An in-depth exploration of the multifaceted roles of EVs in the context of pathogenic single-cell microorganisms

SUMMARY

Extracellular vesicles (EVs) have been recognized throughout scientific communities as potential vehicles of intercellular communication in both eukaryotes and prokaryotes, thereby influencing various physiological and pathological functions of both parent and recipient cells. This review provides an in-depth exploration of the multifaceted roles of EVs in the context of bacteria and protozoan parasite EVs, shedding light on their contributions to physiological processes and disease pathogenesis. These studies highlight EVs as a conserved mechanism of cellular communication, which may lead us to important breakthroughs in our understanding of infection, mechanisms of pathogenesis, and as indicators of disease. Furthermore, EVs are involved in host–microbe interactions, offering insights into the strategies employed by bacteria and protozoan parasites to modulate host responses, evade the immune system, and establish infections.

INTRODUCTION

Infections originating from single-celled microorganisms impact millions of human beings globally, with a particular focus on low- and middle-income nations. These diseases also extend their reach to domestic animals on a global scale (1, 2). While options such as chemotherapy, antibiotics, and immunoprophylaxis exist, sustainable long-term control of most diseases remains challenging. Disease-causing microorganisms fall into two distinct domains: prokaryotes and eukaryotes. Prokaryotic cells lack a true nucleus and membrane-bound organelles, typically containing genetic material in a single circular DNA molecule within the nucleoid region. In contrast, eukaryotic cells feature a well-defined nucleus enclosed by a nuclear membrane and various membrane-bound organelles such as the endoplasmic reticulum and Golgi apparatus. Metabolically, prokaryotes exhibit diverse capabilities such as photosynthesis, chemosynthesis, and fermentation, while eukaryotic metabolism tends to specialize in energy metabolism and cellular replication. Prokaryotes primarily reproduce via binary fission, yielding genetically identical daughter cells, whereas eukaryotes use various modes including mitosis for growth and repair and meiosis for sexual reproduction, involving gamete formation (sperm and egg cells).

Despite the notable differences in their reproduction, metabolism, and cellular structure, both prokaryotes and eukaryotes share a common trait: secretion of extracellular vesicles (EVs) (3). In eukaryotic organisms, EVs are defined as small, membrane-bound structures that are classified into three major types: exosomes, ectosomes, and apoptotic bodies based on their size, biogenesis, and composition (4, 5). Exosomes are 30–100 nm in size, of endocytic origin, and are released after the fusion of multivesicular bodies with the plasma membrane (6, 7). In contrast, ectosomes are more heterogeneous in shape, can be larger (0.1–1 µm) in diameter, and are shed directly from the plasma membrane (8), while apoptotic bodies (0.5–2 µm) originate from the plasma membrane during programmed cell death. In addition, prokaryotic Gram-negative bacteria release their outer membrane, giving rise to outer membrane vesicles (ranging from 10 to 300 nm) and, prokaryotic Gram-positive bacteria shed their cytoplasmic membrane, leading to the formation of membrane vesicles (~20–150 nm) (9).

While EVs have been documented in all domains of life, our understanding of the production, mechanisms, and roles of EVs from single-celled organisms from these domains remains limited. Single-celled prokaryotes are categorized into two primary domains: bacteria and archaea (10). EVs from archaea have been recognized as vital mediators of cell–cell communication, stress responses (especially in the context of environmental stress), and exchange of genetic material between cells, potentially contributing to genetic diversity within archaeal populations (11). However, as archaea are not a significant source of disease in mammals, this review will not delve into archaea and their EVs. Pathogenic bacteria possess traits that are of particular interest, especially concerning their involvement with EVs, such as those related to their interspecies communication and immune evasion strategies. These pathogens have developed specific mechanisms to overcome or evade the host’s immune system, often by producing proteins that inhibit immune cell function or interfere with the host’s ability to recognize and eliminate the bacteria (12). Additionally, many bacterial pathogens produce toxins inflicting damage on host tissues, therefore causing a wide range of symptoms (13).

Single-celled eukaryotes are more difficult to define as they constitute a highly diverse group of microorganisms within the eukarya domain. As mentioned previously, what sets them apart from the prokaryotes is their possession of membrane-bound organelles. Despite their relatively diminutive size and simpler cellular structure compared to multicellular eukaryotes such as plants and animals, single-celled eukaryotes showcase remarkable diversity in morphology, habitat preferences, and ecological roles. Notably, only a fraction of these microorganisms are pathogenic; many play essential roles in various ecosystems and contribute to the ecosystem’s overall stability considering their role as primary producers, i.e., algae and many fungi. Nevertheless, within the realm of single-celled eukaryotes, certain organisms function as pathogens. In this review, we will focus on single-celled organisms found in Euglenozoa, Amoebozoa, Apicomplexa, and Metamonada (3, 14). Especially, EVs of the solely parasitic Apicomplexa are very interesting as many are intracellular and can be maintained inside a host cell to perform complex metabolic functions, which in turn can be beneficial for the prokaryote that is also residing in the same host, which is especially interesting within the gut microbiome.

It is reasonable to assume that all single-celled microorganisms necessitate intraspecific communication to coordinate their development and induce cell proliferation. This would include mechanisms of quorum sensing and responses to environmental cues. EVs are believed to play a pivotal role in these processes as carriers for the transfer of a wide array of cargo between cells, including membrane and cytosolic proteins, lipids, DNA, RNA, and other vital components (6, 15). Moreover, developmental stages among these microorganisms can exhibit variability in response to environmental factors, often exposing them to multiple and sometimes hostile habitats and, due to their lifestyle as infectious agents, to the immune system of different hosts (2, 16). This dynamic environmental influence makes the function of EVs stage-dependent, particularly within the Apicomplexa, a group encompassing several genera of significant veterinary and human medical importance (14, 16). Not all stages of these microorganisms can be replicated in vitro or harvested in vivo, posing significant challenges for EV research in this context.

The challenges posed by single-celled eukaryotes are reminiscent of those encountered in the control of bacterial infections. Bacteria have evolved antibiotic resistance mechanisms, necessitating continuous research and development of new antibiotics (17). Furthermore, the diversity among bacterial species and their varied responses to antibiotics add layers of complexity to treatment strategies (18, 19). In both cases, ongoing research and innovation are imperative to address the intricate and evolving dynamics of these microorganisms and the infections caused by them.

In this review, we will delve into the realm of EVs originating from single-celled organisms (Table 1) and explore how parasitic protozoan and bacterial EVs contribute to pathogenesis. We will illustrate the similarities and differences between EVs originating from a variety of single-celled microorganisms. Lastly, we will highlight their distinct functions and why the ability of pathogen EVs to overcome the hosts’ immune system could be utilized for development of diagnostic and therapeutic applications in the future. Both parasitic protozoan and bacterial EVs contain similarities on the content and function of their EVs; however, there are some aspects that are specific to one group versus the other, which sets them apart. Understanding the similarities and differences between parasitic protozoan and bacterial EVs will help build gaps in the knowledge and provide insights into creation of potential therapeutics. For the purpose of this review, we will not be discussing EVs obtained from viruses, fungi, and multicellular parasites and will only focus on single-celled parasitic protozoan and bacterial EVs. However, comparing parasitic single-celled organisms and bacteria specifically offers a unique opportunity for cross-pollination as both fields are likely unaware of mechanistic work occurring in the other field. A critical assessment of existing literature and approaches is essential, as new techniques and approaches can be adopted from both fields to enhance EV research. Given the differing thinking approaches in bacteriology and parasitology, a collaborative effort would accelerate research progress and facilitate a deeper understanding of EV biology and function.

TABLE 1.

Systematic overview of all prokaryotic and eukaryotic single-celled microorganisms discussed in this article^a

Organism	Genus (genera)
Prokaryotes (bacteria)
Gram-positive	Staphylococcus
	Bacillus
	Listeria
	Streptococcus
Gram-negative	Acinetobacter
	Pseudomonas
	Escherichia
	Klebsiella
	Neisseria
	Helicobacter
	Porphyromonas
Eukaryotes (protozoa)
Apicomplexa	Toxoplasma
	Plasmodium
	Neospora
Euglenozoa	Trypanosoma
	Leishmania
Amoebozoa	Entamoeba
Metamonada	Giardia
	Trichomonas

^a For parasitic protozoa, we used the old classification based on the morphology and locomotion.

