Tsutomu Takeuchi

Entamoeba dispar: Cultivation with sterilized Crithidia fasciculata

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica‐specific monoclonal antibody (4G6) could be grown in either Diamonds BI‐S‐33 medium, newly developed BCSI‐S (Biosate cysteine starch iron‐serum) medium, or casein‐free YI‐S medium in the presence of Crithidia fasciculata (ReF‐1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica‐ and E. dispar‐specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.

Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction

Abstractu2002An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4°C. The present PCR was sensitive enough to detect ten cysts of either of the amebae.

EFFECT OF DINITROANILINE HERBICIDES ON THE GROWTH OF ENTAMOEBA HISTOLYTICA

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 μM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 μM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.

Effect of cytochalasin D on the growth, encystation, and multinucleation of Entamoeba invadens

Abstract The effect of cytochalasin D, a specific inhibitor of microfilaments, on the growth, encystation, and multinucleation of Entamoeba invadens was examined. Cytochalasin D blocked the growth of axenic E. invadens strain IP-1 in a dose-dependent manner, which suggests that the drug is effective against this species of Entamoeba as well as against E. histolytica strain HM1: IMSS as previously demonstrated. Encystation of E. invadens as induced in vitro was also inhibited by cytochalasin D. This is the first evidence of the participation of microfilaments in the encystation process. Concentrations of cytochalasin D effective for the inhibition of encystation were lower than those effective for the inhibition of growth. Trophozoites grown with cytochalasin D became multinucleate; more than three nuclei per cell were observed in 71% of trophozoites grown in the presence of the drug as opposed to only 5% of those grown in the absence of the drug. Also, trophozoites grown with cytochalasin D produced multinucleate cysts following their transfer to encystation medium. Encystation with cytochalasin D was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Also, encystation without cytochalasin D was less frequently observed among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Thus, the multinucleation of trophozoites induced by cytochalasin D had an inhibitory effect on their encystation.

EFFECT OF JASPLAKINOLIDE ON THE GROWTH, ENCYSTATION, AND ACTIN CYTOSKELETON OF ENTAMOEBA HISTOLYTICA AND ENTAMOEBA INVADENS

The effect of jasplakinolide, an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 µM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.

Detection and characterization of DNA polymerase activity in entamoeba histolytica

DNA polymerase activity was detected and characterized in nuclear extracts from trophozoites of Entamoeba histolytica. The activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low. The presence of K+ was inhibitory for the activity and a higher concentration of K+ markedly inhibited the activity. Magnesium ions (Mg+) were absolutely required for activity and its optimal concentration was 6 to 8 mM. The activity was markedly inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, 8, and ε and also by N-ethylmaleimide which is an inhibitor of DNA polymerases, α, γ δ and ε. However, inhibition of the activity by 2′, 3′-dideoxythymidine-5′-triphosphate which is an inhibitor of DNA polymerases β and γ was relatively weak. Thus sensitivity of the E. histolytica enzyme to these inhibitors was similar to that of mammalian DNA polymerases (α, δ and ε) of the α family. Monoclonal antibodies against human DNA polymerase α did not bind to DNA polymerase of E. histolytica.

DNA polymerase activity in encysting Entamoeba invadens.

Abstract Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, we examined the level of DNA polymerase activity in E. invadens during encystation induced in vitro. We first characterized the DNA polymerase activity of trophozoites of E. invadens, comparing it with that of E. histolytica, and found that the activity of E. invadens was lower than that of E. histolytica at pH 2, 4, and 6 and was higher at pH 8 and 10. The activity of E. invadens was completely inhibited by high concentrations of K+. Among inhibitors of mammalian DNA polymerases, aphidicolin and N-ethylmaleimide inhibited the activity, but 2′,3′-dideoxythymidine-5′-triphosphate did not. Thus, the sensitivity of the E. invadens activity to salt and inhibitors of mammalian DNA polymerases was basically the same as that recorded for E. histolytica in our previous results. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs during cyst maturation in the absence of DNA synthesis.

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens.

Abstract The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1–4u2009days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1u2009day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1u2009day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiffu200as reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1u2009day of incubation and appears to be associated with the cyst wall.

Effects of aphidicolin on Entamoeba histolytica growth and DNA synthesis

We have detected and characterized DNA polymerase activity in cell extracts from trophozoites of Entamoeba histolytica and have found that the activity of E. histolytica is inhibited by aphidicolin, which is a specific inhibitor of eukaryotic nuclear replicative DNA polymerases. The present study was aimed to evaluate the effect of aphidicolin on growth and DNA synthesis by this parasite. Aphidicolin blocked the growth of axenic E. histolytica strain HM-1: IMSS. DNA synthesis was also inhibited by aphidicolin when assayed by incorporation of [3H] thymidine into the DNA. The inhibitory effect of aphidicolin on the growth of E. histolytica was abrogated by removal of the drug, and exposure to 3 microg/ml of the drug for at least 48 hr had little effect on the viability. Synchronous growth was observed in the recovery phase after removal of aphidicolin.

Cultivation of Entamoeba dispar: growth-promoting effect of ferredoxin.

We designed a medium (YIGADHA-S) (1) for the trial of axenic cultivation of Entamoeba dispar based on the caseinfree YI-S medium (YI-S) (2,3). YIGADHA-S is different from YI-S in that glucose is replaced by gluconic acid and dihydroxyacetone, and its sterilization is carried out by filtration. Consequently, one strain of E. dispar (CYNO16:TPC) was adapted to this YIGADHA-S and grew stably. However, in this YIGADHA-S culture system, other strains of E. dispar still required a culture associate, such as 10% formalin fixed or autoclaved Pseudomonas aeruginosa or Crithidia fasciculata. Moreover, we recently found that over 18 different cells of animal or plant origin, which were autoclaved (121 8 C, 15 min), could also support the growth of E. dispar. These studies led us to search for a property common to every cell examined (e.g., protozoan, mammalian, and plant cells) for supporting the growth of E. dispar in YIGADHA-S. These cells had either mitochondria, hydrogenosome, or mitochondria and chloroplast. On the other hand, some protozoan cells [ E. histolytica , E. dispar , E. moshkovskii (Laredo), E. invadens (IP-1), Giardia lamblia (Portland I)], and human erythrocytes, which lacked mitochondria or mitochondria-like organelles, did not support the growth of E. dispar in this medium. Based on these findings, we speculated that some substance existing commonly in mitochondria, hydrogenosomes, chloroplasts, and bacteria might support the growth of E. dispar. The present study was attempted to test the growth-promoting effect of a plant nonheme iron–sulfur protein (ferredoxin) on E. dispar in culture. Materials and Methods