Nucleotide sugar transporters of Entamoeba histolytica and Entamoeba invadens involved in chitin synthesis

Chitin, a homopolymer of beta-(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 Ayea4 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.