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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

Discovery and profiling of miRNAs in B. malayi exosome-like vesicles.(A) Comparative abundance of miRNAs in L3 ELV and tissue-derived samples. miRNA discovery and abundance estimation was carried out using the mirDeep2 pipeline. The 20 miRNAs with highest expression in each sample were retained for comparison and abundance was normalized with respect to total miRNA-mapping reads within each sample. Normalized read count is plotted on a log scale for ELV and tissue miRNAs to provide a relative ordering of fractional abundance. Bma-let-7 only appears in the highly expressed subset, and a number of miRNAs with perfect mature sequence identity to host homologs are highlighted (outer blue circle). (B) Sequence conservation between B. malayi ELV-origin miRNAs and the host ( H. sapiens ) miRNA complement. Reduced heat map showing one-to-one homology between a given B. malayi miRNA and its nearest matching human counterpart in terms of percent identity. Bma-let-7, bma-miR-1, bma-miR-9, bma-miR-92, and bma-miR-100b (white asterisks) share 100% identity with a host miRNA, while bma-miR-34 shows high identity with a host miRNA (21/23 nucleotides). This B. malayi miRNA subset (shown in blue) contains potential modulators of host gene expression. (C) Sequence conservation between B. malayi ELV-origin miRNA seed sites and host ( H. sapiens ) miRNA seed sites. miRNAs sharing perfectly conserved seed sites, defined here as nucleotides 2–8 of the mature miRNA, are marked (blue circles).
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pntd.0004069.g005: Discovery and profiling of miRNAs in B. malayi exosome-like vesicles.(A) Comparative abundance of miRNAs in L3 ELV and tissue-derived samples. miRNA discovery and abundance estimation was carried out using the mirDeep2 pipeline. The 20 miRNAs with highest expression in each sample were retained for comparison and abundance was normalized with respect to total miRNA-mapping reads within each sample. Normalized read count is plotted on a log scale for ELV and tissue miRNAs to provide a relative ordering of fractional abundance. Bma-let-7 only appears in the highly expressed subset, and a number of miRNAs with perfect mature sequence identity to host homologs are highlighted (outer blue circle). (B) Sequence conservation between B. malayi ELV-origin miRNAs and the host ( H. sapiens ) miRNA complement. Reduced heat map showing one-to-one homology between a given B. malayi miRNA and its nearest matching human counterpart in terms of percent identity. Bma-let-7, bma-miR-1, bma-miR-9, bma-miR-92, and bma-miR-100b (white asterisks) share 100% identity with a host miRNA, while bma-miR-34 shows high identity with a host miRNA (21/23 nucleotides). This B. malayi miRNA subset (shown in blue) contains potential modulators of host gene expression. (C) Sequence conservation between B. malayi ELV-origin miRNA seed sites and host ( H. sapiens ) miRNA seed sites. miRNAs sharing perfectly conserved seed sites, defined here as nucleotides 2–8 of the mature miRNA, are marked (blue circles).

Mentions: To more fully investigate the nucleic acid contents of these newly discovered vesicles, we carried out RNA-Seq with both L3 ELV and tissue-derived small RNAs. Reads generated by Illumina sequencing were processed and used to seed an miRNA discovery and abundance estimation pipeline using miRDeep2 [47] (read statistics and raw miRNA abundances can be found in S1 Table). To compare ELV and cellular RNA abundance, miRNA expression was normalized to the total miRNA read count within each sample. miRNA discovery and profiling was augmented with data from previously discovered miRNAs in closely related nematode species to help overcome gaps in the B. malayi draft genome assembly (see Methods). Fig 5A compares normalized miRNA expression between ELV and tissue for the 20 most abundant miRNAs in each sample. Although there is considerable conservation in relative miRNAs abundances, there are some notable observations and exceptions.


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)

Discovery and profiling of miRNAs in B. malayi exosome-like vesicles.(A) Comparative abundance of miRNAs in L3 ELV and tissue-derived samples. miRNA discovery and abundance estimation was carried out using the mirDeep2 pipeline. The 20 miRNAs with highest expression in each sample were retained for comparison and abundance was normalized with respect to total miRNA-mapping reads within each sample. Normalized read count is plotted on a log scale for ELV and tissue miRNAs to provide a relative ordering of fractional abundance. Bma-let-7 only appears in the highly expressed subset, and a number of miRNAs with perfect mature sequence identity to host homologs are highlighted (outer blue circle). (B) Sequence conservation between B. malayi ELV-origin miRNAs and the host ( H. sapiens ) miRNA complement. Reduced heat map showing one-to-one homology between a given B. malayi miRNA and its nearest matching human counterpart in terms of percent identity. Bma-let-7, bma-miR-1, bma-miR-9, bma-miR-92, and bma-miR-100b (white asterisks) share 100% identity with a host miRNA, while bma-miR-34 shows high identity with a host miRNA (21/23 nucleotides). This B. malayi miRNA subset (shown in blue) contains potential modulators of host gene expression. (C) Sequence conservation between B. malayi ELV-origin miRNA seed sites and host ( H. sapiens ) miRNA seed sites. miRNAs sharing perfectly conserved seed sites, defined here as nucleotides 2–8 of the mature miRNA, are marked (blue circles).
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pntd.0004069.g005: Discovery and profiling of miRNAs in B. malayi exosome-like vesicles.(A) Comparative abundance of miRNAs in L3 ELV and tissue-derived samples. miRNA discovery and abundance estimation was carried out using the mirDeep2 pipeline. The 20 miRNAs with highest expression in each sample were retained for comparison and abundance was normalized with respect to total miRNA-mapping reads within each sample. Normalized read count is plotted on a log scale for ELV and tissue miRNAs to provide a relative ordering of fractional abundance. Bma-let-7 only appears in the highly expressed subset, and a number of miRNAs with perfect mature sequence identity to host homologs are highlighted (outer blue circle). (B) Sequence conservation between B. malayi ELV-origin miRNAs and the host ( H. sapiens ) miRNA complement. Reduced heat map showing one-to-one homology between a given B. malayi miRNA and its nearest matching human counterpart in terms of percent identity. Bma-let-7, bma-miR-1, bma-miR-9, bma-miR-92, and bma-miR-100b (white asterisks) share 100% identity with a host miRNA, while bma-miR-34 shows high identity with a host miRNA (21/23 nucleotides). This B. malayi miRNA subset (shown in blue) contains potential modulators of host gene expression. (C) Sequence conservation between B. malayi ELV-origin miRNA seed sites and host ( H. sapiens ) miRNA seed sites. miRNAs sharing perfectly conserved seed sites, defined here as nucleotides 2–8 of the mature miRNA, are marked (blue circles).
Mentions: To more fully investigate the nucleic acid contents of these newly discovered vesicles, we carried out RNA-Seq with both L3 ELV and tissue-derived small RNAs. Reads generated by Illumina sequencing were processed and used to seed an miRNA discovery and abundance estimation pipeline using miRDeep2 [47] (read statistics and raw miRNA abundances can be found in S1 Table). To compare ELV and cellular RNA abundance, miRNA expression was normalized to the total miRNA read count within each sample. miRNA discovery and profiling was augmented with data from previously discovered miRNAs in closely related nematode species to help overcome gaps in the B. malayi draft genome assembly (see Methods). Fig 5A compares normalized miRNA expression between ELV and tissue for the 20 most abundant miRNAs in each sample. Although there is considerable conservation in relative miRNAs abundances, there are some notable observations and exceptions.

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