Giardia intestinalis (G. intestinalis), also known as G. lamblia or G. duodenalis, is a single-celled protozoan parasite that is a major cause of waterborne diarrhea worldwide. Giardia has a relatively simple life cycle, with two major life cycle stages: vegetative trophozoites and infectious cysts. Giardia infection is initiated by the ingestion of cysts. Cysts undergo excystation to release excyzoites, which rapidly transition into trophozoites (Ankarklev et al., 2010). Trophozoites adhere to the epithelial surface of the small intestine via ventral adhesive discs (Dawson, 2010). This intimate association and interaction results in clinical symptoms such as diarrhea, abdominal pain, vomiting, nausea, dehydration, and weight loss (Einarsson et al., 2016).

In most cases, Giardia causes infection without invading host cells. During the acute stage of the infection, millions of Giardia trophozoites attach directly to the intestinal epithelial surface. Physical attachment activates downstream biological events that compromise host cells and induce giardiasis. As a non-invasive parasite, Giardia closely adheres to the small intestine and secretes excretory and secretory products (ESPs), including secreted proteins (SPs) and extracellular vesicles (EVs) (Ma'ayeh et al., 2017).

ESPs have been suggested to play crucial roles in Giardia pathogenesis. An earlier study examining Giardia ESPs revealed their functional roles in intestinal absorption and secretion (Samra et al., 1988). Caco-2 and SCBN monolayers exposed to sonicated parasites and culture supernatants showed alterations in F-actin organization (Teoh et al., 2000). Administration of Giardia ESPs to pathogen-free mice elicited Th2 immune responses and eosinophil infiltration (Jimenez et al., 2004). Subsequently, the Giardia secretome was characterized, and host-parasite interactions were investigated (Dubourg et al., 2018, Ma'ayeh et al., 2017). Specific virulence factors, such as cysteine proteases, arginine deiminase, and ornithine carbamoyltransferase (OCT), were thoroughly examined (Banik et al., 2013, Liu et al., 2019a, Liu et al., 2018, Quezada-Lazaro et al., 2022, Stadelmann et al., 2013).

EVs are small membrane-bound vesicles secreted by various types of mammalian cells and disease-causing parasites (Mathivanan et al., 2010, Nievas et al., 2020, Olajide et al., 2023, Zhu et al., 2016). EVs contain proteins, lipids, and nucleic acids that can modulate multiple biological processes (Valadi et al., 2007). Recently, the EVs secreted by Giardia have been investigated. For example, EVs have been reported to facilitate the adherence of parasite to epithelial cells in vitro (Evans-Osses et al., 2017). EVs may affect cell viability (Gavinho et al., 2020). More recently, Giardia EVs have been reported to stimulate pro-inflammatory cytokine production and activate the MAPK signaling cascade in murine macrophages (Zhao et al., 2021a, Zhao et al., 2021b). However, the differences between SPs and EVs, and the impact of EVs on intestinal epithelial cells (IECs) remain unclear.

Therefore, this study aimed to investigate the protein composition of these two fractions and explore the effect of EVs on IECs at the transcriptional level. To this end, we isolated SPs and EVs and performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry to study the enriched proteins in the two secreted fractions. In addition, RNA sequencing was performed to assess the gene expression profiles of IECs in response to EVs. This study illustrates the major protein compositions of EVs and SPs and highlights the potential roles of EVs in Giardia-host cell interactions.