VESICLE BIOGENESIS OF PATHOGENIC PROTOZOA AND BACTERIA

Similar to mammalian systems, EVs of protozoan parasites are represented by either a mechanism of cell-to-cell communication to directly stimulate cells by receptor-mediated contact or through the transfer of genetic material, proteins, or lipids. The actual mechanisms behind the shedding of parasite EVs also include their specific enrichment with molecules associated with the parasite’s biogenesis. Extracellular vesicles are packaged by the endosomal sorting complexes required for transport (ESCRT) (20).

Vesicle biogenesis is enabled either by the release of exosomes originated in intraluminal vesicles contained within multivesicular bodies (MVBs), upon the fusion of MVBs with the cell plasma membrane, or via direct plasma membrane budding of ectosomes from the cell surface (Fig. 1) (21). After EV release, protozoan parasite EVs can interact with a host cell through three different mechanisms. Either (1) direct fusion with the plasma membrane or the host cell, (2) receptor-mediated endocytosis following receptor–ligand interaction between EVs and the host cell, or (3) signaling via direct interaction between receptors and ligands on the recipient cell surface (20).

Fig 1.

Comparison between the release of protozoan parasitic vesicles vs bacterial membrane vesicles. Protozoan parasitic EVs are taken up by the receiving cell through either endocytosis or direct membrane fusion. The parasitic EV then forms an early endosome (EE), where it then develops into a multivesicular endosome (MVE) that contains intraluminal vesicles (ILVs) with ESCRT-III homologs. The protozoan parasitic EVs are then released into the extracellular space through exocytosis. Another way of releasing parasitic EVs is through direct plasma budding (not shown here). Gram-negative bacterial membrane vesicles are released through budding of the outer membrane. The outer membrane of bacteria contains an abundance of proteins and is able to bud when there is a reduction in attachment of lipoproteins to peptidoglycans, resulting in a reduction of membrane proteins on the surface of outer membrane vesicles (OMVs). The OMVs are then released into the extracellular space.

Parasitic single-celled organisms, despite being eukaryotic organisms, often lack a complete set of ESCRT components, which is notable because it challenges the conventional understanding of EV biogenesis, suggesting alternative mechanisms for EV formation in parasitic organisms. On the other hand, bacteria, being prokaryotic organisms, entirely lack ESCRT machinery. This absence is also noteworthy because it raises questions about how bacteria produce EVs without the involvement of ESCRT, which is primarily known to operate in eukaryotic cells (Table 2). The remarkable aspect lies in the evolutionary divergence between pathogenic single-celled parasites and bacteria. Understanding how parasites produce EVs without a complete ESCRT system and how bacteria produce EVs without ESCRT altogether challenges our current knowledge of EV biogenesis. It opens avenues for exploring alternative pathways and mechanisms involved in EV formation across different biological kingdoms, which could have implications for understanding host–pathogen interactions, disease pathogenesis, and development of therapeutic interventions.

TABLE 2.

Direct comparison of bacteria, single-celled parasite biogenesis, EV-cargo, and host immune response

	Bacteria	Protozoa
Biogenesis	Several proposed models	Autonomous ESCRT possible
	Low protein count
	LPS weakening (2, 22)
	PQS in P. aeruginosa (23)
	ESCRT hijacking possible (24)	ESCRT hijacking possible (16)
		ESCRT characterized (9)
		Kinetoplastids (ESCRT I-III)
		Apicomplexa (ESCRT III) (25)
		Amoebozoa (ESCRT III) (26, 27)
	Homologs to ESCRT proteins	Homologs to ESCRT proteins (28)
	Production of OMVs (29, 30)
EV-cargo	Proteins	Proteins
	Surface proteins (31, 32)	Virulence factors (33)
	Toxins (34)	Glycoproteins, glycolipids, and glycopeptides (35)
		Invasion-associated proteins (36)
	Lipids	Lipids
	Fatty acids (37)	Amides, fatty acids, and eicosanoids (38, 39)
	Membrane-bound lipids (37)
	Nucleic acids	Nucleic acids
	mRNA (40)	mRNA (41)
	sRNA (42, 43)	sRNA (41)
	DNA of the host (22)	DNA of the host (44)
Host immune response	Proinflammatory cytokines (42)	Proinflammatory and anti-inflammatory cytokines (45, 46)
	TNF-α, IL-β, IL-8, IL-10127,148, and chemokine CCL2 (42)	Promotion of infection (47)

The ESCRT-pathway

The ESCRT-pathway mediates several cellular processes including cell organelle compartmentalization and plasma membrane maintenance and is a key player in the biogenesis of EVs (21). The ESCRT pathway is composed of around 30 proteins and assembled into five subcomplexes: ESCRT-0, -I, -II, and -III, along with ESCRT proteins Alix and the AAA ATPase Vps4 (48). While many intracellular protozoan parasites are able to hijack their hosts’ ESCRT pathways and utilize them for their own EV production, an autonomous ESCRT system has only been studied in a few parasites (21) and has not been demonstrated in bacteria.

Kinetoplastida is a group of flagellated protists that belong to the Euglenozoa phylum and is one of the few protozoan parasites that has a fully functional ESCRT complex that is active in both extracellular and intracellular stages of infection. Furthermore, a phylogenetic study of the ESCRT system identified several homologs of ESCRT-I, -II, and -III family members in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi, indicating that this system is conserved in Kinetoplastida. Four homologous ESCRT proteins have been fully characterized in T. brucei (TbVps23, TbVps28, TbVps4, and TbVps24) and demonstrated to be important players in endocytosis of ubiquitinated proteins and lysosomal degradation, which occurs in the flagellar pocket (49). Also, ESCRT-II component, Vps36, is critical for the secretion of multivesicular body (MVB)-derived EVs in T. brucei. Analogs of the ESCRT complex families -I, -II, and -III were found in Leishmania (50), but their roles in EV biogenesis remain unexplored. EV proteomic studies have found Rab GTPases, Alix, and other ESCRT homologs to be present within Leishmania EVs (51). Hence, in Kinetoplastida, many of the ESCRT family members are conserved, and ESCRT proteins have been found in their EVs.

In the Apicomplexa, the ESCRT system has only been implicated in EV biogenesis in Plasmodium falciparum. This parasite infects red blood cells, which have lost the majority of their organelles (25). While P. falciparum lacks ESCRT-0, -I, and -II sub-complexes, the ESCRT-III sub-complex plays an important role in the formation of EVs from infected red blood cells (25). In contrast, Toxoplasma gondii can infect any nucleated cell, and instead of relying on producing its own ESCRT machinery, it co-opts the host version. Thus, the effector protein TgGRA14, a dense granule protein, is associated with the recruitment of the host’s ESCRT machinery (52). In addition to TgGRA14, there may be other parasitic proteins that help in the recruitment of the host’s ESCRT machinery since the Alix protein, a major player in the ESCRT pathway, is not affected by TgGRA14 disruption; however, TSG101 recruitment relies on TgGRA14 activity (52). While both P. falciparum and T. gondii only contain ESCRT-III components, they are both obligate intracellular parasites, which allow them to utilize the host’s ESCRT system to further benefit the parasite’s infiltration and spread.

There is also evidence that extracellular parasites also produce EVs. Entamoeba histolytica is a parasitic member of the Amoebozoa phylum that utilizes the ESCRT machinery in order to remodel its membrane. Entamoeba histolytica performs phagocytosis by contacting macrophages through the ESCRT machinery for initial adherence. Several ESCRT-III proteins (EhVps2, EhVps20, and EhVps32) and ESCRT-I proteins (EhVps23) have been localized in the parasite’s plasma membrane during initial contact with E. histolytica (23). Giardia duodenalis is another extracellular protozoan parasite from the Metamonada phylum with reduced ESCRT machinery. Similar to P. falciparum and T. gondii, G. lamblia contains some ESCRT-III orthologs but lacks several genes encoding ESCRT-I, -II, and some subunits of ESCRT-III (53). While G. lamblia lacks certain ESCRT machinery, the overexpression of the GIVps4a mutant or GIVps4a antisense mRNA leads to a reduction in intraluminal vesicle formation on the peripheral vacuole (53).

