A Detailed Gene Expression Map of Giardia Encystation

Abstract

Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

Dual RNA Sequencing Reveals Key Events When Different Giardia Life Cycle Stages Interact With Human Intestinal Epithelial Cells In Vitro

“…Genes encoding kinases, regulators of the cell cycle, and arginine metabolism and cytoskeletal proteins were the main down-regulated genes in the interacting trophozoites ( Peirasmaki et al., 2020 ) ( Figure 7 ). This can also be seen in the encysting cells ( Figure 7 and Table S4 ), but the most down-regulated genes in 7 h encysting cells during host cell interactions are up-regulated genes during encystation ( Rojas-López et al., 2021 ); ORFs 5638, 5435, and 2421 cyst wall proteins 1 to 3, 4245 and 11151 hypothetical proteins, 114495 and 137701 NEK kinases, 10552 Ephrin-like receptor, 14626 Oxidoreductase, 112341 Transmembrane protein, 27806 Myb-like protein, 113610 GlcNAc-PI synthesis, and 88581 synaptic glycoprotein ( Table S4 ). This suggests that encysting cells are inhibited in the encystation process during the interaction with Caco-2 cells, and follow-up experiments showed that fewer cysts are produced when 7 h encysting cells are transferred to DMEM or to interacting Caco-2 cells ( Figure S9 ).…”

Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics

“…Although reduced encystation efficiency was noted in the M1 and M2 isolates studied here, the underlying molecular rationale is not known as it is still unclear how encystation is initiated at the molecular level ( Rojas-López et al, 2021 ). The encystation process is complex, as evidenced by the extensive and coordinated gene regulatory program that is associated to formation of viable cysts, which holds true for the early part of the process as well ( Rojas-López et al, 2021 ). Thus, the substantial transcriptomic and proteomic changes the parasite needs in order to tolerate MTZ may be in their sum too costly to afford creation of transmission stages.…”

“…In the case of G. intestinalis, evolution of MTZ resistance appears to be strongly associated with a significant and multimodal fitness cost (reduced growth rate, attachment, encystation, and animal infectivity), which might explain why it took decades for drug resistance to develop. Although reduced encystation efficiency was noted in the M1 and M2 isolates studied here, the underlying molecular rationale is not known as it is still unclear how encystation is initiated at the molecular level (Rojas-López et al, 2021). The encystation process is complex, as evidenced by the extensive and coordinated gene regulatory program that is associated to formation of viable cysts, which holds true for the early part of the process as well (Rojas-López et al, 2021).…”

A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia

“…Giardia's cyclin dependent kinase 2 (CDK2) is upregulated at G2+M (46) and has been shown to activate MYB2 through phosphorylation (47). Two recent RNAseq studies also identified an encystation-upregulated cyclin (GL50803_15532) (13) and G2-dependent TF GLP1 (GL50803_7272) (46). In the future, it would be interesting to explore how GLP1, GLP4, CDK2, and cyclin work together to initiate encystation.…”

“…Giardia's cyst wall is composed of 37% cyst wall proteins (CWPs) and 63% carbohydrate filaments which are mainly made up by a [D-GalNAc-β(1-3)-D-GalNAc] n homopolymer (10)(11)(12). The expression levels of CWP1-3 are upregulated at 2-4 h post induction of encystation (13)(14)(15). The enzymes involved in the synthesis of β(1-3) GalNAc homopolymer, including GlcN 6-P isomerase (G6PI-B), GlcN 6-P N-acetylase (GNPNAT), phosphoacetylglucosamine mutase (PGM), UDP-GlcNAc pyrophosphorylase (UAP), UDP-GlcNAc 4'-epimerase (UAE) are upregulated at 10-18h post induction of encystation (10,11,13,15).…”