Extracellular vesicles in intestinal protozoa: hidden mediators of host-parasite communication

Abstract

Intestinal protozoan parasites, including, Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica, and Blastocystis sp. pose significant global health challenges, particularly in developing regions with limited access to sanitation and healthcare resources. Extracellular vesicles (EVs) have emerged as critical factors for communication in the complex interplay between intestinal protozoan parasites and their hosts. This review explores the diverse roles of parasite-derived EVs, with a focus on their biogenesis, molecular cargo, and host interactions. Studies show that G. duodenalis EVs disrupt tight junctions in intestinal epithelia and promote Th1 immune responses. Blastocystis sp. EVs modulate cytokine production, increasing IL-6 and TNF-α while reducing IL-10 and IL-4. E. histolytica EVs inhibit immune cell recruitment by downregulating STAT6 signaling and suppressing IL-4 and IL-13. Additionally, Cryptosporidium-infected epithelial cells release EVs enriched in GP60 and CpRom1 proteins that stimulate host immune responses via the TLR4/IKK pathway. Beyond pathogenesis, the specific and stable cargo of parasitic EVs supports their utility as biomarkers and therapeutic agents. Ongoing research into their roles in microbiome modulation and immune regulation may inform future diagnostics and treatments for parasitic infections.

Introduction

Intestinal parasitic infections (IPIs) affect over one billion people globally, with children and individuals in impoverished regions being disproportionately impacted [1, 2]. These infections are caused by a range of intestinal parasites, including protozoa such as Giardia duodenalis and Cryptosporidium spp., as well as helminths like Ascaris lumbricoides and hookworms. Together, these parasites continue to represent a significant global public health burden, particularly in areas with limited access to clean water, sanitation, and healthcare. The consequences of these infections are wide-ranging, including acute symptoms such as diarrhea and malnutrition, as well as long-term effects like stunted growth in children and impaired cognitive development [2, 3]. Hookworm infections can lead to iron deficiency anemia, significantly reducing physical work capacity and impairing academic performance. In immunocompromised individuals, especially those with HIV/AIDS, parasite infections such as cryptosporidiosis can lead to serious, life-threatening consequences. In high-income countries, outbreaks are frequently associated with foodborne or waterborne transmission, highlighting the influence of globalized food systems and travel in spreading these infections [3, 4]. Although intestinal parasite infections have a comparatively low direct fatality rate, they exert a significant economic and social burden through diminished production, healthcare costs, and persistent adverse health effects. Initiatives to manage these illnesses emphasize enhancing access to potable water, sanitation, and health education, in conjunction with widespread deworming efforts in endemic areas [5].

During parasite invasion, pattern recognition receptors (PRRs) on immune system cells including macrophages and dendritic cells identify Microbe-associated molecular patterns (MAMPs). Recognizing this MAMPs triggers the secretion of cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), which initiate inflammatory reactions. Type I interferons have been demonstrated to have a protective function in mitigating inflammation during schistosomiasis, a disease induced by waterborne parasitic worms. These cytokines diminish granuloma formation and mitigate tissue damage, which could otherwise intensify disease severity [6]. Th2 cells are particularly mediate the main adaptive immune response in combating IPIs. These cells produce cytokines like IL-4 and IL-13, which stimulate the production of immunoglobulin E (IgE) and the activation of eosinophils to target the parasites. In some cases, regulatory mechanisms are also activated to prevent excessive inflammation and tissue damage [7].

