Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.
PLoS Negl Trop Dis. 2010 Feb 16;4(2):e607. doi: 10.1371/journal.pntd.0000607.
Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment.
抑制包囊形成可以被视为一种潜在有用的机制，以阻止在自然条件下溶组织内阿米巴的传播。不幸的是，体外无法实现阿米巴包囊形成，也没有可抑制囊形成的药物。体内诱导包囊形成的腔道条件也不清楚，但细胞应激，如暴露于免疫细胞或肠道微生物群产生的活性氧，可能会参与其中。某些二价阳离子作为参与脱囊的酶的辅助因子的作用也已被描述。在这项研究中，我们表明，在痕量几种阳离子存在的情况下，用双氧水处理滋养体培养物，会转化为小型的球形和折光性结构，这些结构对不同的去污剂具有抗性。扫描和透射电子显微镜下的超微结构分析显示出多核结构（有些有四个核），具有光滑、厚实的膜和多个空泡。用Calcofluor White 染色以及使用麦胚凝集素的 ELISA 结合测定表明存在 N-乙酰葡萄糖胺（几丁质）的聚合物，几丁质是天然囊壁的主要成分。通过 RT-PCR 也证实了葡萄糖胺 6-磷酸异构酶的过表达，这可能是几丁质合成途径中的限速酶。这些结果表明，当暴露于我们的处理时，溶组织内阿米巴滋养体激活了包囊形成途径。
