca 2C transients, and which leads to invasion, has not been resolved but probably involves one of the calmodulin-like domain protein kinases (CDPK), previously described in T. gondii [36] and P. falciparum [37].…”