Parasites are adept at evading immune detection and modulating host responses. For instance, helminths release molecules that can inhibit immune cell activation, effectively dampening the immune response [8, 9]. Toxoplasma gondii, a protozoan parasite, has been shown to manipulate host immune signaling to enhance its survival [10, 11]. These evasion strategies present significant challenges in developing vaccines or long-lasting treatments. In protozoan parasites, extracellular vesicle (EV) secretion can originate directly from the organellar compartments of parasite or be mediated through parasite-infected or antigen-stimulated host cells in response to physiological stressors under both in vitro and in vivo conditions [12]. These EVs are regarded as critical communication mediators, enabling interactions between parasite cells and between the parasite and its host, thereby promoting parasite growth by altering the functionality of targeted host tissues [13]. Given the preceding discussion on epidemiology and immune evasion, EVs provide a unique intersection between parasitic pathogenesis and host immune modulation. Consequently, EVs derived from protozoa could serve as a valuable source of clinical biomarkers for parasitic infections. The biomolecules carried within vesicles maintain their inherent stability, structure, and sequence. Furthermore, alterations in the type of protein found on outside or within extracellular vesicles can indicate the condition of the cells that produce them, rendering these structures excellent biomarkers for various diseases. While several protozoan species inhabit the human gut, this review primarily focuses on the most clinically and epidemiologically significant ones: Giardia duodenalis, Blastocystis sp., Entamoeba histolytica, and Cryptosporidium spp.

Exosomes: structure and function

Exosomes are small EVs (30–150 nm), produced and released by nearly all cell types. They are enclosed by a lipid bilayer containing diverse biomolecules such as proteins, lipids, RNA, and DNA [14]. These vesicles play a vital role in intercellular communication by delivering molecular cargo to recipient cells, thereby influencing cellular behavior and functions. Exosomes are found in various bodily fluids, including blood, urine, saliva, and cerebrospinal fluid, indicating their ubiquity in physiological and pathological processes [15].

Exosomes originate within cells through the inward budding of the cell membrane to form multivesicular bodies (MVBs). These MVBs eventually fuse with the plasma membrane, releasing exosomes into the extracellular environment. These MVBs contain intraluminal vesicles (ILVs) formed by inward budding of the endosomal membrane. Two primary mechanisms drive ILV formation: Endosomal Sorting Complex Required for Transport (ESCRT)-dependent and -independent pathways. In the ESCRT-dependent pathway, the ESCRT machinery is crucial in ILV formation, consisting of four complexes (ESCRT-0, -I, -II, and -III) that sequentially sort cargo into ILVs and mediate membrane scission [16]. However, in some cases, ILV formation depends on ceramide-enriched lipid raft domains rather than ESCRT proteins [17]. MVBs, upon formation, either fuse with lysosomes for degradation or merge with the plasma membrane, resulting in the release of ILVs as exosomes into the extracellular environment (Fig. 1).

Fig. 1.

The precise mechanisms of exosome biogenesis for intestinal protozoa not fully understood, but this intricate procedure can be delineated into three primary stages: initially, the formation of early and late endosomes occurs; subsequently, intraluminal vesicles (ILVs) are generated within the late endosomes, resulting in the creation of multivesicular bodies (MVBs); finally, through the joining of MVBs with the plasma membrane, ILVs are released via exocytosis, thereby being designated as exosomes. Biogenesis of exosomes can occur through two primary mechanisms: Endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent. ESCRT is a unique multi-subunit complex that facilitates the accumulation of ubiquitinated proteins and the inwardly budding and fragmentation of ILVs

Lipid raft domains and ceramide-rich regions play a crucial role in exosome biogenesis in many parasites, particularly protozoa, where ESCRT-independent mechanisms are prominent. For instance, G. duodenalis exhibits a lack of ESCRT whole set components, is devoid of tetraspanins, and is unable to produce ceramide by itself [18]. This parasite seems to possess a distinctive method for the formation of exosome-like vesicles. Research indicates that the biogenesis of exosomes takes place in peripheral vacuoles (PVs), with their formation and secretions relying on the ESCRT-associated protein ceramide, Vps4a, and Rab 11. Since Giardia lacks a Golgi complex, the proteins are released and transported through the endoplasmic reticulum to the cell membrane or to organelles like PVs [19]. The Rab 11 protein plays a role in differentiated cells and division, and appears to be linked to the interaction between the endoplasmic reticulum and the vesicle populations. Rab 1 and Rab 2 a/b proteins, associated with exocytic trafficking of vesicles, seem to play a role in excretion of exosome [20].

