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Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

Zamanian M, Fraser LM, Agbedanu PN, Harischandra H, Moorhead AR, Day TA, Bartholomay LC, Kimber MJ - PLoS Negl Trop Dis (2015)

Bottom Line: Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes.The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced.Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers.

No MeSH data available.


Related in: MedlinePlus

Electron microscopy confirms secretion of exosome-like vesicles in intra-host stages of B. malayi.TEM images of L3 (A and B) and adult female (C) ELV preparations are shown. L3 vesicles take on a distinct morphology often reported in the literature. Adult isolations are more heterogenous and may require further optimization to achieve uniform vesicle preparation. White arrows show canonical L3 ELVs (B) and putative adult ELVs (C). This provides evidence for the release of exosome-like vesicles in the human-infective L3 stage of the parasite and much of the rest of the work we report is focused on vesicles derived from this larval stage.
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pntd.0004069.g001: Electron microscopy confirms secretion of exosome-like vesicles in intra-host stages of B. malayi.TEM images of L3 (A and B) and adult female (C) ELV preparations are shown. L3 vesicles take on a distinct morphology often reported in the literature. Adult isolations are more heterogenous and may require further optimization to achieve uniform vesicle preparation. White arrows show canonical L3 ELVs (B) and putative adult ELVs (C). This provides evidence for the release of exosome-like vesicles in the human-infective L3 stage of the parasite and much of the rest of the work we report is focused on vesicles derived from this larval stage.

Mentions: In order to ascertain whether exosomes are released by B. malayi, extracellular vesicles were isolated from parasites incubated in culture media using a filtration and ultracentrifugation protocol. We focused our initial discovery efforts on larval and adult stage parasites. L3, adult male, and adult female B. malayi were incubated in vitro for 24 hour periods under standard culture conditions, and purified vesicle preparations were evaluated with electron microscopy (EM). Infectious stage L3 parasites in culture release abundant 50–120 nm microvesicles consistent with the classical “deflated ball” morphology of mammalian and non-mammalian exosomes reported in the literature [36] (Fig 1A & 1B). We refer to these as exosome-like vesicles (ELVs) throughout this manuscript, in recognition that they cannot be unequivocally designated as exosomes, rather than another class of extracellular vesicles, because their biogenesis has not been determined. Preparations from adult stage B. malayi were more heterogenous and dilute, not allowing for the definitive categorization of putative exosome-like vesicles (Fig 1C). This, despite the fact a much higher mass of total parasite tissue was used for adult preparations as compared to larval preparations. These data suggest ELV release to be a predominantly larval phenomenon in B. malayi, a working hypothesis supported by analysis of RNA associated with the vesicles. We therefore chose to focus our subsequent experiments on L3 stage parasites. A compelling overall hypothesis for the function of B. malayi ELVs is that they mediate the secretion and trafficking to host cells of effector molecules that facilitate parasitism and the observation that ELV secretion occurs primarily in those parasite stages that infect the host and establish parasitemia is consistent with this narrative.


Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

Zamanian M, Fraser LM, Agbedanu PN, Harischandra H, Moorhead AR, Day TA, Bartholomay LC, Kimber MJ - PLoS Negl Trop Dis (2015)

Electron microscopy confirms secretion of exosome-like vesicles in intra-host stages of B. malayi.TEM images of L3 (A and B) and adult female (C) ELV preparations are shown. L3 vesicles take on a distinct morphology often reported in the literature. Adult isolations are more heterogenous and may require further optimization to achieve uniform vesicle preparation. White arrows show canonical L3 ELVs (B) and putative adult ELVs (C). This provides evidence for the release of exosome-like vesicles in the human-infective L3 stage of the parasite and much of the rest of the work we report is focused on vesicles derived from this larval stage.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581865&req=5

pntd.0004069.g001: Electron microscopy confirms secretion of exosome-like vesicles in intra-host stages of B. malayi.TEM images of L3 (A and B) and adult female (C) ELV preparations are shown. L3 vesicles take on a distinct morphology often reported in the literature. Adult isolations are more heterogenous and may require further optimization to achieve uniform vesicle preparation. White arrows show canonical L3 ELVs (B) and putative adult ELVs (C). This provides evidence for the release of exosome-like vesicles in the human-infective L3 stage of the parasite and much of the rest of the work we report is focused on vesicles derived from this larval stage.
Mentions: In order to ascertain whether exosomes are released by B. malayi, extracellular vesicles were isolated from parasites incubated in culture media using a filtration and ultracentrifugation protocol. We focused our initial discovery efforts on larval and adult stage parasites. L3, adult male, and adult female B. malayi were incubated in vitro for 24 hour periods under standard culture conditions, and purified vesicle preparations were evaluated with electron microscopy (EM). Infectious stage L3 parasites in culture release abundant 50–120 nm microvesicles consistent with the classical “deflated ball” morphology of mammalian and non-mammalian exosomes reported in the literature [36] (Fig 1A & 1B). We refer to these as exosome-like vesicles (ELVs) throughout this manuscript, in recognition that they cannot be unequivocally designated as exosomes, rather than another class of extracellular vesicles, because their biogenesis has not been determined. Preparations from adult stage B. malayi were more heterogenous and dilute, not allowing for the definitive categorization of putative exosome-like vesicles (Fig 1C). This, despite the fact a much higher mass of total parasite tissue was used for adult preparations as compared to larval preparations. These data suggest ELV release to be a predominantly larval phenomenon in B. malayi, a working hypothesis supported by analysis of RNA associated with the vesicles. We therefore chose to focus our subsequent experiments on L3 stage parasites. A compelling overall hypothesis for the function of B. malayi ELVs is that they mediate the secretion and trafficking to host cells of effector molecules that facilitate parasitism and the observation that ELV secretion occurs primarily in those parasite stages that infect the host and establish parasitemia is consistent with this narrative.

Bottom Line: Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes.The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced.Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers.

No MeSH data available.


Related in: MedlinePlus