Despite lacking a conventional endolysosomal pathway, Giardia lamblia possesses the capability to generate and discharge exosome-like vesicles, which are similar in size, shape, and protein and lipid composition to exosomes found in other eukaryotic cells (53). Additionally, the presence of intraluminal vesicles within some peripheral vacuoles during the stationary phase (54) suggests a connection between the formation of intraluminal vesicles and general EV release, as they are both dependent on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide (53).

In addition, the formation of EVs increases during host cell exposure for Trichonomonas vaginalis. Several proteins involved in the ESCRT complex of T. vaginalis have been identified, including TvTSP1, TvMIF, and TvTCTP proteins. Additionally, various Rab proteins, such as Rab4, Rab5, Rab11, Rab35, Rab27a, and Rab27b, have been implicated in different stages of exosome release across different cell types. Furthermore, T. vaginalis possesses 48 members of the GP63 protease family, although their precise role in infection remains incompletely understood, with proposals suggesting their significance in the infection process (55, 56), especially VPS32, which plays a vital role in both EV biogenesis and cargo sorting in this parasite and significantly influences parasite attachment to prostate cells, thus impacting T. vaginalis pathogenesis (56). Moreover, overexpression of VPS32 leads to an increase in the release of exosomes and ectosomes, indicating its pivotal role in EV biogenesis (57).

This indicates an involvement of ESCRT proteins in the biogenesis of EVs. As our understanding of ESCRT proteins and their parasite homologs deepens, it will be easier to determine whether protozoa ubiquitously use this pathway. As with any eukaryotic pathogen, parasite-specific proteins may be a significant druggable target. It is, therefore, relevant to determine which parasites and lifecycle stages are dependent on the host vs parasite ESCRT machinery.

Bacterial vesicle secretion

Although our knowledge of the origins of protozoan parasite EVs has greatly expanded, it is still not well-understood how bacterial membrane vesicles are produced. However, in contrast to protozoan parasitic cells, the role of proteins in vesicle formation within bacteria remains less substantiated. While it was the general belief that the ESCRT pathway was exclusive to eukaryotic cells, recent works have shown that bacterial protein PspA is homologous to ESCRT-III and belongs to the same family (58); however, it is not suggested to be a part of vesicle formation.

Bacterial membrane vesicles are directly budded from the membrane, suggesting that the integrity of the membrane likely influences their formation. It is worth noting that both Gram-positive and Gram-negative bacteria possess distinct membrane structures. Gram-positive bacteria are defined by a robust layer of peptidoglycans in their cell wall, which can act as a barrier limiting access to their cytoplasmic membrane (38). In contrast, Gram-negative bacteria possess a distinctive dual asymmetric membrane structure. The outer membrane contains lipopolysaccharides, porins, and various proteins, contributing to their unique physiology and interactions with their environment (33).

Due to the presence of thick peptidoglycans, it was previously believed that Gram-positive bacteria did not secrete vesicles. However, in 1990, Dorward and Garon conducted a survey of several Gram-positive strains, such as B. subtilis, B. cereus, S. aureus, and S. sanguis (44). Their findings indicated that B. subtilis and B. cereus produced vesicles, while no vesicle formation was reported for S. aureus and S. sanguis in their log-phase growth (44). Interestingly, in recent years, vesicle production from S. aureus has been highlighted (59, 60). Additionally, Gram-positive bacteria such as Bacillus anthracis and Listeria monocytogenes have also been reported to produce vesicles (61, 62).

As previously mentioned, the substantial peptidoglycan layer in Gram-positive bacteria poses a challenge for vesicle budding. While the mechanism behind Gram-positive bacteria membrane vesicles is still unclear, it is reasonable to consider that degradation of the peptidoglycan cell wall is essential for secretion of vesicles. Previously, it was reported that the expression of the endolysin within a prophage resulted in peptidoglycan weakening, which, in turn, prompted the cytoplasmic membrane to protrude through the compromised peptidoglycan layer, giving rise to vesicles (63). However, it is also important to note that the weakening of peptidoglycans leads to decrease in membrane integrity, which further leads to the death of the cell. In bacteria such as S. aureus and S. pneumoniae, peptidoglycan-degrading enzymes were found in the vesicles, indicatng that these enzymes might play a role in vesicle formation (60, 64).

Gram-negative bacteria are distinguished by their dual-membrane structure, which consists of the inner and outer membranes with the periplasmic space positioned in between (33). The bacterial outer membrane displays an intriguing asymmetry: the inner leaflet is made up of phospholipids, while the outer leaflet consists of a dynamic mixture of proteins, phospholipids, and lipopolysaccharides (LPS) (33). This intricate composition has given rise to several hypotheses regarding the factors that influence the biogenesis of OMVs. While there is no single explanation for OMV formation, currently there are several overarching mechanisms.

Bacterial outer membranes are decorated with several critical lipoproteins such as porins, outer membrane proteins (Omp), and bacterial surface antigens (33). In 1976, Hoekstra conducted comprehensive biophysical analyses on both the outer membrane vesicles and the parental outer membrane (65). The results revealed a notable contrast: while the parent outer membrane comprised approximately 40% proteins, the OMVs exhibited a significantly lower content of membrane proteins, ranging from 10% to 15% (65). These findings suggest that reduced attachment of lipoproteins to the peptidoglycans triggers membrane bulging, potentially leading to biogenesis of vesicles (Fig. 1). Recent research on pathogenic bacteria, specifically A. baumanii, suggests that deleting the ompA gene, responsible for outer membrane protein A production, boosts vesicle production (66). This reinforces the connection between reduced outer membrane proteins and increased vesicle formation.

Lipopolysaccharide (LPS) is a key component of the Gram-negative bacteria outer membrane. Structurally, LPS comprises three main components, lipid A (an anchor embedded in the membrane), the core polysaccharide (inner and outer core), and the O-antigen portion (31). In 2014, Schwechheimer et al. investigated LPS accumulation and OMV formation. They found that deleting the rfaC and rfaG genes led to increased LPS levels, causing strains to exhibit a larger amount of OMV produced compared to the wild-type (67). This highlights that the LPS also plays a role in OMV formation.

Within the realm of P. aeruginosa, there exists a belief that the Pseudomonas quinolone signals (PQSs) play a crucial role in OMV formation. The pqsA mutant, responsible for quinoline/quinolone production, demonstrated a significant reduction in OMV production (26). Significantly, the addition of biologically relevant quantities of PQS to the media resulted in the emergence of OMVs, underscoring the indispensable contribution of PQS to OMV generation within P. aeruginosa (26). Furthermore, PQS serves as a protein-inducing membrane curvature, implying its potential role in aiding the process of OMV formation (68).

The outer membrane of bacteria has long been recognized for its ability to form extracellular vesicles known as outer membrane vesicles; however, recent reports have introduced an intriguing dimension to our current understanding of vesicles released from Gram-negative bacteria, revealing that bacteria possess the ability to co-secrete their inner and outer membrane, giving rise to outer–inner membrane vesicles (O-IMV) (9). Kadurugamuwa and Beveridge initially proposed that enzymes such as autolysin might degrade peptidoglycans, potentially allowing the inner membrane to release with the outer membrane (37). This process paves the way for the creation of outer–inner membrane vesicles, which was later experimentally demonstrated by (69, 70). The exact mechanistic details for O-IMV biogenesis is yet to be fully understood.

EV-CARGO OF PATHOGENIC PROTOZOA AND BACTERIA

EVs have been shown to contain proteins, lipids, and nucleic acids, and their exact content can vary depending on the microorganism’s life cycle and environment, including the host cell type. Determining which biomolecules exactly are packaged within EVs is often difficult, and hence an appropriate isolation method also needs to be chosen (71). Specifically, two problems arise. First, the separation between EVs of the microorganism and its host often is not possible. Second, microorganisms, whose life cycle includes varying growth patterns and different cellular niches, likely produce different EVs dependent on the stage (71). However, there are common key features that can be identified and compared between protozoan parasite EVs and bacterial OMVs (Fig. 2).

