Overview of extracellular vesicles in pathogens with special focus on human extracellular protozoan parasites

Extracellular vesicles (EVs) are lipid-bilayered membrane-delimited particles secreted by almost any cell type, involved in different functions according to the cell of origin and its state. From these, cell to cell communication, pathogen-host interactions and modulation of the immune response have been widely studied. Moreover, these vesicles could be employed for diagnostic and therapeutic purposes, including infections produced by pathogens of diverse types; regarding parasites, the secretion, characterisation, and roles of EVs have been studied in particular cases. Moreover, the heterogeneity of EVs presents challenges at every stage of studies, which motivates research in this area. In this review, we summarise some aspects related to the secretion and roles of EVs from several groups of pathogens, with special focus on the most recent research regarding EVs secreted by extracellular protozoan parasites.

Key words:
extracellular vesicles; protozoa; parasites; infectious diseases; arthropods; helminths

Authorship

Johan Alvarado-Ocampo

Planning

designing

conceiving

writing

original draft

Universidad de Costa Rica, Facultad de Microbiología, Centro de Investigación en Enfermedades Tropicales, San José, Costa RicaUniversidad de Costa RicaCosta RicaSan José, Costa RicaUniversidad de Costa Rica, Facultad de Microbiología, Centro de Investigación en Enfermedades Tropicales, San José, Costa Rica

Elizabeth Abrahams-Sandí

writing

revision

editing

Universidad de Costa Rica, Facultad de Microbiología, Departamento de Parasitología, San José, Costa RicaUniversidad de Costa RicaCosta RicaSan José, Costa RicaUniversidad de Costa Rica, Facultad de Microbiología, Departamento de Parasitología, San José, Costa Rica

Lissette Retana-Moreira ⁺+ Corresponding author: lissette.retanamoreira@ucr.ac.cr

writing

revision

editing

https://orcid.org/0000-0001-5215-582X

+ Corresponding author: lissette.retanamoreira@ucr.ac.cr

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (1)
Tables (1)

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Explored general roles of extracellular vesicles (EVs) derived from extracellular protozoa in pathogenesis, parasite-parasite communication, and its relationship with immune effectors. (A) Entamoeba histolytica EVs are possible involved in en/excystment processes and have effects over neutrophils; (B) Giardia duodenalis EVs provoke alteration of Caco-2 cells tight junctions and enterobacteria, promote adhesion of the parasite and induce a proinflammatory outcome; (C) free living amoeba (i.e., Acanthamoeba sp., Naegleria fowleri) derived-EVs are uptaken by glial cells and other mammalian cells and are also associated with a proinflammatory chemokine/cytokine production; (D) Trichomonas vaginalis induce the production of nitric oxide (NO) in macrophages and stimulate adhesion of the parasites to ectocervical cells. Figure created with BioRender.com.

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TABLE
Isolation and characterisation methods commonly employed for the collection of extracellular vesicles (EVs) from extracellular protozoan pathogens, including sizes ranges reported in literature

Explored general roles of extracellular vesicles (EVs) derived from extracellular protozoa in pathogenesis, parasite-parasite communication, and its relationship with immune effectors. (A) Entamoeba histolytica EVs are possible involved in en/excystment processes and have effects over neutrophils; (B) Giardia duodenalis EVs provoke alteration of Caco-2 cells tight junctions and enterobacteria, promote adhesion of the parasite and induce a proinflammatory outcome; (C) free living amoeba (i.e., Acanthamoeba sp., Naegleria fowleri) derived-EVs are uptaken by glial cells and other mammalian cells and are also associated with a proinflammatory chemokine/cytokine production; (D) Trichomonas vaginalis induce the production of nitric oxide (NO) in macrophages and stimulate adhesion of the parasites to ectocervical cells. Figure created with BioRender.com.

