High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions.

Dimitra Peirasmaki,Staffan G Svärd,Showgy Y Ma’Ayeh,Sara Campos,Feifei Xu,Jingyi Liu,Marcela Ferella

doi:10.3389/fgene.2020.00913

Abstract

Giardia intestinalis colonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) in Giardia, which might relate to the establishment of infection and disease induction. Giardia trophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles during Giardia-host cell interactions.

Highlights

Diarrheal disease is still a major cause of death in children under 5 years of age, it is both preventable and treatable (Kotloff et al, 2013)
RNA sequencing was performed on Giardia intestinalis trophozoites incubated with and without differentiated Caco-2 cells, in three biological replicates
An average of 40.4 million paired-end reads were obtained for the starting trophozoite population (DMEM pre-incubated trophozoites), amongst which 79.4% mapped to the Giardia WB reference genome (Xu et al, 2020)

Summary

Introduction

Diarrheal disease is still a major cause of death in children under 5 years of age, it is both preventable and treatable (Kotloff et al, 2013). It is the leading cause of malnutrition in young children (Kotloff et al, 2013). If the disease becomes chronic, symptoms of malabsorption (e.g., malaise and weight loss) become more prominent (Farthing, 1997; Ortega and Adam, 1997; Gascón, 2006) and children can become stunted (Rogawski et al, 2017)

Methods

Results

Conclusion