Their structure protects enclosed biomolecules from enzymatic degradation, ensuring the delivery of functional molecules to target cells under various conditions, including hypoxia and stress. Their stability, specificity in cargo delivery, and ability to cross biological barriers (e.g., the blood-brain barrier) make them attractive for diagnostics and drug delivery. Exosomes have been explored as carriers for targeted therapies in cancer, neurodegenerative diseases, and cardiovascular disorders [16]. They have also attracted considerable attention in research for their potential as both biomarkers and therapeutic agents. These nano vesicles contribute to maintaining homeostasis, supporting stem cell activity, and facilitating angiogenesis. Additionally, their ability to transfer genetic material and proteins enables them to modulate immune responses and aid in cellular waste clearance [21].

Virtually all cell types are capable of secreting parasite-derived exosomes. Multiple vesicles are produced by membrane budding in cells, and they are then released into the extracellular space when the vesicular outer membrane fuses with the cell membrane [22]. Upon external release, these vesicles attach to and merge with target cells through endocytosis, leading to the opening or degradation of the vesicular membrane within the target cell and the ensuing discharge of their contents which causes regulatory functions.

The life cycles of parasites exhibit complexity, with the release of exosomes occurring at various developmental stages. Exosomes derived from parasitized cells transport a variety of antigens and immunomodulatory factors, including nucleic acids, drug-resistance proteins, enzymes, and lipids. The EVs possess at least three mechanisms to interact with target cells: (1) They might remain adhered to the cell membrane, interacting with the cell receptor to initiate signaling cascades; (2) they can be taken on via receptor-mediated endocytosis; or (3) they may merge with the target cell’s membrane [23]. The stability of the enclosed nucleic acids is increased by the lipid bilayer composition of these exosomes, which prevents nucleases from breaking down RNA and DNA. Exosomes increase the effectiveness of delivering nucleic acids to specific cells and tissues, allowing them to enter cells quickly and having immunoregulatory effects [24, 25]. Antigens and proteins within these tiny particles help deliver antigens and work together to influence host immune responses [26]. Exosome-mediated transfer of toxic virulence and drug-resistant compounds increases parasite toxicity and promotes survival of the parasite in the host [27, 28]. Parasites can avoid host immunological responses by modulating innate immune system proteins with exosomal proteases. Exosomal lipids interact with immune cells and contribute to disease progression by binding to specific receptors [27]. Therefore, exosomes function as significant carriers of signaling molecules and are essential for communication between hosts and parasites. The following sections explain the crosstalk of extracellular vesicles between host and pathogenic intestinal protozoa.

Blastocystis Sp.

The interaction between gut parasites and the mammalian immune system has led to the development of complex parasite–host interactomes. In summary, these interspecies relationships may exhibit mutualistic, commensal, or parasitic characteristics, along with various intermediate scenarios, without a clear boundary delineating the results of the host–parasite interaction. Although Blastocystis sp. is recognized as the most prevalent protist associated with humans, its potential beneficial or detrimental effects on the host immune system remain a subject of ongoing debate [29]. Blastocystis sp. is classified within the stramenopile group and is recognized as a prevalent single-celled intestinal parasite affecting humans and various animal species [30]. Analysis of the small subunit (SSU) rRNA genes of Blastocystis sp. has identified 40 subtypes (ST1-ST17, ST21, ST23-ST44), which may represent distinct species, and have been detected in both humans and a wide range of animal hosts [31, 32]. Among these, ST1-ST10, ST12 and ST14 have been documented in human cases, with ST1-ST4 representing the predominant strains, accounting for over 90% of human Blastocystis isolates [33]. It is important to note that the prevalence of subtypes varies significantly across different regions [34]. Furthermore, these subtypes exhibit considerable differences in biological characteristics, including drug resistance, immune system response, pathogenicity, and their impact on the microbiota [30].