TABLE Isolation and characterisation methods commonly employed for the collection of extracellular vesicles (EVs) from extracellular protozoan pathogens, including sizes ranges reported in literature

Parasite form	Isolation method	EV diameter (nm)	Technique	Reference
Trichomonas vaginalis trophozoites	DC + sucrose gradient	~ 50 - 100	TEM	¹⁴⁰140. Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, et al. Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host: parasite interactions. PLoS Pathog. 2013; 9(7): e1003482. doi: 10.1371/journal.ppat.1003482. https://doi.org/10.1371/journal.ppat.100...
	DC	50 - 100	TEM	¹⁴⁸148. Olmos-Ortiz LM, Barajas-Mendiola MA, Barrios-Rodiles M, Castellano LE, Arias-Negrete S, Avila EE, et al. Trichomonas vaginalis exosome-like vesicles modify the cytokine profile and reduce inflammation in parasite-infected mice. Parasite Immunol. 2017; 39(6): e12426. doi: 10.1111/pim.12426. https://doi.org/10.1111/pim.12426...
	DC	30 - 150	TEM	¹⁴²142. Ong SC, Cheng WH, Ku FM, Tsai CY, Huang PJ, Lee CC, et al. Identification of endosymbiotic virus in small extracellular vesicles derived from Trichomonas vaginalis. Genes. 2022; 13: 531. doi: 10.3390/genes13030531. https://doi.org/10.3390/genes13030531...
	DC	100 - 1000	TEM	¹⁴¹141. Nievas YR, Coceres VM, Midlej V, de Souza W, Benchimol M, Pereira-Neves A, et al. Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction. Cell Mol Life Sci. 2018; 75: 2211-26. doi: 10.1007/s00018-017-2726-3. https://doi.org/10.1007/s00018-017-2726-...
	DC	380 / 63	DLS
	DC + density gradient	108 - 146	DLS	¹⁴⁴144. Rada P, Hrdý I, Zdrha A, Narayanasamy RK, Smutná T, Horácková J, et al. Double-stranded RNA viruses are released from Trichomonas vaginalis inside small extracellular vesicles and modulate the exosomal cargo. Front Microbiol. 2022; 13: 893692. doi: 10.3389/fmicb.2022.893692. https://doi.org/10.3389/fmicb.2022.89369...
DC	~ 105	NTA	⁽⁴⁶⁾
Giardia duodenalis trophozoites	DC	20 - 25 / 50 - 100	TEM	⁽¹³⁵⁾
	DC	22,8 / 85,2	DLS	⁽¹³⁵⁾
	DC	60 - 150	TEM	⁽¹²⁶⁾
	DC	150 - 350	NTA	⁽¹²⁶⁾
	DC	100 - ~ 200	TEM	⁽¹³⁰⁾
	DC	143,5	NTA	⁽¹³⁰⁾
	DC	50 - 90 / 117 - 282	TEM	⁽¹²⁸⁾
	DC	82,6 / 238,5	NTA	⁽¹²⁸⁾
	DC	187,6 / 67,7	NTA	¹³¹131. Gavinho B, Sabatke B, Feijoli V, Rossi IV, da Silva JM, Evans-Osses I, et al. Peptidyl arginine deiminase inhibition abolishes the production of large extracellular vesicles from Giardia intestinalis, affecting host-pathogen interactions by hindering adhesion to host cells. Front Cell Infect Microbiol. 2020; 10: 417. doi: 10.3389/fcimb.2020.00417. https://doi.org/10.3389/fcimb.2020.00417...
	ExoEasy maxi kit (QIAGEN)	CaCl₂ treatment: 210 Bile treatment: 270	NTA	¹³⁷137. Siddiq A, Dong G, Balan B, Harrison LG, Jex A, Olivier M, et al. A thermo-resistant and RNase-sensitive cargo from Giardia duodenalis extracellular vesicles modifies the behaviour of enterobacteria. J Extracell Biol. 2023; 2: e109. doi: 10.1002/jex2.109. https://doi.org/10.1002/jex2.109...
Entamoeba histolytica trophozoites and/or cysts	Total exosome isolation (Invitrogen)	< 200	TEM	¹⁵⁷157. Díaz-Godínez C, Ríos-Valencia DG, García-Aguirre S, Martínez-Calvillo S, Carrero JC. Immunomodulatory effect of extracellular vesicles from Entamoeba histolytica trophozoites: regulation of NETs and respiratory burst during confrontation with human neutrophils. Front Cell Infect Microbiol. 2022; 12: 1018314. doi: 10.3389/fcimb.2022.1018314. https://doi.org/10.3389/fcimb.2022.10183...
	Total exosome isolation (Invitrogen)	< 50 - 600 Peak: 483	NTA
