Entamoeba invadens: Enhancement of Excystation and Metacystic Development by Cytochalasin D

et al. 2002

Parasitology Research

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The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.

Section: Discussionmentioning

confidence: 98%

“…The transfer of cysts from an encystation medium to a growth medium is necessary for the induction of excystation of E. invadens (Makioka et al 2001b). This means that the signal of the medium change is transduced from the cyst wall to nuclei to trigger excystation.…”

Section: Discussionmentioning

confidence: 99%

Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens

Makioka

Kumagai

et al. 2002

Parasitology Research

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“…Treatment of trophozoites with 0.25-0.5 lM of latrunculin A, an actin inhibitor [Makioka et al, 2001], resulted in the immobilization of trophozoites and detachment from the slide glass. The ingestion of the FITC-yeast particle was also significantly reduced (data not shown).…”

Section: Inhibition Of Microtubules Actin or Myosin Affects The Kinmentioning

confidence: 99%

“…To test the effects of phagosome acidification and degradation, 4-6 3 10 5 trophozoites of E. histolytica or E. dispar were pretreated for 1 h with 10 lM concanamycin A, a specific inhibitor for V-ATPase; 20-200 lM of nocodazole, 100-200 lM of oryzalin, inhibitors for microtubule polymerization [Makioka et al, 2000], 0.25-0.50 lM of latrunculin A, an inhibitor of actin polymerization [Makioka et al, 2001] or 20-40 mM of BDM, an inhibitor of myosin ATPase [Borlak and Zwadlo, 2004] or 200 lM of E64, a CP inhibitor in 13 3 100 mm 2 screwcapped Pyrex glass tubes at 35 8 C in BI-S-33 or YIGADHA-S medium, respectively.…”

Section: Treatment Of Parasites With Inhibitorsmentioning

confidence: 99%

Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenicEntamoeba histolytica and the non-pathogenicEntamoeba dispar

Mitra

Yasuda

Cell Motil. Cytoskeleton

et al. 2005

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Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.

“…The numbers of nuclei per metacystic amoeba stained with modified Kohn at 5, 24 and 72 h of incubation were counted and the percentage of amoebae in each class (1-to 7-nucleate) was determined reorganization of the actin cytoskeleton is necessary for these excystation events. We demonstrated that the actin-modifying drugs latrunculin A and jasplakinolide inhibited the excystation and metacystic development of E. invadens (Makioka et al 2001b). Regarding the relation between the reorganization of the actin cytoskeleton and PKC, Santiago et al (1994) demonstrated an interaction of E. histolytica trophozoites with the fibronectin-induced reorganization of the actin cytoskeleton and an increase in proteolytic activities through the activation of PKC pathways.…”

Section: Discussionmentioning

confidence: 93%

Involvement of signaling through protein kinase C and phosphatidylinositol 3-kinase in the excystation and metacystic development of Entamoeba invadens

Makioka

Kumagai

et al. 2003

Parasitology Research

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Using an axenic excystation system in vitro, we examined the effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the PKC inhibitors staurosporine, chelerythrine chloride and calphostin C in a concentration-dependent manner during incubation, compared with the controls. As cyst viability was not affected by these inhibitors, reduced excystation was not due to their direct toxic effects on cysts. Metacystic development, when determined by the number of nuclei in the amoebae, was delayed by these PKC inhibitors, because the percentage of 1-nucleate amoebae was lower than in controls at day 3 of incubation. Wortmannin, a potent inhibitor of PI3K, also inhibited excystation and metacystic development of E. invadens in a concentration-dependent manner, compared with the controls. These results indicate that signaling through PKC and PI3K contributes to the excystation and metacystic development of E. invadens.