The gut microbiota, which includes bacteria, fungus, viruses, and several other microbes, has a symbiotic connection with the host and has co-evolved alongside it [35, 36]. Human-associated Blastocystis sp. are a genetically heterogeneous component of the intestinal microbiome [37]. Wu et al. demonstrated variability in pathogenicity between and within ST4 and ST7 subtypes [38]. Most immunological research examining the relationship between Blastocystis sp. and the host immune system has focused on ST4 and ST7. ST4 is the predominant subtype among European populations, as shown by metagenomic studies of the human gut [39, 40]. In contrast, ST7 is rarely encountered in populations, despite its isolation from human sources. Therefore, it is important to recognize that studies on the potential pathogenicity of ST7 may offer limited insights into the interactions between the more prevalent Blastocystis sp. subtypes found in the human gut, such as ST1, ST3, and, to a lesser extent, ST2 and ST4, and the human immune response [30].

A variety of general gastrointestinal symptoms, such as diarrhea, stomach pain, nausea, bloating, vomiting, and anorexia, are associated with Blastocystis sp. infections. Additionally, it may cause less common dermatological issues such as urticaria and severe itching [41, 42]. However, the parasite has mostly been found in asymptomatic people, suggesting that it may not be invasive [43]. Mice infected with Blastocystis have shown edema and infiltration of immune cells in the cecum and colon area [44]. An experiment demonstrated that Blastocystis leads to IL-8 release by colon T84 cell populations in a time-dependent way, leading to the invasion of immune cells into the lamina propria, which results in injury to tissue and digestive problems [45].

EVs derived from Blastocystis were recognized in H and ST7 B isolates, exhibiting a cup-like appearance and a mean size ranging from 50 to 240 nm, which aligns with the characteristics of EVs from other parasites [46]. Norouzi et al. reported that exosomes derived from Blastocystis sp. ST1 led to an increase in TNF-α and IL-6 levels while decreasing IL-10 and IL-4 levels [43]. Also, ST2 and ST3-derived EVs resulted in a reduction of IL-10, and those from ST3 also caused an increase in IL-6 levels. Similarly, Leonardi et al. showed increase of IL-1β expression by THP1 cell lines after incubation with a high amount of ST7 EVs [47].

There is ongoing debate regarding the infectious potential of Blastocystis, its clinical significance, and its potential impacts on the host immune response [30]. The pathogenicity of Blastocystis sp. is contingent upon various factors, including its relationship with the human gut microbiome, the specific subtype present, and the regulatory or modulatory roles of the human immune response [48]. The gut microbiome is crucial for the health and disease condition of the host. Reducing the gut microbiota is linked to immune system dysregulation, which is play a critical role in the onset of autoimmune disorders [49]. Blastocystis sp. resides in the human gut without causing infection; however, this dynamic may alter if there is a disturbance in the immune system or the balance of the gut microbiome [50]. Blastocystis sp. EVs suppress the proliferation of a helpful gut microbiota; diminish the survival of a human gastrointestinal cell model, and elevating the expression of tumor-inducing inflammatory markers in that same model. These are associated with dysbiosis, inflammation, and damage to the host gut epithelium, manifestations commonly observed in Blastocystis-associated diseases [47] (Fig. 2).

Fig. 2.

The effects of Blastocystis sp. EVs on host. Blastocystis-derived exosomes carry a range of biomolecules that disrupt epithelial integrity, modulate immune responses, and disturb gut microbiota. These interactions contribute to pro-inflammatory cytokine release, gut dysbiosis, and tissue damage, collectively leading to host-pathogen imbalance and disease progression

Giardia duodenalis

Giardiasis is a zoonotic disease caused by the flagellated protozoan G. duodenalis, also known as G. intestinalis or G. lamblia. This parasite can infect a wide range of hosts, including both domestic and wild mammals. There are eight separates genetically combinations of this protozoan identified. The zoonotic combinations A and B influence humans, while assemblages C through H are exclusive to animals [20, 51]. The parasite’s cyst, excreted in feces, is essential for both transmission and survival in the environment. In contrast, trophozoites colonize the host’s gut. Due to their exceptional resistance to environmental conditions, particularly in water, cysts are a major source of waterborne diarrheal disease outbreaks [52].

