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....
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.
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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제안 방법
z 1999). In this study, extracellular and intracellular calcium delators, calci um ionophore, calcium channel blockers, and calmodulin antagonist were used to reduce the Ca2+ in T. gondii. When tachyzoites were co-treated with the Ca2+ ionophore A23187 and calcium chelator EGTA, invasion into HeLa cells was reduced at 4 hr and 18 hr PL Treatment of A23187 and EGTA effectively suppressed tachyzoite invasion compared to tachyzoites treated with A23187 or EGTA alone.
Therefore, the present experiment was undertaken to determine the influence of Ca2+ in T. gondii during host cell invasion and intracellular replication by using calcium modulators and calmodulin antagonist. Moreover, changes of intracellular Ca2+ and calmodulin in tachyzoites were observed by confocal and electron microscopy.
대상 데이터
gondii were treated with 1 μM calmidazolium in RPMI 1640 for 1 hr, fixed with 2% glutaraldehyde for 3 hrz and then post-fixed with 1% osmium tetroxide. Specimens were sequentially dehydrated in 20%, 50%, 70% and 100% ethanol, and embedded in LR white. Ultrathin sections on a nickel grid were incubated with NH4C1z 0.
데이터처리
The Mann-Whitney U test was used for statistical analysis. All experimental data are presented as means ± standard deviation.
성능/효과
The results of the present study suggest that intra cellular Ca2+ changes in T. gondii, especially due to calcium ionophores and extracellular calcium, are involved in the invasion and intracellular replication of T. gondii, and further suggest that calmodulin, which is located in anterior portion of T. gondii tachyzoitesz might also be related to host cell invasion. The influence of the intracellular Ca2+ depletion of host cells on the infectivity of T.
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