Abstract
Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1, cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.
Highlights
Giardia intestinalis is a parasitic protist that infects the small intestines of mammals, including humans (Thompson, 2000)
Between 82 and 84% of each raw read library were successfully mapped to the human genome, and at least 90% of the mapped reads fell within annotated coding domains (12,266 in total), which were transcribed across all experimental Caco-2 cell samples
RNA levels of the same Differentially Transcribed Genes (DTGs) were compared between parasitized proliferating and differentiated Caco-2 cells and this showed significant differences between the two groups at each time point (1.5 h P = 0.0006, 3 h P = 0.0096 and 4.5 h P = 0.0015) with a noticeable higher fold change for most of the genes in differentiated intestinal epithelial cells (IECs) (See heat map, Figure S2)
Summary
Giardia intestinalis is a parasitic protist that infects the small intestines of mammals, including humans (Thompson, 2000). Trophozoites possess a unique structure, the ventral disc, which allows parasite attachment to intestinal epithelial cells (IECs) This begins a process of interaction with host cells, leading to cellular damage, change in cell size and shape (Chávez et al, 1995; Teoh et al, 2000; Humen et al, 2011; Maia-Brigagão et al, 2012), cell cycle arrest (Stadelmann et al, 2012), induction of apoptosis (Chin et al, 2002; Panaro et al, 2007), villus atrophy (Buret et al, 1990; Panaro et al, 2007), digestive enzyme deficiencies (Humen et al, 2011) and electrolytes imbalance (Buret et al, 1990), which collectively lead to dysfunctional intestinal epithelial barrier (Cotton et al, 2011). The information on disease mechanisms and parasite pathogenic factors are still incomplete
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