Giardiasis induces intestinal barrier failure through epithelial cell death and the breakdown of the apical junctional structures [53]. The strong adhesion of G. duodenalis trophozoites to intestinal epithelial cells via its adhesive disk diminishes the absorbent area of the small intestine and leads to a generalized reducing of the intestinal epithelial microvilli. This results in disaccharidase deficiency and malabsorption of electrolytes, water, and other nutrients [54, 55]. Additionally, the host’s CD8 + T lymphocytes are activated by secretory/excretory products of parasite, which results in decreased intestinal brush edge area, disaccharidase failure, and increased crypt/villus ratios in giardiasis [56].

G. duodenalis has experienced reductive evolution, resulting in a simplified endomembrane system relative to other parasites, lacking an endosomal/lysosomal system, Golgi complex, peroxisomes, and mitochondria [57]. The absence of a Golgi complex causes proteins to be easily released and transported from the endoplasmic reticulum (ER) to the cell membrane or to organelles, such as peripheral vacuoles (PVs). This parasite possesses PVs that function as both endosomes and lysosomes, serving as specialized endocytic cellular components [19]. Certain encystation secretion vesicles (ESVs) found in G. duodenalis serve to control the release of components from the cyst wall and share similarities with the Golgi complex.

Similar to other protozoa, Giardia can release EVs that are involved in cell signaling and may influence the pathological process of giardiasis as well as the host’s immune response. Evans-Osses et al. showed that Giardia duodenalis trophozoites can secrete microvesicles (MVs) in response to varying pH levels and calcium, which act as triggers for vesicle release. Electron microscopy revealed that EVs range in size from 60 to 150 nm. Additionally, nanoparticle tracking analysis indicated the presence of larger vesicles with a peak average size of 201.6 nm [58]. They also showed how cholesterol affects MV release by demonstrating that different concentrations of methyl-β-cyclodextrin (MβCD) reduced MV formation. The external addition of MVs restored parasite attachment to host cells, which had been impaired by cholesterol depletion. This suggests that MVs play a crucial role in the adhesion of Giardia duodenalis to intestinal epithelial cells [58, 59]. Proteomic analysis of EV population from Giardia trophozoites revealed the presence of pathogenesis-related proteins, including giardins, variable surface proteins (VSPs), arginine deiminase, ornithine carbamoyltransferase, and cathepsin B [60]. The lipidome of Giardia trophozoites EVs are mainly include cardiolipins, ceramides and cholesterols [61]. These lipids are contributed to the parasite growth, immune cells activation and pathogenesis.

Giardia EVs exhibit an antibacterial impact on commensal gut bacteria, including Enterobacter cloacae and Enterococcus faecalis. Moreover, exposure to Giardia EVs enhanced mobility of non-invasive commensal strains E. coli HB101 and E. cloacae [62]. In addition, Giardia EVs disrupted the integrity of epithelial junctions in vitro and potentially elucidating the frequency of post-infectious disorders following disease elimination [62, 63]. Faria et al., proposed the use of Giardia EVs as a cell-free vaccine for giardiasis. They found that Giardia EVs stimulate pro-inflammatory signaling pathways, including SAPK/JNK and ERK1/ERK2, along with the NF-kB route. Furthermore, these EVs enhance the maturation of human monocyte-derived dendritic cells (Mo-DCs) and significantly promote the proliferation of T cells with a Th1 phenotype [64]. In another study, Giardia EVs affected HIF-1 and cAMP signaling pathways of Caco-2 cell lines [65]. These studies highlight the potential impact of Giardia EVs on intestinal epithelial cells (IECs) and their role in disease pathology (Fig. 3).