Fig 2.

Comparison between the content of protozoan parasitic vesicles vs bacterial membrane vesicles. Protozoan parasitic EVs are made up of a phospholipid bilayer and contain different types of proteins. For example, parasite specific proteins, ESCRT proteins (TSG101 and Alix), cytoskeleton proteins (actin and tubulin), and heat shock proteins (HSP70). Parasitic EVs also contain DNA along with different types of RNA (miRNA, tRNA, rRNA, and sRNA). On their outer membrane, they contain common tetraspanins found on the surface of many EVs (CD63, CD81, and CD9). Gram-negative bacterial membrane vesicles contain many components both on the outside and inside of the vesicle. The outer layer of the bacterial membrane vesicle is generally composed of a mixture of phospholipids and LPS, while the inner membrane is composed only of phospholipids. On the outside and inside of their membrane, they contain DNA and RNA (mRNA, tRNA, rRNA, and sRNA). Bacterial membrane vesicles contain outer membrane proteins on its surface, along with cytosolic proteins within. Some other components found inside bacterial membrane vesicles are peptidoglycans and toxins.

Proteins

While studies focus on host–parasite communication in order to better understand disease, studies on proteomic profiles of EVs from single-celled organisms are still in the beginning stage. However, proteins important for parasite invasion were commonly shown across several types of Plasmodium EVs, showing the parasites’ adaptability of EVs to different environments (5, 25). Similarly, Gram-positive and Gram-negative bacterial OMVs can carry a variety of proteins facilitating their interactions within their respective environments. However, their derivation is vastly different. Proteins included in the ESCRT pathways have been discussed in more detail in the second section and will not be discussed again here.

The functional role of EVs derived from Kinetoplastids has been studied most intensively; however, the number of identified proteins not involved in the ESCRT pathway or homologous to proteins involved in this pathway is still small. A proteomic analysis of EVs from Leishmania spp. revealed that the protein cargo of EVs differ quantitatively in response to changes in temperature and pH, making EVs a decisive factor in the parasites’ response to different environmental cues (72, 73). Virulence factors such as GP63/leishmania, membrane proteins, heat-shock proteins, and redox enzymes such as tryparedoxin peroxidase are among some of the proteins identified in Leishmania EVs (73). The surface protease GP63 has also been identified in EVs from macrophages of L. mexicana exposed to parasite promastigotes (74). Extracellular vesicles from T. cruzi include major glycoproteins, glycolipids, and glycopeptides of the parasite surface (35). Many of the proteins found in the EVs secreted by T. cruzi are involved in metabolism, parasite survival, signaling, host and parasite interaction, and nucleic acid binding (28). Furthermore, Trichomonas vaginalis may use EVs with protein cargo to manipulate the host defense responses, similarly to the secretion of virulence factors via EVs by pathogenic bacteria (45).

Giardia duodenalis EVs also contain heat-shock proteins (Hsp70), in addition to cysteine proteases, β- and α-tubulin, variant surface proteins, and other proteins that guarantee parasite survival (75, 76). Proteomic analysis of Entamoeba histolytica showed that their EVs were enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins, further giving their EVs prominent roles in intercellular communication that regulates parasite growth and development (77).

Extracellular vesicles from P. falciparum contain both host and parasite proteins, including some malaria antigens (MSP1, -3,–7, −9, and Hsp70) and serine-repeat antigen (78). These EVs also contain proteins that are essential for red blood cell invasion (AMA-1, EBA-175, and MSP-1) (79). The proteomics results revealed P. falciparum EVs to contain invasion-associated proteins, while T. gondii releases EVs that contain CD63 and Hsp70 (80). By using proteomics, EVs released from T. gondii tachyzoites have also been shown to carry parasite specific proteins such as P30 (surface antigen), microneme proteins, dense granule antigens, glycosylphosphatidylinositol, ubiquitin, and cyclophilin (81). Studies on EV production by the slow replicating, cyst-forming life cycle stage, the bradyzoite, have yet to be conducted, which is likely due to the shortage of materials; however, such studies could reveal differences in EV content and therefore function. These stages are long-lived and found intracellularly within neurons, which themselves rely on significant EV cell–cell communication; therefore, production of EVs by these parasites has the potential to manipulate chronicity of infection or alter cell function within the brain.

Both Gram-positive and Gram-negative bacterial membranes host an array of proteins on their surfaces, facilitating interactions within their respective environments, similar to some of the protozoan parasites (38, 82). When enclosed within OMVs, proteins find protection behind the membrane, an advantage over extracellular secretion, making this a favorable form of protein release (83).

Numerous proteins can be secreted within vesicles, encompassing toxins, outer membrane proteins for Gram-negative bacteria, periplasmic proteins, and signaling molecules. Vesicles released by Gram-negative bacteria stem from the outer membrane of the bacteria, and consequently, it is apparent that some of the outer membrane proteins will also be encompassed within these vesicles. Proteomic analyses of OMVs from various Gram-negative bacteria, including N. meningitidis, P. aeruginosa, E. coli, A. actinomycetemcomitans, A. baumanii, and V. cholerae, have been conducted (84–89). These investigations unveiled a diverse array of cargo within these vesicles, encompassing not only outer membrane proteins, porins, and efflux pumps but also several periplasmic and soluble proteins, representing a wide spectrum of protein classes. In addition to the proteins, the OMVs released by P. aeruginosa encapsulate a substantial amount (86%) of the hydrophilic signaling molecule PQS, marking a significant presence within these vesicles (26). Furthermore, OMVs secreted by various Gram-negative bacteria, including A. actinomycetemcomitans, V. cholerae, pathogenic E. coli, P. syringae (a plant pathogen), P. aeruginosa B. anthracis, and H. pylori, encapsulate their respective virulence factors, which aids in facilitation of OMV pathogenesis (62, 90–95).

While Gram-negative bacteria release a relatively small fraction (approximately 0.1% of the total population) of vesicles in the form of outer–inner membrane vesicles (O-IMV), proteomics studies have revealed the presence of cytosolic and membrane proteins within these vesicles (69, 70). Turnbull et al. (39) reported that O-IMVs produced by P. aeruginosa also incorporate endolysin, an enzyme that aids in the breakdown of peptidoglycans (39). Similar to their Gram-negative counterparts, membrane vesicles released by Gram-positive bacteria contain a diverse array of proteins, including membrane proteins and toxins. Proteomic analysis of opportunistic Gram-positive bacteria S. aureus shows that it contains the toxin Staphylococcal enterotoxin, which facilitates in pathogenesis (60). In the case of pathogenic Streptococcus pneumoniae, electron microscopy-immunogold staining was employed to show that vesicles contained pneumolysin, a protein toxin found within the bacterial cytoplasm (96). Although pneumolysins could be identified through electron microscopy, its prominence was relatively reduced in immunoblotting analyses. Interestingly, it was more noticeably observed in both the whole cell lysate and the supernatant. L. monocytogenes, an opportunistic Gram-positive bacterium, produces vesicles containing its virulence factors, including phosphatidylinositol-specific phospholipase C, an enzyme that hydrolyzes phosphatidylinositol lipids and aids in host invasion, and listeriolysin O (LLO), a pore-forming toxin (61). These studies emphasize the significance of these vesicles for pathogenic bacteria, as they transport crucial cargo that contributes to pathogenesis, and also show that while both bacterial OMVs and protozoan parasite EVs can contain proteins specific to the cell type from which they are released, bacterial use of toxins gives the organisms another opportunity for host cell manipulation, which protozoan parasites are missing.

