Chitin, a homopolymer of β-(1,4) linked N-acetylglucosamine (GlcNAc), is a major component of cyst wall in the protozoan parasites Entamoeba histolytica (Eh) and Entamoeba invadens (Ei). The Entamoeba chitin synthase makes chitin at the vesicular membrane rather than the plasma membrane in fungi, even though the chemistry of chitin synthesis is most likely the same. However, the role of nucleotide sugar transporter(s) (NSTs) that are involved in chitin synthesis in Entamoeba are not yet established. In this study, we have identified the putative UDP-GlcNAc transporter (EiNst5) of Ei by BLASTP analysis using the amino acid sequence of EhNst3, the UDP-GlcNAc transporter of Eh. Heterologous expression of both EhNst3 and EiNst5 was found to complement the function of Yea4p (UDP-GlcNAc transporter of S. cerevisiae) in YEA4 null mutant and increased the cell wall chitin content. Like Yea4p in S. cerevisiae, Myc-epitope tagged EhNst3 and EiNst5 were localized to the endoplasmic reticulum in Δyea4 cells. The EiNST5 transcript was up-regulated during the in vitro encystation and oxidative stress in E. invadens. Similar up-regulation was also seen for EhNST3 under oxidative stress in E. histolytica. Down-regulation of EiNst5 expression using gene-specific dsRNA significantly reduced cyst formation during in vitro encystation in E. invadens. Our observations suggest for the first time the involvement of EhNst3 and EiNst5 in chitin synthesis and so in encystation of Entamoeba.

几丁质是一种与β-（1,4）连接的N-乙酰氨基葡萄糖（GlcNAc）的均聚物，是原生动物寄生虫组织解脂虫（Etamoeba histolytica ）（Eh）和入侵Entamoeba invadens（Ei）中囊壁的主要成分。该阿米巴甲壳素合成酶甲壳素使得在囊泡膜而不是真菌细胞膜，即使几丁质合成的化学性质很可能是相同的。然而，尚未确定参与Entamoeba中的几丁质合成的核苷酸糖转运蛋白（NST）的作用。在这项研究中，我们使用EhNst3的氨基酸序列，即EhNst3的UDP-GlcNAc转运蛋白的氨基酸序列通过BLASTP分析鉴定了Ei的推定UDP-GlcNAc转运蛋白（EiNst5）。呃。EhNst3和EiNst5的异源表达被发现可以补充Yea4p（酿酒酵母的UDP-GlcNAc转运蛋白）在YEA4无效突变体中的功能，并增加细胞壁几丁质含量。像Yea4p在酿酒酵母，Myc的表位标记的EhNst3和EiNst5分别定位到在Δ内质网是啊4个细胞。EiNST5转录本在体外侵染和氧化应激中被上调。在溶组织性大肠杆菌中氧化应激下，EhNST3也有类似的上调。使用基因特异性dsRNA下调EiNst5表达可显着减少体外囊肿形成入侵大肠杆菌（E. invadens）。我们的观察结果首次提示EhNst3和EiNst5参与甲壳素的合成，从而参与了Entamoeba的侵染。