Molecular and cellular interactions of four giardia duodenalis isolates with the murine intestinal epithelial cell line IEC-6 in vitro

Giardia duodenalis is a protozoan parasite that causes diarrhoea in humans and adds to the disease burden in the developing world. Giardia cysts are the infectious agent from which trophozoites emerge and colonise the small intestines causing the disease, giardiasis. Clinical outcomes range from asymptomatic infection to acute or chronic diarrhoea. The genotypes A and B infect humans; their association with symptoms is inconclusive and disease mechanisms are incompletely understood. Trophozoite attachment to enterocytes via the ventral disc and the release of parasite proteases and metabolic enzymes are recognised disease mechanisms. Giardia isolates vary in virulence but the exact virulence-associated factors remain unknown. Although host histopathological and immunological responses to different isolates have been elucidated over the years, information on molecular events underpinning host responses are lacking. Likewise, a gap exists in the knowledge of molecular events underpinning the establishment of infection by different isolates. Therefore, we selected isolates with differing virulence, WB, P-1, NF (assemblage A) and GS/M (assemblage B), to study the interaction with the intestinal epithelial cell line, IEC-6 in vitro. We first investigated Giardia proteases using zymography (SDS-polyacrylamide gels with gelatine) to assess any correlation with virulence, as published data suggests. The four isolates released five cysteine proteases on contact with IEC-6 cells indicating their involvement in pathogenicity. Nevertheless, proteolysis patterns (i.e. protease numbers and molecular weights) were similar in all isolates challenging the association of proteases with virulence. Representational difference analysis (RDA) was used to identify up-regulated genes in the Giardia isolates and IEC-6 cells during interaction. RDA showed that the four isolates recruit the same genetic machinery to infect cells; however, they expressed different variable surface proteins (VSPs), high cysteine membrane proteins and varied in the response to oxidative stress. These collectively were suggested as factors attributing for the difference in isolates virulence. The isolates modulated the transcription of genes associated with different cellular processes in IEC-6 cells. The transcriptomes of all parasitised IEC-6 cells were indicative of the production of reactive oxygen species (ROS), the host innate defence molecules. At two hours of interaction, parasitised cells up-regulated the expression of genes associated with the promotion of cell survival, ROS attenuation, and tissue repair; however, at six hours genes associated with the induction of cell death (i.e. apoptosis, mediated by caspase-3) were induced. Oxidative stress and hypoxia were pronounced in cell transcriptomes (at 6h) and were proposed as inducers of apoptosis and disease mechanisms in giardiasis. IEC-6 cells also transcribed the immune modulators, transforming growth factor beta-1 and ubiquitin-like protein Fubi, highlighting the possibility of these mediating the inhibition of cytokine production during giardiasis. Parasitised IEC-6 cell responses varied between isolates in the range of up-regulated genes associated with tissue repair and oxidative stress response possibly attributable to differences in trophozoite capabilities to attach and induce tissue damage, and neutralise host ROS, respectively. This study adds to the knowledge of disease mechanisms in giardiasis. The RDA work suggests a range of new treatment targets for giardiasis and areas of interest for future studies