In that regard, both bacterial OMVs and EVs released by protozoan parasites are thought to acquire a protein corona upon exposure to host cells or biological fluids. This corona, formed by binding of proteins to the surface of the vesicles, varies in composition depending on factors such as the species of the microorganism, growth conditions, and the surrounding environment. Studies indicate that the protein corona influences the interactions of both bacterial OMVs and protozoan EVs with host cells, affecting processes such as immune modulation, adhesion, internalization, and cargo delivery. Despite the limited knowledge regarding the protein corona in bacteria and protozoans, fostering an interdisciplinary collaboration between these fields could yield significant insights, accelerating the elucidation of specific proteins involved and their functional implications, thereby immensely benefiting both areas of research.

Lipids

Lipids are the least analyzed metabolites, although they are fundamental components outlining EVs, and their involvement has been suggested in EV formation, characteristics, and biological functions (20, 97–99) in eukaryotes and prokaryotes alike.

Apicomplexan life stages are highly dependent on large and specific amounts of lipids as well as lipid homeostasis to sustain intracellular development, membrane/organelle biogenesis, and parasite survival and propagation (100). Parasites also require lipids as communication tools to trigger active host cell invasion and egress mechanisms, and they adapt their lipid metabolism to changing host and nutrient environments (101–103). This is intriguing as the parasites can change their mode of lipid acquisition to adapt to environmental conditions such as selective starvation in vitro (104). This flexibility includes the so-called “make and take” method, whereby lipids are produced by a combination of de novo–produced lipids (“make”) and those scavenged from the host (“take”) (104, 105). Within the parasite’s metabolic pathways, lipids interact with proteins such as adhesins, proteases, receptors, vesicle trafficking-promoting particles, and molecules involved in membrane fission or fusion. One of the most important metabolic adaptations of Apicomplexan parasites in the physiological context lies in fatty acid synthesis (FAS), which can be adapted to the environment, as outlined previously (104). Fatty acids are essential for the formation of membranes (106) and therefore for the multiplication of viable parasite cells, especially asexual stages that are then able to infect new host cells or hosts.

Furthermore, EVs carry parasite-derived cargo to the host (107, 108) and therefore carry important pathogenesis factors in a variety of infectious diseases (105). Exosomes and ectosomes are capable of directly transporting lipids, such as amides, fatty acids, and eicosanoids, from producing to recipient cells, potentially causing changes in the immune response and the metabolism of the recipient cells (109). A detailed analysis of the lipid cargo transported by protozoan parasite EVs could also give insights into the mechanisms of the infection process and, more generally, parasite biology. High-resolution mass spectrometry-based lipid measurement and data analysis for profiling EV proteins and lipids were recently applied to analyze human plasma, serum (110), and cancer cells (111). Although the cargo of cells commonly used for in vitro culture has been analyzed intensively in recent years, limited data on EV cargoes of protozoans exist, and further investigations will be necessary to understand the roles of lipids in the host–pathogen interactions.

However, the lipidome of Giardia lamblia has already been characterized and primarily shows sphingolipids including sphingoid bases, ceramides, glycosphingolipids, and sphingomyelin (112). Recent lipidomic analyses have revealed dynamic changes in the lipid and fatty acid profiles of Giardia throughout its life stages and that ceramide, a crucial component of sphingolipid biosynthesis, plays a significant role as a bioactive lipid in signaling pathways that regulate essential cellular responses such as apoptosis, cell differentiation, and cell cycle arrest across various cell types (57).

Unlike protozoan parasites, the bacterial cell membrane consists of a diverse array of amphiphilic lipids, including traditional glycerophospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), and lysyl-phosphatidylglycerol (LPG) (113). Additionally, the membrane incorporates phosphorus-free lipids such as for Gram-negative bacteria, ornithine lipids (OLs), glycolipids (GLs), and other unique components (113). While bacterial vesicles originate from the parent bacterial membrane, it is crucial to emphasize that not all lipid components are integrated into the vesicle’s membrane, and, conversely, a minor lipid component was also identified in the vesicle that was not part of the parent bacterial membrane (94).

The composition of membrane vesicles is critical for maintaining a fluid structure. Hoekstra and coworkers first reported, in detail, the lipid composition of the membrane vesicle vs the parent bacterium, where it was highlighted that vesicles were enriched in a higher proportion of unsaturated fatty acids compared to the parent E. coli membrane, while the proportion of saturated fatty acids was equivalent in both compartments (65). Analysis of the phospholipid composition of P. aeruginosa revealed significant differences compared to that of the parent membrane. Outer membrane vesicles predominantly consisted of PE at 59.7%, whereas PG dominated the bacterial membrane at 63.0% (114). Due to its conical shape, PE has the ability to induce membrane curvatures, thereby playing a role in facilitating the formation of membrane vesicles. Interestingly, in contrast to OMVs of E. coli, vesicles from P. aeruginosa were discovered to contain elevated levels of saturated fatty acids (114). This enhances the rigidity of the vesicles, potentially facilitating their budding from the more rigid sections of the membrane. These observations emphasize the diversity in lipid profiles found in OMVs from different organisms.

Since OMVs bud directly from the outer membrane of the Gram-negative bacteria, they inherently contain LPS, which is a unique characteristic of Gram-negative bacteria (37, 65, 93, 114). Furthermore, advancements in lipidomics have yielded valuable insights into the lipid composition of OMVs and the impacts of various treatments on the OMV lipid components (115–118).

Lipids play a crucial role in the composition of Gram-positive bacterial membrane vesicles, comparable to Gram-negative bacteria. Similarly, vesicles from Gram-positive bacteria can contain lipids that might not be present in the parent bacterial membrane. Rivera et al. (62) highlighted this disparity, revealing that while stearic and palmitic acids constituted the major lipid components in both the membrane vesicles and the parent membrane of Bacillus anthrax, additional lipids such as caprylic, capric, tridecanoic, and tricosanoic acids were uniquely present in the vesicles but absent in the parent bacterial cell membrane (62). The vesicles of Listeria monocytogenes exhibit a higher content of unsaturated fatty acids and are enriched with PE, sphingolipids, and triacylglycerols compared to the parent membrane (61). The roles that such selectively enriched vesicle membrane lipids might play in the interactions between bacteria and hosts still need to be elucidated. While many of the lipids found in bacterial OMVs and protozoan parasite EVs may differ, both Listeria monocytogenes vesicles and Giardia lamblia EVs contain sphingolipids, an important component of membrane structure.

When we look at both bacterial outer membrane vesicles (OMVs) and parasitic protozoan EVs, we find that sphingolipids, which are important lipids in eukaryotic cells and usually not found much in bacteria, are actually abundant in OMVs. This observation suggests a potential role for sphingolipids in modulating the levels of bioactive lipids, potentially influencing interactions with gut epithelial cells, which are known to respond to bacterial sphingolipids (118, 119). Similarly, sphingolipids are implicated in EV biogenesis of certain protozoan parasites, such as G. intestinalis, although it remains unclear if this phenomenon extends to all pathogenic protozoa (21). Despite these findings, the biosynthetic pathways responsible for sphingolipid production in either bacteria or parasites have yet to be fully elucidated.

Nucleic acids

Despite the apparent lack of miRNA genes in protozoans (120), their EVs have been repeatedly found to contain a cargo of small RNAs, often acquired by the parasites from their host organism. In recent years, different RNA subtypes contained in EVs have been extensively studied with respect to their potential in altering the gene expression of the recipient cells. While there is a variety of RNA subtypes, in the case of T. gondii, EV miRNA has been the most studied. To evade the host’s immune responses, T. gondii transform themselves into tissue cysts and modulate host cell cycle and apoptosis. During T. gondii infection, an elevated expression of miR-155–5p in EVs from infected dendritic cells triggers a miR-155–5p-dependent pro-inflammatory response upon uptake by macrophages, which was shown by analysis of exosomal miRNA expression profiles (121). EVs from dendritic cells infected with the RH strain of T. gondii revealed an upregulation of miR-196b-5p (122). EVs of T. gondii also contain miR-21. When these EVs are added to glioma cells, a decrease in anti-tumor gene expression was observed (123). Although the study’s utilization of a single, infrequently employed cell line in vitro presents a limitation, the authors addressed this by incorporating an in vivo xenograft mouse model. This model not only demonstrated restricted tumor growth but also enabled an analysis of altered microRNA expression profiles, particularly focusing on mature exosomal miRNAs within microglial cells. This ability for T. gondii-stimulated miR-21 to alter cell growth and even susceptibility to tumors has significant clinical implications, while it may also reveal links between the biology of infections and the development of other diseases. Hence, EVs derived from Toxoplasma parasites play a multifaceted role in host–parasite interactions by carrying host miRNAs. These EVs contribute to the evasion of host immune responses and the modulation of host cell processes, including cell cycle and apoptosis. Importantly, the presence of host miRNAs within parasite-derived EVs suggests a complex interplay between parasite and host factors, potentially influencing the pathogenesis of Toxoplasma infection and highlighting the intricate nature of host–parasite interactions at the molecular level.