Fig. 3.

Effects of Giardia duodenalis exosomes on the host. Giardia can influence the host through the secretion of exosomes in several ways. These exosomes carry Giardia virulence factors that can initiate giardiasis. Additionally, they disrupt the intestinal barrier and promote the motility of pathogenic bacteria, leading to chronic inflammation and post-infection disorders. Proinflammatory cytokines, together with the exosomal lipidome, can stimulate a Th1 immune response, ultimately contributing to gut dysfunction

Entamoeba histolytica

Entamoeba histolytica, the causative agent of amebiasis, represents a significant public health concern, particularly in regions with poor sanitation. E. histolytica exhibits a two-phase life cycle, comprising a predominantly dormant cyst stage and a metabolically active trophozoite stage. Upon ingestion of cysts through contaminated food or water, the parasites pass through the small intestine, where they excyst and transform into trophozoites. Upon reaching the colon, trophozoites proliferate and may occasionally cause severe infection, leading to tissue damage and colonization of the colon or liver. For reasons that remain unclear, trophozoites can also encyst and be excreted via the feces, contaminating external environments and potentially infecting new hosts [66]. It has been shown that E. histolytica-derived extracellular vesicles (EhEVs) are enriched in typical exosome marker proteins, which include proteins related to vesicle creation, cell signaling, metabolism, and cytoskeletal functions. Furthermore, these EVs demonstrated a selective ability to carry out small RNAs (sRNA), which preserved within the vesicles from RNase therapy. EhEVs also affect intercellular interaction among parasites, modify encystation effectiveness, and specially immunomodulation [67] (Fig. 4).

Fig. 4.

Immunomodulatory Effects of E. histolytica-derived EVs (EhEVs). EhEVs inhibit the production of Th2 cytokines by downregulating the STAT6 transcription factor. They also suppress the expression of genes involved in recruiting immune cells to the site of infection. Furthermore, EhEVs can inhibit NETosis in neutrophils and reduce M2 polarization of macrophages. These effects promote parasite survival and impair anti-parasite immunity

Macrophages enhance the initial host reaction when faced with amoebic infection through the release of proinflammatory cytokines and the recruitment of additional immune cells [68, 69]. Toll-like receptor (TLR) 2/4/6 and TLR 9 identify lipopeptidophosphoglycan (LPPG) on the surface of amoebas and amoebic DNA, respectively, inducing a significant host immunological response [70]. Chowdhury et al. demonstrated that treatment with EhEV modified the energetics in THP-1 macrophage cell lines. The reduction in energy production by EhEVs in THP-1 macrophage is primarily attributed to the inhibition of oxidative phosphorylation (OXPHOS) and mitochondrial material. The metabolic profile of M1 macrophages is distinct from that of M2 macrophages. For instance, M1 macrophages primarily depend on glycolysis, while M2 macrophages predominantly encourage OXPHOS to supply their energy requirements [71]. It is now widely recognized that immunometabolism plays a crucial role in guiding the function of immune cells, and this topic has been thoroughly investigated [72–74]. Parasitic organisms exploit changes in the metabolism of immune cells to influence their activation, thereby modulating parasitic survival and progression. Macrophages infected with Trypanosoma cruzi exhibit diminished levels of glycolysis and oxidative species in comparison to M1 macrophages [75].

In addition, THP-1 macrophages activated with EhEVs inhibit the IL-4 response by downregulating pSTAT6 [76]. IL-4 and IL-13 are pivotal in allergic inflammation and parasitic infections, functioning via the STAT6 signaling pathway [77]. Moreover, EhEVs reduced the expression of MCP2, MCP3, IL-8, eotaxin-2/CCL24, and eotaxin-3/CCL26. These factors are essential for the recruitment of immune cells to infection site and host immunity against parasite [76]. However, there is a lack of sufficient research on the impact of EhEVs on macrophage polarization. Therefore, further investigation is required to gain a deeper understanding of this phenomenon.

