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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

Isolation of Small RNAs from larval and adult B. malayi ELV fractions.Bioanalyzer data are shown for RNAs isolated from L3, adult male, and adult female Brugia preparations. L3 ELVs contain significant amounts of small RNAs in the 25–200 nt range (25 and 200 nt reference peaks labeled), while adult male and female vesicle preparations yield fewer RNAs. Vesicle fractions were prepared from 300 L3 and 30 adults in 24 hr culture incubations. Despite the much higher total tissue amounts used in adult culture, we detect much higher levels of small RNAs in L3-derived ELVs.
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pntd.0004069.g004: Isolation of Small RNAs from larval and adult B. malayi ELV fractions.Bioanalyzer data are shown for RNAs isolated from L3, adult male, and adult female Brugia preparations. L3 ELVs contain significant amounts of small RNAs in the 25–200 nt range (25 and 200 nt reference peaks labeled), while adult male and female vesicle preparations yield fewer RNAs. Vesicle fractions were prepared from 300 L3 and 30 adults in 24 hr culture incubations. Despite the much higher total tissue amounts used in adult culture, we detect much higher levels of small RNAs in L3-derived ELVs.

Mentions: We probed larval and adult microvesicle preparations for the presence of small RNA species. Exosomes have been found to contain both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs) in a diverse range of species and cell types. Of particular interest to us was the potential presence of small non-coding RNAs, including microRNAs (miRNAs), that could potentially mediate parasite-parasite communication or modulate host gene expression. Small RNA species were preferentially isolated from putative ELV-containing pellets and examined with an Agilent Bioanalyzer. The microvesicle fractions of L3 B. malayi (24 hr incubations of 300 worms) revealed an abundance of small RNA species in the 25–200 nt range (Fig 4). Much less RNA was detected from incubations of adult male and female B. malayi (24 hr incubations of 30 adult worms), despite the much higher mass of tissue in adult stage culture media. This lack of correlation between total parasite tissue material and RNA yield, coupled to the differential quality of larval and adult microvesicle preparations as evaluated by EM, further indicates that ELV release is primarily a characteristic of larval-stage parasites and perhaps more biologically relevant to early parasite infection.


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)

Isolation of Small RNAs from larval and adult B. malayi ELV fractions.Bioanalyzer data are shown for RNAs isolated from L3, adult male, and adult female Brugia preparations. L3 ELVs contain significant amounts of small RNAs in the 25–200 nt range (25 and 200 nt reference peaks labeled), while adult male and female vesicle preparations yield fewer RNAs. Vesicle fractions were prepared from 300 L3 and 30 adults in 24 hr culture incubations. Despite the much higher total tissue amounts used in adult culture, we detect much higher levels of small RNAs in L3-derived ELVs.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004069.g004: Isolation of Small RNAs from larval and adult B. malayi ELV fractions.Bioanalyzer data are shown for RNAs isolated from L3, adult male, and adult female Brugia preparations. L3 ELVs contain significant amounts of small RNAs in the 25–200 nt range (25 and 200 nt reference peaks labeled), while adult male and female vesicle preparations yield fewer RNAs. Vesicle fractions were prepared from 300 L3 and 30 adults in 24 hr culture incubations. Despite the much higher total tissue amounts used in adult culture, we detect much higher levels of small RNAs in L3-derived ELVs.
Mentions: We probed larval and adult microvesicle preparations for the presence of small RNA species. Exosomes have been found to contain both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs) in a diverse range of species and cell types. Of particular interest to us was the potential presence of small non-coding RNAs, including microRNAs (miRNAs), that could potentially mediate parasite-parasite communication or modulate host gene expression. Small RNA species were preferentially isolated from putative ELV-containing pellets and examined with an Agilent Bioanalyzer. The microvesicle fractions of L3 B. malayi (24 hr incubations of 300 worms) revealed an abundance of small RNA species in the 25–200 nt range (Fig 4). Much less RNA was detected from incubations of adult male and female B. malayi (24 hr incubations of 30 adult worms), despite the much higher mass of tissue in adult stage culture media. This lack of correlation between total parasite tissue material and RNA yield, coupled to the differential quality of larval and adult microvesicle preparations as evaluated by EM, further indicates that ELV release is primarily a characteristic of larval-stage parasites and perhaps more biologically relevant to early parasite infection.

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