The RNA cargo of EVs produced by various Trypanosomatids is mostly represented by specific fragments of rRNA and tRNA transcripts (124–126). EVs from Trypanosoma cruzi contain a variety of different small RNAs, predominantly ribosomal RNA (rRNA) and transfer RNA (tRNA) with over a million reads each (124). Of the different tRNAs, Glu (glutamic acid) and Val (L-valine) were most abundant (124). Similar to T. gondii, T. cruzi EVs contained significant amounts of miR-155 and miR-21, along with miR-146 (127). These miRNAs were further analyzed as potential biomarkers for the detection of Chagas’ disease at an early stage (127). Extracellular vesicle researchers are also trying to use Plasmodium vivax miRNA to help detect malaria. Extracellular vesicles derived from P. vivax-infected plasma showed elevated levels of miR-150–5p and miR-15b-5p, as compared to serum from uninfected patients (128). These miRNAs are known to be linked to cellular processes, for example, TGF-β signaling that can broadly affect host immune responses to infection (128).

RNA sequencing of Giardia EVs revealed different types of small RNAs (sRNA) with an abundance of ribosomal-small RNA (rsRNA) inside EVs, followed by messenger-small RNA (msRNA) and transfer-small RNA (tsRNA) (129). In addition to the protein content, the RNA content of EVs can also vary based on the developmental stage of Giardia (124, 126). While there were many similarities in the small non-coding RNA content of EVs from either Leishmania braziliensis and Leishmania donovani, there were also differences. Only L. braziliensis EVs contain transcripts derived from small interfering RNAs (siRNAs), while EVs from both species contain many tRNA fragments (126).

Trichomonas vaginalis EVs also contain a variety of small RNAs. RNA-seq analysis revealed that tsRNA, particularly 5' tRNA halves, constitute the predominant type of small RNA within these vesicles. Furthermore, it was determined that the tsRNAs present in T. vaginalis EVs originate from specific processing of tRNAs within the cells, and their selective packaging suggests a preference for certain types of RNA molecules (130). The potential impact of these tsRNAs, a newly recognized class of small regulatory RNAs, on modulating host cellular responses warrants further investigation. However, the RNA cargo from T. vaginalis was observed to be efficiently internalized by human cells via lipid raft-dependent endocytosis (131), presenting a novel avenue for parasite–host communication.

Furthermore, the sRNA contents of EVs isolated from E. histolytica showed that these EVs, depending on the secreting cells’ developmental stage, are capable of modulating the encystation process and amebic EVs contained RNAi pathway effector proteins as well as small RNAs. Their function is similar to that of Plasmodium falciparum-infected erythrocyte-secreted EVs packaged with functional miRNA–Argonaute 2 complexes (132), as mentioned previously, and modulates gene expression in target endothelial cells, which helps the parasite evade the host cell immune response (77).

The significance of sRNA components in bacterial interspecies communication and virulence is well-described (40, 133). Membrane vesicles can contain RNA serving various purposes in pathogenesis, communication, and protection against extracellular RNase degradation. The diversity of RNA found in OMVs from different organisms such as E. coli, Porphyromonas gingivalis, Vibrio cholerae, Neisseria gonorrhoeae, Acinetobacter baylyi, and Acinetobacter baumannii is remarkable (44, 86, 134–137). These RNA types encompass mRNA, rRNA, sRNA, and tRNA. Gram-positive membrane vesicles from S. aureus have also been found to contain RNA (42). In the mentioned studies, RNA was reported to be present in the lumen and on the surface of the vesicles; however, the exact mechanism responsible for the presence of RNA-loaded vesicles remains enigmatic. A plausible explanation suggests that if protein synthesis occurs proximate to the membrane during vesiculation, mRNA could potentially become encapsulated within the vesicles (47). Overall, while many different types of RNAs have been identified in both bacterial OMVs and protozoan parasite EVs, further studies need to be carried out to better understand the roles of these RNA subtypes when taken up by an uninfected cell. Although a broad range of RNA subtypes have been found in vesicles, protozoan parasite EV and bacterial OMV studies have focused more on small RNAs.

While EVs have been known to contain DNA, there seems to be limited research on the DNA content of protozoan parasite EVs. A recent experiment observed that DNA from Leishmania EVs is enriched in drug-resistant genes, similar to the parasite’s original DNA (138). Amplification of the mrpA gene was also observed in both parasitic and EV DNA (138). Extracellular vesicles from P. falciparum contain plasmodial genomic DNA that, when taken up by the host cell, is able to elicit host cytosolic immune cell receptors (139). The full capacity of protozoan parasite EV DNA in the parasite–host interplay still needs to be explored.

Contrary to protozoan parasite EVs, DNA has been better studied in bacterial OMVs. Bacterial vesicles can transport DNA either within their interior or as surface-associated cargo. As mentioned previously, Kadurugamuwa and Beveridge (37) identified DNA within OMVs released by P. aeruginosa, but the mechanism allowing DNA to breach the inner membrane barrier and be enclosed within OMVs remained enigmatic. It was not until (69, 70) proposed that Gram-negative bacteria have the capacity to concurrently release both outer and inner membranes, forming O-IMVs that encapsulate DNA, that this phenomenon was mechanistically explained. The presence of lysed bacteria in the medium during the formation of new OMVs can also result in encapsulation of DNA (44, 140, 141).

In addition to the lumen, P. aeruginosa OMVs also carry DNA on their surface and have the capacity to transfer their DNA into eukaryotic cells (43). This discovery not only underlines OMVs as potential vehicles for long-range DNA delivery and transfer but also highlights their multifunctional nature. Helicobacter pylori and A. actinomycetemcomitans have also been reported to contain surface-associated DNA (142–144).

While in the 1990s it was assumed that DNA was exclusively present in Gram-negative bacteria OMV and not in Gram positive bacteria (44), the presence of DNA within OMVs secreted by Ruminococcus species, a genus of Gram-positive gut bacteria, was demonstrated later (145). After treatment with DNase I, the DNA was completely removed, suggesting that the DNA present on these OMVs is primarily surface-associated rather than originating from the cytoplasm. Staphylococcus aureus membrane vesicles also contained DNA, with some being part of the lumen and some being surface-associated, and came off after treatment with DNase (42). Though the field of Gram-positive bacterial membrane vesicles is still under scrutiny, it appears that these vesicles share structural similarities with Gram-negative vesicles and also carry nucleic acids both on their surfaces and internally. It will be important to see whether the transfer of bacterial DNA to the host, as seen in the case of Gram-negative bacterial OMVs, holds true for other bacterial species too.

HOST IMMUNE RESPONSE

The production of EVs by pathogens plays a major role in modulating the host’s immune response and may be responsible for successful infection. For example, Leishmania EVs contain virulence factors that can modulate macrophage activity and inhibit the secretion of pro-inflammatory cytokines, thereby preventing activation of effector cells and parasite killing (73). One virulence factor present in Leishmania EVs is a metalloprotease glycoprotein of 63 kDa (GP63), which regulates NF-кB and other transcription factors (74, 146). GP63 is expressed in many Leishmania species and can degrade a broad range of substrates, making it an important factor for the parasite’s resistance to host defense mechanisms (147). Indeed, parasites deficient in GP63 were able to establish an infection, with the consequence of decrease in lesion sizes in vivo (147). Macrophages exposed to EVs from L. shawi had increasing levels of intracellular toll-like-receptor (TLR) 9 and cytoplasmic NOD-like-receptor (NOD) 1, suggesting activation of the immune system (148). Macrophages stimulated with Leishmania EVs also exhibited an upregulation in IL-1β, a pro-inflammatory cytokine, along with a downregulation of TNF-α, a regulator of inflammation, promoting a balanced immune response by activating both pro-inflammatory and anti-inflammatory cytokines (148).