Upon the invasion of tissue by the amoeba, the inflammatory reaction is triggered, leading to the recruitment of numerous neutrophils, which, in some situations, seems to help in protection alongside the INF-mediated Th1 response [78]. Viable E. histolytica trophozoites can stimulate the development of neutrophil extracellular traps (NETs), which in turn can increase neutrophil lysis [79]. E. histolytica trophozoites are among the most powerful microbial triggers for NETosis, a process necessitating reactive oxygen species (ROS) [80]. ROS produced by amoebic trophozoites may be transported to neutrophils via parasite EVs, potentially affecting neutrophil respiratory burst and NET production. However, the role of EVs as carriers of ROS has received limited investigation [81]. The endocytosis of amoebic EVs by neutrophils leading to a substantial decrease in the oxidative burst and NETosis induced by ionophore A23187, PMA, or the amoebae as stimuli [82]. The active transfer of components across cells is a necessary feature of the NETosis process in amoebiasis. During close interaction between the two cell types, this process requires the transfer of ROS from the amoeba to the neutrophil and the transmission of myeloperoxidase (MPO) from the neutrophil to the amoeba surface [83].

Díaz-Godínez et al., have shown that amoebic EVs containing reactive oxygen species (ROS) did not alter baseline ROS levels and induced minimal NETosis in unstimulated neutrophils. This suggests that contact-dependent communication processes are necessary in addition to parasite ROS for amoeba-induced NETosis. In addition, the findings of this study indicate that EVs primarily exert an inhibitory impact on NETosis. Consequently, the introduction of EVs from either amebae or neutrophils, which resulted in a reduction of ROS production in activated neutrophils, or EVs derived from the co-culture, which had no impact on ROS levels, led to a postponement in the initiation of NETosis and the quantity of NETs released, irrespective of the stimulus applied. The findings indicate that EVs derived from inactive neutrophils and amoebae exert minimal or no influence on inactive neutrophils. In contrast, these EVs appear to have an immunomodulatory impact on stimulated neutrophils, leading to a downregulation of respiratory burst and NET creation. Research indicates that the EVs from resting cells typically exert inhibitory properties on adjacent cells, including the modulation of ROS creation and cell death, thereby contributing to organismal homeostasis [82].

Cryptosporidium spp.

Cryptosporidium spp. are obligate intracellular parasites that cause cryptosporidiosis, a gastrointestinal disorder that affects both humans and animals. The parasite is a common cause of childhood diarrhea worldwide and is associated with impaired child development. Additionally, it poses a life-threatening risk to individuals with HIV/AIDS and transplant recipients [84]. The genus Cryptosporidium encompasses nearly 40 valid species, which exhibit marked differences in host range. Among these, over 10 species, mainly C. hominis and C. parvum, have been reported in humans, with a variety of genotypes being zoonotic [85]. The oocyst is a parasitic stage of Cryptosporidium spp. that is excreted in the feces of infected individuals, facilitating the spread of the parasite to others. Oocysts possess a robust “shell,” known as the oocyst wall, composed of polysaccharides, lipids, and specific proteins referred to as Cryptosporidium oocyst wall proteins (COWPs). This structure confers significant resistance against external and chemical-based stresses. Oocysts exhibit resistance to common water disinfection treatments, which can lead to extensive problems affecting thousands of individuals [84]. Although Cryptosporidium poses a significant threat to human health, there is still a lack of effective drugs, and vaccines have yet to be developed.

There is a lack of sufficient studies investigating the characteristics and effects of Cryptosporidium spp. EVs. One study demonstrated that exosomes secreted by Cryptosporidium-infected epithelial cells induce inflammation in primary spleen cells. These exosomes effectively stimulate a host immune response [86]. A study by Bertuccini et al. showed the expression of immunostimulatory antigens glycoprotein GP60 and membrane protein CpRom1 in Cryptosporidium-derived EVs [87]. Following Cryptosporidium spp. infection, epithelial cells enhance exosome secretion via the stimulation of TLR4/IKK signaling pathways. Furthermore, the interaction between exosomes and Cryptosporidium sporozoites has been associated with an anti-parasitic effect [88].

