Investigating the role of Giardia intestinalis extracellular vesicles in host-parasite communication in 2D human jejunum organoid model systems

Sammanfattning

Diarrheal diseases remain a global burden on children’s health, accounting for 800,000 child deaths under the age of five each year. A notable contributor to this impact is the protozoan parasite Giardia intestinalis. Unlike other protozoan pathogens, G. intestinalis disrupts the intestinal epithelium without inducing large-scale inflammatory responses, leaving its virulence mechanisms poorly understood. It has been suggested that the parasite utilizes a complex secretome, comprised of a wide variety of secreted proteins and extracellular vesicles, to modulate host expression and damage the intestinal environment. The vesicles have been gaining particular interest in recent years, as they deliver cargo that induces cellular changes when taken up by recipient cells. To clarify their role during infection, this study aimed to distinguish the vesicles’ functions from free proteins, while exploring the protein alpha-1 giardin as a potential mediator of the parasite’s secretion. This was achieved by comparing the secretome of the wild type WB and an alpha-1 giardin knockout mutant via Coomassie-stained SDS-PAGE. Following this, the vesicles and free proteins of the wild type secretome were separated using the ExoEasy MaxiKit to study their effects on host cells individually, using human jejunum organoid monolayers as a model system. Subsequently, cytokine expression of IL-8, CXCL-1, and CCL-20 was quantified with qPCR. Though the statistical significance of the results was limited by the small sample size and a large variance, consistent trends in the cytokine response were observed. Free proteins elicited a stronger response than the vesicles, which suggests that the vesicles are less immunogenic. This may reflect a functional divergence in the secretome, wherein the free proteins induce host responses that the vesicles potentially silence or modulate. This creates complex interactions between the host and the secretory products of G. intestinalis. Furthermore, the alpha-1 giardin knockout displayed a diminished secretome, evidenced by reduced band intensity onSDS-PAGE and confirmed by lower protein concentrations measured with a Qubit fluorometer, indicating that alpha-1 giardin could be involved in the secretion of G.intestinalis. Taken together, these findings elucidate distinct roles of the extracellular vesicles and secreted proteins, providing a deeper understanding of the vesicles’ function during infection. This study also identifies alpha-1 giardin as a potential mediator of the secretion machinery, making it a candidate biomarker for novel therapeutics.