EVs from P. falciparum-infected red blood cells were found to contain invasion-associated proteins. In vitro studies showed that P. falciparum EVs are internalized by monocytes and macrophages, allowing them to deliver cargo into the cytosol and prompt an innate humoral immune response (149). The addition of EVs from T. gondii, G. duodenalis, and T. cruzi onto macrophages elicits a pro-inflammatory response through the increased production of NO, IL-12 IL-6, and TNF-α (46, 80, 150, 151). This enhanced cytokine production was also supported by the activation of TLR2 and NLRP3 signaling, indicating the start of an innate immune response (151). T. cruzi EVs also inhibit C3 of the complement pathway, allowing the parasite to evade the host’s immune response and promote infection (152, 153). Along with a pro-inflammatory response, the addition of T. gondii EVs onto macrophages resulted in a significant increase in the production of IFN-γ, along with a significant decrease in IL-10 production (80). Serum levels after in vivo injections of T. gondii EVs led to production of antibodies against T. gondii and an increase in survival, giving rise to the potential of utilizing protozoan parasite EVs as vaccines (80). Overall, these studies suggest that EV production by protozoan parasites is not a silent process and can stimulate an inflammatory environment, although deliberate manipulation of immune responses via secreted parasite proteins contained within EVs is potentially happening in parallel. Thus, as with many parasitic processes, the balance of host avoidance and activation is part of the successful mechanism of parasitism.

For bacteria, the interaction of OMVs and host cells is a well-recognized phenomenon, and these vesicles can be internalized through a variety of mechanisms, including clathrin-mediated, caveolin-mediated, lipid-raft-mediated, or macropinocytosis-mediated pathways (154). While these routes can also be used for protozoan parasite EVs, the mechanism depends on the cell type, and for many central nervous system (CNS) cells, the exact method has yet to be identified. OMVs contain LPS and toxins, which makes them a great contender for eliciting an immune response. Previously, N. meningitis OMVs were incubated with human neutrophils, and it was reported that neutrophils release proinflammatory cytokines (155). A similar response was noted with several periodontal bacteria, where OMVs from T. denticola, P. gingivalis, and T. forsythia tested against several immune cells (156). It was noted that all oral bacterium-linked OMVs induced an inflammasome immune response. Specifically, a cytokine response of TNF-α, IL-β, IL-8, and IL-10 production was reported (156). Although the immune response to O-IMV has not yet been studied, it is reasonable to hypothesize that a similar response may be elicited, given the shared outer membrane structure containing LPS and potential toxins.

The immune response to Gram-positive vesicles has not been extensively studied; however, there are reports suggesting that these vesicles can also elicit an immune response. S. aureus OMVs were tested against human lung epithelial cells, and it was reported that a cytokine response was present, specifically with the production of IL-8, IL-6, and leukocyte recruiting chemokine CCL2 (42). L. monocytogenes vesicles are known to contain the pore-forming toxin (61); however, recent studies have shown that these vesicles can accumulate in lysosomes (157).

FUTURE APPLICATIONS

Early identification of microbial infections is often difficult, and therefore diagnosis is frequently delayed. Due to their ability to traverse biological barriers, their extended circulation within the host and their efficient immunostimulation, EVs offer considerable potential as diagnostic as well as therapeutic targets (158). EVs have emerged as promising disease biomarkers due to their bioactive cargos, offering potential for expedited diagnosis in parasite and bacterial infections. It is essential to comprehend the diversity of EV cargo and delineate disparities between biomarkers and their applications to steer pathogenic EV research toward validating EVs as biomarkers (159). However, the suggestion that plasma EVs in malaria could function as multifunctional biomarkers has sparked scientific investigations, prompting a series of functional studies to explore their potential pathogenic role. For therapeutic applications, EV isolation may necessitate more stringent and large-scale methodologies compared to diagnostic purposes (160, 161). The spectrum of potential developments ranges from diagnostic markers, immunotherapy, and development of precise drug delivery systems to tools for targeting hereditary diseases (151, 162).

Exosomes have already demonstrated significant potential as diagnostic and prognostic biomarkers for a range of diseases, with established application in the identification of neurodegenerative diseases (163) and for cancer prognosis (164, 165). Their distinctive lipid bilayer composition and the abundance of adhesion molecules make them optimal carriers for targeted drug delivery (166). Moreover, EVs have a low immunogenicity and capacity to traverse the blood–brain barrier, further enhancing their suitability for drug delivery applications (27, 167). Considering that protozoan parasites, bacteria, and other single-celled organisms often outpace or otherwise overcome the host’s immune system during infections, it is worth noting that pathogen-derived EVs may hold untapped potential in the realm of drug delivery. Additionally, exosomes have been found to facilitate tissue regeneration by stimulating cell proliferation and differentiation, both crucial components of a parasite’s life cycle (20).

Recent research has unveiled a fascinating phenomenon where EVs derived from drug-resistant P. falciparum can transmit drug resistance to susceptible parasites through the transfer of episomal DNA. This mechanism plays a pivotal role in facilitating the transmission of drug resistance during human infections. Furthermore, EVs have been implicated in the regulation of parasite cell differentiation (168). Just prior to P. falciparum exiting from erythrocytes, there is a notable surge in the release of these EVs, which might be worth exploring. This surge is particularly significant as it coincides with sexual commitment and differentiation, both crucial phases in the Apicomplexan life cycle. Moreover, the introduction of purified parasites of the same species stimulates the differentiation of asexual P. falciparum stages into gamonts, thereby perpetuating their life cycle (149, 169). Likewise bacterial OMVs were shown to play a dual role during infection as they can be potent vehicles for toxin transmission to host cells, while also strongly contributing to the immunogenic responses. As such, they present potential in both increasing the pathogenicity of infectious agents as well as for acting as promoters of immunogenicity.

Though single-celled microorganisms are not yet routinely used for diagnostics or therapeutics in infectious diseases, there are two possible strategies to transition into this approach. First, create an exosome-based vaccine by presenting antigens through inclusion of bacterial proteins in EVs. Second, using infectious biomarkers for detecting viral mRNA and proteins in secreted exosomes, especially for protozoan parasites (162). To harness the potential of EVs for commercial and clinical use, it has become imperative to develop efficient, cost-effective methods for large-scale EV production. These techniques aim to streamline their isolation, facilitating high-throughput biomarker research and enabling EV-based therapies (167).

OMVs have demonstrated their utility in the pharmaceutical field, evidenced by the development and approval of an OMV-based vaccine by the American Food and Drug Administration (170). Furthermore, their compact size and the array of surface proteins, encompassing virulence factors, render them an attractive prospect for the creation of infection vaccines (115). Beyond vaccine candidates, OMVs can serve as carriers for antibiotics, facilitating drug delivery for treating infectious diseases (171). Engineered OMVs show promise in cancer treatment, with studies demonstrating their therapeutic potential. Gujrati et al. (172) engineered OMVs containing antibodies targeting HER2 for cancer cell specificity, coupled with cytotoxic payloads such as siRNA (172). Standalone OMVs, even without toxic payloads, have proven effective in reducing tumor growth, emphasizing their significance in cancer therapeutics (173). Additionally, OMVs loaded with doxorubicin, a chemotherapeutic agent, present a successful avenue for cancer therapy (34).

Previously, liposomes encapsulating tobramycin, an antibiotic used to treat S. aureus infections, have shown success (174). Since it has been reported that S. aureus membrane vesicles interact with host cells to induce cell death (175), there is potential that engineered S. aureus membrane vesicles void of the toxin could also potentially be loaded with antibiotics as an effective treatment against the infection. Rivera et al. (62) conducted an experiment where mice were inoculated with B. anthrax membrane vesicles. The results showed that mice exposed to the OMVs had a significantly prolonged survival rate of 13 days compared to the control mice, which survived only 2 days when exposed to the bacteria. This suggests that these membrane vesicles have potential as a vaccination strategy (62). Similar results were seen in membrane vesicles released from S. pneumoniae (96). These studies emphasize that, like Gram-negative OMVs, Gram-positive membrane vesicles also possess the potential to serve as vaccine candidates.

