Calmodulin Distribution and the Actomyosin Cytoskeleton in <i>Toxoplasma gondii</i>

Pezzella–D'Alessandro, Nathalie; Moal, H. Le; Bonhomme, A.; Valere, Audrey; Klein, Christophe; Gómez‐Marín, Jorge Enrique; Pinon, J. M.

doi:10.1177/002215540104900404

Cited by 26 publications

(28 citation statements)

References 22 publications

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“…14). It is therefore noteworthy that recent immunofluorescence studies of the calmodulin-actomyosin complex in intracellular T. gondii disclose an apically focused pattern very similar to that found for the V-PPase in this study (46) and that earlier studies demonstrated an anteriorally displaced concentration of intracellular Ca 2ϩ within unidentified compartments, possibly acidocalcisomes, in the same organism (47). Knowing that microneme discharge, one of the first events in host cell invasion, requires the mobilization of intracellular Ca 2ϩ stores (48), it is conceivable that acidocalcisomes serve as a source of this Ca 2ϩ .…”

Section: Discussionsupporting

confidence: 87%

“…Knowing that microneme discharge, one of the first events in host cell invasion, requires the mobilization of intracellular Ca 2ϩ stores (48), it is conceivable that acidocalcisomes serve as a source of this Ca 2ϩ . Acidocalcisomes may thereby provide the Ca 2ϩ required for activating the calmodulin-dependent myosin light chain kinases that regulate the actomyosin motor that governs parasite motility and host cell invasion (46). If this were the case, the V-PPase would provide the motive force for acidocalcisomal Ca 2ϩ accumulation by H ϩ /Ca 2ϩ antiport.…”

Section: Discussionmentioning

confidence: 99%

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Isolation and Characterization of TgVP1, a Type I Vacuolar H+-translocating Pyrophosphatase fromToxoplasma gondii

Drozdowicz¹,

Shaw

Nishi

et al. 2003

Journal of Biological Chemistry

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Here we report the isolation and characterization of a type I vacuolar-type H ؉ -pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire Nterminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmidborne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K ؉ -activated pyrophosphate (PP i ) hydrolysis and PP i -energized H ؉ translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 M had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I 50 for the inhibition of TgVP1 activity in vitro (0.9 M) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisomelike vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.Vacuolar-type H ϩ -translocating inorganic pyrophosphatases (V-PPases) 1 are primary electrogenic proton pumps that derive their energy from the hydrolysis of inorganic pyrophosphate (PP i ) (1). Long considered to be restricted to plants and certain photosynthetic bacteria, V-PPases have recently been identified in a wide range of organisms, including prokaryotic extremophiles and the kinetoplastid protists Trypanosoma and Leishmania, the causative agents of Chagas' disease and leishmaniasis, and the apicomplexan protists Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis (reviewed in Ref.2). The discovery of V-PPases in these parasitic protists has attracted much attention. The seemingly complete absence of V-PPases from their animal hosts has given rise to the exciting possibility that this enzyme might serve as an effective drug target for a nu...

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of TgVP1, a Type I Vacuolar H+-translocating Pyrophosphatase fromToxoplasma gondii

Drozdowicz¹,

Shaw

Nishi

et al. 2003

Journal of Biological Chemistry

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“…The relationship between passively forced conoid extrusion and infectivity of T. gondii would be investigated. The actin and myosin filaments that influence the motilities of tachyzoites are also located at the apical pole, and calmodulin is located at the anterior region of T. gondii tachyzoites during the extracellular state (Pezzella-D'Alessandro et al, 2001). In our experiment, calmodulin and actin were observed at the anterior end of tachyzoites (Figs.…”

Section: Discussionmentioning

confidence: 99%

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

et al. 2004

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Abstract:Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca 2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca 2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca 2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca 2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.

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“…Conoid extrusion induced by Ca 2+ ionophores can be inhibited by CD, indicating a participation of actin filaments (Mondragón and Frixione, 1996). Previous studies have shown actin localized at the apical end (Yasuda et al, 1988;Pezzella-D'Alessandro et al, 2001) and as part of the subpellicular network (Patrón et al, 2005). As the conoid is directly related to the subpellicular network cytoskeleton (Nichols and Chiappino, 1987;Patrón et al, 2005), it was reasonable for us to evaluate the role of cytoskeleton proteins under the conoid extrusion induced by ethanol.…”

Section: +mentioning

confidence: 97%

Induction and regulation of conoid extrusion inToxoplasma gondii

Carmen

Mondragón

González

et al. 2009

Cellular Microbiology

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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.

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Calmodulin Distribution and the Actomyosin Cytoskeleton in Toxoplasma gondii

Cited by 26 publications

References 22 publications

Isolation and Characterization of TgVP1, a Type I Vacuolar H+-translocating Pyrophosphatase fromToxoplasma gondii

Isolation and Characterization of TgVP1, a Type I Vacuolar H+-translocating Pyrophosphatase fromToxoplasma gondii

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Induction and regulation of conoid extrusion inToxoplasma gondii

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