Induction and regulation of conoid extrusion inToxoplasma gondii

Abstract

SummaryCell invasion by the intracellular parasite Toxoplasma gondii occurs through an active process that involves dynamic events, such as gliding motility and conoid extrusion, followed by a sequential secretion from specialized secretory organelles. Increase of intracellular Ca2+ by ionophores induces conoid extrusion, although in an irreversible way, thus limiting the characterization of the regulatory pathways. In this report we studied the effect of different activating conoid conditions to characterize the regulatory mechanisms involved. Exposure of tachyzoites to ethanol, a well-known activator of microneme secretion through the increase of intracellular Ca 2+, induced conoid extrusion without affecting parasite viability nor its in vitro invasive capability, in a process that could be completely reverted and repeatedly reactivated. A temporal relationship between conoid extrusion and microneme secretion was here studied. Under this condition, signal transduction pathways and the precise role of the parasite cytoskeleton were characterized. Our results indicate that phospholipase C, Ca 2+ released through channels sensitive to inositol-3-phosphate and ryanodine, as well as myosin together with actin filaments, but not microtubules, all participate in conoid extrusion. Specific inhibitors for serine-threonine kinases blocked conoid extrusion; in contrast, calmodulin inhibitors did not affect the induction. A regulatory model for conoid activation is here proposed.

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle

“…Using Ca 2ϩ ionophores, Ca 2ϩ -chelating agents, and ethanol, a link between Ca 2ϩ and conoid extrusion was postulated (8,46), although no direct parallel Ca 2ϩ measurements were reported. The effect of Ca 2ϩ on gliding motility was studied on trypsin-permeabilized tachyzoites (47) or by measuring Ca 2ϩ oscillations in extracellular tachyzoites loaded with the Ca 2ϩ dye Fluo-4/AM (7,35).…”

“…Previous studies on the role of Ca 2ϩ fluctuations in gliding motility (7), conoid extrusion (8), microneme secretion (9 -11), host cell invasion (7,12), and egress (13) were done using indirect methods, such as labeling extracellular parasites with fluorescent dyes and following Ca 2ϩ changes during their gliding motility (7) and using Ca 2ϩ ionophores and other exogenous agents to elevate Ca 2ϩ in extracellular parasites stimulating conoid extrusion (8) or microneme secretion (9 -11). Another indirect strategy was the use of intracellular or extracellular Ca 2ϩ chelators to prevent host cell invasion (7,12) or egress (13).…”

Dynamics and 3D organization of secretory organelles of Toxoplasma gondii

“…We consider this proposition reasonable, as microneme secretion and conoid extrusion are both dependent on calcium and can be induced by calcium ionophores (Carruthers and Sibley, 1999;Lovett and Sibley, 2003;Mondragon and Frixione, 1996;González Del Carmen et al, 2009). Thus, micronemes would require that the conoid be extruded in order to dock and fuse with a specific domain at the plasma membrane.…”

SPM1 Stabilizes Subpellicular Microtubules in Toxoplasma gondii

“…The conoid is composed of tubulin sheets (42) that spiral to form a cone-shaped organelle that can extend beyond the APR or retract within it to be surrounded by the basket of subpellicular microtubules (64). Conoid extension is thought to facilitate host cell invasion and is dependent upon calcium signaling (18,57,58,66).…”