Still, to date, there is only one current licensed bacterial-derived extracellular EV-based vaccine against meningococcal disease, which has been already proven to have great potential in using bacteria-derived EVs for vaccine-based research. Protozoan parasite-derived EVs may also provide promising vaccine agents that induce immunity against parasitic infections, as already shown in animal models, but the challenges regarding particle components associated with the host organism have make a successful development difficult to date.

CONCLUDING REMARKS AND OUTLOOK

Extracellular vesicles derived from pathogenic single-celled microorganisms share common features related to their roles in intercellular communication and potential pathogenic effects. It is crucial to acknowledge the distinct differences that set these microorganisms apart. These disparities are hardly surprising, given the marked contrasts, extending even to the composition and permeability of the cell walls in single-celled prokaryote and single-celled eukaryote membranes. Consequently, their membrane-bound or derived vesicles also diverge significantly in terms of their structure and functionality. Protozoan parasite-derived EVs play pivotal roles in their life cycles, encompassing crucial processes such as differentiation and transmission. In contrast, bacterial OMVs display variations between Gram-positive and Gram-negative bacteria, with notable disparities in EV biogenesis. While autonomous ESCRT machinery is exclusive to pathogenic parasites and ameba, this mechanism remains to be fully understood in bacteria. Nevertheless, some bacteria and protozoan parasite species can hijack the host’s ESCRT pathway for EV production. Furthermore, these microorganisms share similarities in the cargo transported by EVs. Appreciating these shared attributes and disparities is essential for comprehending the precise mechanisms governing host–pathogen interactions and holds promise for potential therapeutic interventions.

Yet, many questions remain unanswered. For instance, could the variations in EV biogenesis be a contributing factor to the cohabitation of eukaryotic and prokaryotic single-celled pathogens within the same host organ? With the evolving understanding of the host’s microbiota, a new horizon for exploration emerges. As a stable consortium of bacteria takes root early in the host’s life, these species collaborate to effectively modify and metabolize substrates within specific niches of the host’s mucosa. Some of these resident bacteria offer benefits to the host, impacting areas such as nutrition, immune development, and protection against other pathogens. Parasitic protozoans infecting mucosal surfaces could interact with these local bacterial communities, thus implying that eukaryotes and prokaryotes engage in communication. Whether EVs play a role in this interplay remains a mystery. Additionally, the realm of EV research in single-celled pathogenic microorganisms should also focus on potential additive effects to comprehensively decipher these complex interactions.

Drawing conclusions in this context might be more straightforward for bacteria, which typically reproduce through binary fission, maintaining a relatively consistent lifecycle. In contrast, parasitic protozoa may have complex life cycles that encompass a range of stages, including asexual, sexual, and environmental phases, which can occur within a host cell or its external environment. While the mechanisms of cellular invasion by intracellular parasites have been well-documented, the role of EVs in these processes has yet to be explored. Cellular migration outside of the host cell and subsequent invasion events are crucial for the completion of these parasites' life cycles. It is worth noting that bacterial infection via manipulation of host cell signaling through EVs has been suggested, and the secretion of toxins to interfere with host cell functions has been linked to EVs. However, the following question arises: what about the involvement of EVs in the formation of biofilms, which serve as protective reservoirs of bacteria that can continually release and infect host cells?

Another question underlying comparative EV studies is as follows: can we actually compare EVs of single-celled organisms, as they are generated by very different biogenesis pathways? As proven by this article, we believe so. Not only because the generating of EVs through budding is very similar between bacteria and protozoan parasites, but also because all single-celled microorganisms commonly use EVs to interact with their surroundings. Furthermore, the use of specific cargo to modulate the host immune response is very similar. Still this also remains the most prevalent challenge in developing new adaptations to a clinical or diagnostic context, showing that comprehensive EV studies across phyla could be beneficial for understanding single-celled pathogens.

However, the widespread use of EVs as a communication mechanism across diverse biological domains highlights their significance as a universal cell language. While the regulatory role of EVs in biology is still unfolding, their potential in infectious disease is particularly thrilling, especially concerning detection and vaccine development. Exploring the fundamental biology of EV communication employed by microorganisms in an infectious setting is going to be a captivating area of study in the future.

Biographies

Anna Sophia Feix earned her Master's degree in Biology from the University of Vienna, specializing in animal anatomy, morphology, ultrastructure, biodiversity, and systematics. Subsequently, she completed her PhD in Parasitology at the University of Veterinary Medicine Vienna in 2022, focusing on characterizing the sexual stages of Cystoisospora suis. This work was recognized with Early Career Research Awards in both 2021 and 2022. Her academic pursuits led her to Sweden's University of Gothenburg and the Czech Academy of Sciences in Budweis. Presently, she works as a Postdoctoral Researcher at the University of Veterinary Medicine Vienna, concentrating on extracellular vesicles of Apicomplexan parasites.

Emily Z. Tabaie is currently a PhD candidate at the University of California, Riverside, specializing in Biomedical Sciences. Having earned her B.S. in Cell and Molecular Biology from Seattle University in 2019, she delved into comparing the free energy of unfolding in psychrophilic and mesophilic proteins during her undergraduate tenure. Transitioning to her doctoral studies in 2020, Emily focuses on the intricate communication between astrocytes and neurons during chronic T. gondii infection, leveraging extracellular vesicles. Her research probes how EVs from infected neurons modulate astrocytic gene expression and function, driven by her fascination with neurobiology and the disruptions parasitic infections and neurodegenerative diseases impose on brain homeostasis. With four years of dedicated research in this domain, Emily is poised to defend her thesis in the summer of 2024.

Aarshi N. Singh is a Ph.D. candidate in the Department of Chemistry at Lehigh University working in Dr. Nathan J. Wittenberg's lab. With a B.S. in Biochemistry and Chemical Biotechnology, along with industry experience in immunological assay development, her research focuses in bioanalytical chemistry. Her primary research objective is to identify heterogeneity of single bacterial outer membrane vesicles to enhance understanding of these nano-sized particles. Additionally, she works on creating bacterial membrane mimics for drug development purposes.

Nathan J. Wittenberg is an Associate Professor of Chemistry at Lehigh University, where he has been since 2016. He received a B.S. in chemistry from the University of Minnesota and a Ph.D. in chemistry from the Pennsylvania State University. He completed postdoctoral training at the University of Edinburgh (chemistry), the University of Minnesota (chemistry; electrical and computer engineering), and the Mayo Clinic (neurology). Prof. Wittenberg leads a research group with interests in analytical and biophysical chemistry related to lipid membrane structure and function. His group works on projects related to lipid oxidation, lipid-protein interactions in the nervous system, and the biochemical analysis of bacterial outer membrane vesicles.

Emma H. Wilson is Professor of Biomedical Science and Associate Dean of the Graduate Division within the Department of Biomedical Sciences at the University of California, Riverside. She commenced her academic journey in the United Kingdom, where she completed her doctoral studies in 2002 before transitioning to a Postdoctoral position at the University of Pennsylvania, USA. Her laboratory currently emphasizes research on the immune response within the brain subsequent to Toxoplasma gondii infection. Recognized for her exemplary mentorship, she has been consistently acknowledged as a "Marvelous Mentor" by biomedical students over several consecutive years.

Anja Joachim is an expert in veterinary parasitology and graduated from the University of Veterinary Medicine Hannover, Germany, in 1992. She pursued postgraduate and postdoctoral training across Sydney, Hannover, Leipzig, and Copenhagen. In 2003, she ascended to Full Professor for Parasitology at the University of Veterinary Medicine, Vienna. She also holds positions in various professional organizations, including serving as President of the European Veterinary Parasitology College from 2009 to 2012 and as a member of the Executive Committee of the World Association for the Advancement of Veterinary Parasitology since 2023.