Despite the growing body of research on EVs in various protozoan parasites, there remains a significant gap in the literature concerning the EVs of other intestinal protozoan parasites including Cyclospora, Cystoisospora, and Isospora species. To date, no studies have specifically investigated the characteristics, composition, or biological effects of EVs secreted by these coccidian parasites in human. However, Feix et al. (2025) investigated the production and composition of EVs across different developmental stages of Cystoisospora suis. They employed in vitro cultivation methods to isolate and analyze EVs from various parasite stages, including sporozoites, merozoites, and gamonts. Their analyses revealed stage-specific variations in EV shedding, with notable differences in the expression of proteins associated with the Apicomplexa phylum and those involved in vesicle shedding. Lipid profiling indicated that while fatty acid concentrations remained consistent across stages, the levels of sterol lipids, sphingolipids, and glycerolipids varied. These findings suggest that C. suis modulates its EV composition throughout its life cycle, potentially reflecting adaptations to different host environments or functional requirements at each stage [89].

Concluding remarks

A deeper understanding of EV biology in under-explored protozoan species offers crucial insights into infection mechanisms, survival strategies, and immune evasion tactics (Table 1). Such knowledge may not only advance parasitic biology but also inform the development of novel diagnostic tools and targeted therapeutic strategies. For instance, the stability and specific cargo of EVs make them promising candidates for early detection biomarkers, while their ability to cross biological barriers suggests utility in vaccine and drug delivery. EV-based vaccines, such as those explored in Giardia duodenalis, represent a framework that could be extended to other protozoa.

Table 1.

Comparative summary of EV characteristics in major intestinal protozoa

Parasite	EV Biogenesis Features	Immune Targets / Modulation	Diagnostic / Therapeutic Potential
Blastocystis sp.	Originates from vesicle secretion organelles; 50–240 nm	↑ IL-6, TNF-α; ↓ IL-10, IL-4 (pro-inflammatory skewing)	Potential subtype-specific biomarkers; linked to gut inflammation
Giardia duodenalis	Lacks Golgi; EVs from peripheral vacuoles; 60–201 nm	Activates Th1 cells; disrupts tight junctions; affects microbiota	Vaccine potential shown in preclinical models; lipidome is diagnostic
Entamoeba histolytica	Contains exosome markers; affects parasite communication	Inhibits M2 macrophage polarization; ↓ IL-4/IL-13 via STAT6 suppression	Promising in immune modulation studies; sRNA content is stable marker
Cryptosporidium spp.	EVs from infected host epithelial cells	Stimulates TLR4 pathway and splenocyte activation	GP60 and CpRom1 proteins in EVs suggest use in immunodiagnostics
Cystoisospora suis	Stage-specific EV variation; sterol/lipid profile shifts	Limited data in humans; immune impact not well studied	Lipid and protein markers under investigation for stage-specific roles

Despite these promising avenues, several gaps remain. The molecular mechanisms governing EV-host interactions, the functional heterogeneity of EVs across parasite life stages, and the long-term consequences of EV-driven immune modulation are not fully understood. Additionally, the role of EVs in mediating microbiome changes and their contribution to disease progression requires further study.

Finally, the dynamic interplay between parasitic EVs and the gut microbiome could influence not only infection outcomes but also the risk of autoimmune and inflammatory disorders. Addressing these gaps through integrative omics and functional studies may reshape future diagnostic and therapeutic strategies in parasitology.

Author contributions

MF and ML, Design and writing original draft; FR, FA, and SR, review & editing; TK, AS, KHN and EH, Project administration, Conceptualization, and Supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Student Research Committee, Tabriz University of Medical Sciences under grant number 76251.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.