The Transcriptional Response to Encystation Stimuli in Giardia lamblia Is Restricted to a Small Set of Genes

Abstract

The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a ␤(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors.The differentiation of Giardia lamblia cells to cysts is a key step in the simple life cycle of this ubiquitous intestinal parasite. Induced trophozoites, the flagellated, motile, and attachment-competent stage, exit the cell cycle at the G 2 -M transition (1) and begin to synthesize the components of an extracellular matrix, the cyst wall (CW). Synthesis and export of the cyst wall proteins (CWPs) to specialized organelles, termed encystation-specific vesicles (ESVs), is completed between 8 and 10 h postinduction (p.i.). After proteolytic cleavage of the CWP2 C terminus, the CWPs are sorted into two fractions which are deposited sequentially and polymerize on the surface of the cyst (18). The first layer of the CW, which eventually completely encloses the parasite (19,24), is secreted in the last minutes of differentiation simultaneously with nuclear division and morphological transformation of the cell (cyst formation). In vitro, the entire encystation process typically takes 20 to 24 h in our hands and can be induced by modifying medium components and/or the concentrations of bile, fatty acids (cholesterol), or lactic acid (3,10,15,22). Because the condition(s) which induce encystation in vivo are not known, all factors and components mentioned above are implicated since their concentrations vary in the small intestine, as does the pH, which increases from ϳ6 in the duodenum to 7.5 to 8.0 in the distal ileum. Although all available data indicate that differentiation is induced by one or several environmental signals, the mechanism(s) for reception and transduction have not been characterized. Upregulation...

UV irradiation responses in Giardia intestinalis

“…In recent years several studies have been performed studying transcriptional changes during different stress conditions in Giardia (for example DTT, drugs, differentiation, host-interaction and oxidative stress) (Birkeland et al, 2010;Morf et al, 2010;Muller et al, 2008;Raj et al, 2014;Ringqvist et al, 2011;Spycher et al, 2013). However, this is the first report on transcriptional profiling on Giardia after UV irradiation.…”

Comparative biochemistry of Giardia, Hexamita and Spironucleus: Enigmatic diplomonads

“…The best understood diplomonad life cycle is that of G. intestinalis, where encystment to give the infectious agent, a process essential for survival and involving massive turnover of trophozoite components, only requires transcriptional up-regulation of a relatively small cohort of core genes numbering 18 [49] during the initial stages of encystations, but >300 during the later steps to complete differentiation [50]. This process of transmission and pathogenesis has been probed by proteomics [51].…”

The generation gap: Proteome changes and strain variation during encystation in Giardia duodenalis

“…Teardropped trophozoites, which are flagellated and binucleate (4N), become increasingly rounded, lose the ability to attach to host cells, http internalise their flagella, disassemble median bodies and become immotile cysts with 4 nuclei and 16N ploidy [3,4]. Trophozoites bound for encystation are shunted out of the cell-cycle at the G 2 -M transmission to begin the process, which takes approximately 20-24 h [5]. Early encystation is characterised by cyst wall (CW) formation and the presence of encystation-specific vesicles (ESV) within the first 8-10 h post induction (p.i).…”