	Total exosome isolation (Invitrogen)	125	NTA	¹⁵⁶156. Sharma M, Morgado P, Zhang H, Ehrenkaufer G, Manna D, Singh U. Characterization of extracellular vesicles from Entamoeba histolytica identifies roles in intercellular communication that regulates parasite growth and development. Infect Immun. 2020; 88: 00349-20. doi: 10.1128/IAI.00349-20. https://doi.org/10.1128/IAI.00349-20...
Acanthamoeba sp. trophozoites	DC	PYG medium: 31,9 - 467 Glucosed medium: 33,7 - 303,2	TEM	¹⁶⁵165. Gonçalves DS, Ferreira MS, Liedke SC, Gomes KX, de Oliveira GA, Leão PEL, et al. Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells. Virulence. 2018; 9(1): 818-36. doi: 10.1080/21505594.2018.1451184. https://doi.org/10.1080/21505594.2018.14...
	DC	PYG medium: 56,1 - 68,4 / 150,4 - 223,0 / 402,9 - 659,4 Glucosed medium: 173,2 - 234,8 / 585,1 - 746,5	DLS
	DC	28°C incubation: 184,6 ± 50,80 / 50,29 ± 8,49 37°C incubation: 111,3 ± 19,8	DLS	¹⁶⁰160. Moreira LR, Ramírez DV, Linares F, Ledezma AP, Garro AV, Osuna A, et al. Isolation of Acanthamoeba T5 from water: characterization of its pathogenic potential, including the production of extracellular vesicles. Pathogens. 2020; 9: 144. doi: 10.3390/pathogens9020144. https://doi.org/10.3390/pathogens9020144...
	Ultrafiltration: Amicon ultracentrifugation filters (Merck Millipore) + Total exosome isolation (Invitrogen)	Peak: 118	NTA	¹⁶⁴164. Lin WC, Tsai CY, Huang JM, Wu SR, Chu LJ, Huang KY. Quantitative proteomic analysis and functional characterization of Acanthamoeba castellanii exosome-like vesicles. Parasit Vectors. 2019; 12: 467. doi: 10.1186/s13071-019-3725-z. https://doi.org/10.1186/s13071-019-3725-...
	DC	101 - 150 / 151 - 200	NTA	¹⁶³163. Costa AO, Chagas IAR, de Menezes-Neto A, Rêgo FD, Nogueira PM, Torrecilhas AC, et al. Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba. Cell Biol Int. 2021; 45: 1060-71. doi: 10.1002/cbin.11551. https://doi.org/10.1002/cbin.11551...
Naegleria fowleri trophozoites	DC	43,88 / 207,95	TEM	¹⁶⁹169. Retana-Moreira L, Espinoza MFS, Camacho NC, Cornet-Gomez A, Sáenz-Arce G, Osuna A, et al. Characterization of extracellular vesicles secreted by a clinical isolate of Naegleria fowleri and identification of immunogenic components within their protein cargo. Biology. 2022; 11: 983. doi: 10.3390/biology11070983. https://doi.org/10.3390/biology11070983...
		216 ± 83	NTA
		227,13 ± 37,98 / 206,29 ± 37,08 / 24,24 ± 9,18	DLS
	Size exclusion chromatography	22,4 - 955	DLS	¹⁷²172. Lê HG, Kang JM, Võ TC, Yoo WG, Na BK. Naegleria fowleri extracellular vesicles induce proinflammatory immune responses in BV-2 microglial cells. Int J Mol Sci. 2023; 24: 13623. doi: 10.3390/ijms241713623. https://doi.org/10.3390/ijms241713623...
	DC	156,8 ± 13,4 / 141 ± 8,3	NTA	¹⁶⁸168. Lertjuthaporn S, Somkird J, Lekmanee K, Atipimonpat A, Sukapirom K, Sawasdipokin H, et al. Extracellular vesicles from Naegleria fowleri induce IL-8 response in THP-1 macrophage. Pathogens. 2022; 11: 632. doi: 10.3390/pathogens11060632. https://doi.org/10.3390/pathogens1106063...
	DC + Size exclusion chromatography	Overall average of five strains: 152,6	NTA	¹⁷⁰170. Russell AC, Bush P, Grigorean G, Kyle DE. Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri. Front Microbiol. 2023; 14: 1264348. doi: 10.3389/fmicb.2023.1264348. https://doi.org/10.3389/fmicb.2023.12643...

When data is not presented as a range, it corresponds to a mean or a NTA peak; DC: differential centrifugation; /: indicates different EV subpopulations.

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Overview of extracellular vesicles in pathogens with special focus on human extracellular protozoan parasites

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[1] + Corresponding author: lissette.retanamoreira@ucr.ac.cr