Establishment of axnic culture of non-invasive Entamoeba dispar and study on its biological characterization

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We developed an axnic cultivation system for Entamoeba dispar which is difficult to grow in usual axnic medium of Entamoeba histolytica without intestinal bacteria or some protozoa such as Crithidia fasciculata.Since Clark (1995) reported successful axnic cultivation of strain SAW 760 RR clone A of E.dispar, other strains also have been tried for the axnic cultivation. However, E.dispar strain which could be adapted to axnic culture conditions is only the SAW 760 RR clone A,up to the present.In this research project, we found one metabolic intermediate (6-phosphogluconate : 6PG) which could promote the growth of three strains, at least, of E.dispar. This finding followed our study on growth promoting activities in the extract of Pseudomonas aeruginosa. The basic monoxemic culture system of E.dispar with P.aeruginosa needed modification of the medium for axnic Entamoeba histolytica (BI-S-33 medium) as follows : the addition of acetone or dihydroxyacetone, removal or replacement of gluco … More se with maltose or hydrlized starch and sterilization by filtration. In this system, the number of E.dispar ameba isolates (over 12 strains) usually reached 2-3 x 10^5/ml at those growth peaks, constantly.We tried modification of this system and design a new medium for cultivation of E.dispar axnically. Basic medium for the axnic culture was casein-free YI-S (Diamond, 1995) by which Clack (1995) reported successful axnic cultivation of E.dispar by supplementation of gastric mucin.A E.dispar growth promoting activity could be found in the polysaccharide rich fraction of P.aeruginosa, and it was more effective in gluconate and dihydroxyacetone supplemented YI-S medium. Though gluconate kinase and glucose-6-phosphate dehydrogenase (G6PDH) for producing 6PG could not be detected in the extract of E.dispar, gluconate seemed to be metabolized by E.dispar in the presence of P.aeruginosa extract. And, interestingly, it was found that 6PG rich fraction made from glucose-6-phosphate (G6P), NADP and crude extract of Crithidia fasciculata which has extremely high G6PDH ativity and also gluconate kinase activity, has the growth promoting effect. It was further confirmed that this growth promoting effect was attributed to 6PG by using commercial G6PDH and G6P.These results are still preliminary but seem clear and reproducible, so we are now studying the role of 6PG in the axnic growth of E.dispar and the related glycolytic pathway such as Entner-Doudoroff pathway which was reported to be present in xenic E.histolytica (Hilker and White, 1959). Less