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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

Particle tracking analysis reveals prolific larval Brugia exosome-like vesicle release rate.Profile of ELVs isolated from culture media incubated with 300 L3 parasites for successive 24 hr incubations. The size distribution of L3-derived ELVs from Day 1 (left), Day 2 (center) and Day 3 (right) incubations are shown (mean ± SD). Calculated vesicle release rates are provided in tabular format. ELV rate of release and size specificity decay in a time-dependent manner in vitro. * re-scaled based on dilution for comparison to 0–24 hour (1:20) dilution.
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pntd.0004069.g002: Particle tracking analysis reveals prolific larval Brugia exosome-like vesicle release rate.Profile of ELVs isolated from culture media incubated with 300 L3 parasites for successive 24 hr incubations. The size distribution of L3-derived ELVs from Day 1 (left), Day 2 (center) and Day 3 (right) incubations are shown (mean ± SD). Calculated vesicle release rates are provided in tabular format. ELV rate of release and size specificity decay in a time-dependent manner in vitro. * re-scaled based on dilution for comparison to 0–24 hour (1:20) dilution.

Mentions: To more accurately resolve the dynamics of ELV release in L3 B. malayi, we used a nanoparticle tracking analysis (NTA) system to measure vesicle output in a 72 hr in vitro time course. Media was collected from 300 worms after three successive 24 hr incubation periods, vesicles were purified by ultracentrifugation as before and individual vesicle preparations were analyzed via NanoSight LM10 as shown in Fig 2 (sample recording: S1 Video). Day 1 (0–24 hr in culture) preparations reveal a prolific ELV release rate (> 9,000 ELVs/parasite/min) with a very narrow size distribution centered at ∼90 nm. Day 2 (24–48 hr in culture) preparations show an essentially equivalent rate of release, but a stark broadening of the size distribution. Day 3 (48–72 hr in culture) preparations are associated with significantly lower levels of release (<4,000 ELVs/parasite/min) and an even wider multimodal size distribution. These data suggest an overall time-dependent decay in vesicle rate of release and size specificity, which correlates to decreased L3 viability in vitro. The release of considerable quantities of precisely-sized ELVs in viable worms (Days 1–2) is followed by the release of smaller quantities of a broader size range of particles that potentially include larger membrane vesicles and apoptotic blebs (Days 2–3). This suggests an active and regulated mechanism of ELV release in healthy and viable L3 stage parasites, as opposed to a passive mode of noisy cellular deterioration.


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)

Particle tracking analysis reveals prolific larval Brugia exosome-like vesicle release rate.Profile of ELVs isolated from culture media incubated with 300 L3 parasites for successive 24 hr incubations. The size distribution of L3-derived ELVs from Day 1 (left), Day 2 (center) and Day 3 (right) incubations are shown (mean ± SD). Calculated vesicle release rates are provided in tabular format. ELV rate of release and size specificity decay in a time-dependent manner in vitro. * re-scaled based on dilution for comparison to 0–24 hour (1:20) dilution.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004069.g002: Particle tracking analysis reveals prolific larval Brugia exosome-like vesicle release rate.Profile of ELVs isolated from culture media incubated with 300 L3 parasites for successive 24 hr incubations. The size distribution of L3-derived ELVs from Day 1 (left), Day 2 (center) and Day 3 (right) incubations are shown (mean ± SD). Calculated vesicle release rates are provided in tabular format. ELV rate of release and size specificity decay in a time-dependent manner in vitro. * re-scaled based on dilution for comparison to 0–24 hour (1:20) dilution.
Mentions: To more accurately resolve the dynamics of ELV release in L3 B. malayi, we used a nanoparticle tracking analysis (NTA) system to measure vesicle output in a 72 hr in vitro time course. Media was collected from 300 worms after three successive 24 hr incubation periods, vesicles were purified by ultracentrifugation as before and individual vesicle preparations were analyzed via NanoSight LM10 as shown in Fig 2 (sample recording: S1 Video). Day 1 (0–24 hr in culture) preparations reveal a prolific ELV release rate (> 9,000 ELVs/parasite/min) with a very narrow size distribution centered at ∼90 nm. Day 2 (24–48 hr in culture) preparations show an essentially equivalent rate of release, but a stark broadening of the size distribution. Day 3 (48–72 hr in culture) preparations are associated with significantly lower levels of release (<4,000 ELVs/parasite/min) and an even wider multimodal size distribution. These data suggest an overall time-dependent decay in vesicle rate of release and size specificity, which correlates to decreased L3 viability in vitro. The release of considerable quantities of precisely-sized ELVs in viable worms (Days 1–2) is followed by the release of smaller quantities of a broader size range of particles that potentially include larger membrane vesicles and apoptotic blebs (Days 2–3). This suggests an active and regulated mechanism of ELV release in healthy and viable L3 stage parasites, as opposed to a passive mode of noisy cellular deterioration.

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