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Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.

Nowacki FC, Swain MT, Klychnikov OI, Niazi U, Ivens A, Quintana JF, Hensbergen PJ, Hokke CH, Buck AH, Hoffmann KF - J Extracell Vesicles (2015)

Bottom Line: Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences.Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5' tsRNAs and fourteen 3' tsRNAs).Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions.

View Article: PubMed Central - PubMed

Affiliation: IBERS, Aberystwyth University, Aberystwyth, UK.

ABSTRACT

Background: Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos.

Results: Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30-100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5' tsRNAs and fourteen 3' tsRNAs).

Conclusions: The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions.

No MeSH data available.


Related in: MedlinePlus

Most of the 35 known miRNAs released by schistosomula are found in comparable abundance between EV-enriched and EV-depleted fractions. Outputs from the miRDeep2.pl script within the miRDeep2 package were used to identify known platyhelminth miRNAs in schistosomula E/S samples. Manual curation against the recent literature (49,51,53) was also performed to identify previously described miRNAs that have not yet been uploaded to miRBase. A total of 35 known miRNAs were subsequently identified. (a) Known sma-miRNAs (26) found at similar abundance between EV-enriched (dark bars) and EV-depleted (light bars) fractions. (b) Known sma-miRNAs (9) found at a higher relative proportion in the EV-enriched (black bars) or the EV-depleted (grey bars) fraction (i.e. containing reads only in the EV-enriched or EV-depleted fractions after normalization). The asterisk (*) represents sma-miRNAs (sma-mir-76 and sma-mir-2149) identified by seed sequence similarity to Schmidtea mediterranea sme-miRNAs. Bar charts represent the miRNA log2 normalized read counts for each sample where the indicated sma-miRNA was found (see Materials and methods). Raw and processed sma-miRNA read data are found in Supplementary file 3.
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Figure 0004: Most of the 35 known miRNAs released by schistosomula are found in comparable abundance between EV-enriched and EV-depleted fractions. Outputs from the miRDeep2.pl script within the miRDeep2 package were used to identify known platyhelminth miRNAs in schistosomula E/S samples. Manual curation against the recent literature (49,51,53) was also performed to identify previously described miRNAs that have not yet been uploaded to miRBase. A total of 35 known miRNAs were subsequently identified. (a) Known sma-miRNAs (26) found at similar abundance between EV-enriched (dark bars) and EV-depleted (light bars) fractions. (b) Known sma-miRNAs (9) found at a higher relative proportion in the EV-enriched (black bars) or the EV-depleted (grey bars) fraction (i.e. containing reads only in the EV-enriched or EV-depleted fractions after normalization). The asterisk (*) represents sma-miRNAs (sma-mir-76 and sma-mir-2149) identified by seed sequence similarity to Schmidtea mediterranea sme-miRNAs. Bar charts represent the miRNA log2 normalized read counts for each sample where the indicated sma-miRNA was found (see Materials and methods). Raw and processed sma-miRNA read data are found in Supplementary file 3.

Mentions: Among the 35 known sma-miRNAs identified in the schistosomula E/S products (Fig. 4), sma-bantam, sma-miR-10, sma-miR-3479 and sma-miR-n1 were all previously detected in sera obtained from individuals (mice, humans or rabbits) chronically infected with S. mansoni (49, 50). While none of the newly identified 170 sma-miRNAs have yet been detected in infected sera, their presence should be investigated due to their high abundance in schistosomula E/S products (Fig. 5 and Supplementary file 3). Nevertheless, our findings now indicate a potential mechanism, larval excretion/secretion of known and novel miRNAs, by which these sma-miRNAs are released from schistosomes and accumulate in host blood. The discovery that sma-bantam, sma-miR-10, sma-miR-3479 and sma-miR-n1 (among numerous novel sma-miRNAs) are found in similar abundance between EV-enriched and EV-depleted E/S fractions (Figs. 4a and 5a) would suggest that, in addition to EV encapsulation, serum-detectable sma-miRNAs could be stabilized by protein interactions. Alternatively, the comparable abundance of these (and other, Supplementary file 3) miRNAs found within EV-enriched and EV-depleted E/S fractions could also be indicative of vesicle lysing during the 72 h in vitro cultures. Until a time in which these 2 scenarios are resolved (using differential proteinase K and RNase A processing of EVs), quantitative reverse-transcription PCR detection of the most abundantly excreted/secreted 72 h schistosomula sma-miRNAs (sma-mir-new-215, sma-mir-new-271 and sma-mir-new-717; bold miRNAs highlighted in Fig. 5a and b) in infected mouse sera is currently being planned to confirm their presence in EV-enriched and EV-depleted host biofluids.


Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.

Nowacki FC, Swain MT, Klychnikov OI, Niazi U, Ivens A, Quintana JF, Hensbergen PJ, Hokke CH, Buck AH, Hoffmann KF - J Extracell Vesicles (2015)

Most of the 35 known miRNAs released by schistosomula are found in comparable abundance between EV-enriched and EV-depleted fractions. Outputs from the miRDeep2.pl script within the miRDeep2 package were used to identify known platyhelminth miRNAs in schistosomula E/S samples. Manual curation against the recent literature (49,51,53) was also performed to identify previously described miRNAs that have not yet been uploaded to miRBase. A total of 35 known miRNAs were subsequently identified. (a) Known sma-miRNAs (26) found at similar abundance between EV-enriched (dark bars) and EV-depleted (light bars) fractions. (b) Known sma-miRNAs (9) found at a higher relative proportion in the EV-enriched (black bars) or the EV-depleted (grey bars) fraction (i.e. containing reads only in the EV-enriched or EV-depleted fractions after normalization). The asterisk (*) represents sma-miRNAs (sma-mir-76 and sma-mir-2149) identified by seed sequence similarity to Schmidtea mediterranea sme-miRNAs. Bar charts represent the miRNA log2 normalized read counts for each sample where the indicated sma-miRNA was found (see Materials and methods). Raw and processed sma-miRNA read data are found in Supplementary file 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 0004: Most of the 35 known miRNAs released by schistosomula are found in comparable abundance between EV-enriched and EV-depleted fractions. Outputs from the miRDeep2.pl script within the miRDeep2 package were used to identify known platyhelminth miRNAs in schistosomula E/S samples. Manual curation against the recent literature (49,51,53) was also performed to identify previously described miRNAs that have not yet been uploaded to miRBase. A total of 35 known miRNAs were subsequently identified. (a) Known sma-miRNAs (26) found at similar abundance between EV-enriched (dark bars) and EV-depleted (light bars) fractions. (b) Known sma-miRNAs (9) found at a higher relative proportion in the EV-enriched (black bars) or the EV-depleted (grey bars) fraction (i.e. containing reads only in the EV-enriched or EV-depleted fractions after normalization). The asterisk (*) represents sma-miRNAs (sma-mir-76 and sma-mir-2149) identified by seed sequence similarity to Schmidtea mediterranea sme-miRNAs. Bar charts represent the miRNA log2 normalized read counts for each sample where the indicated sma-miRNA was found (see Materials and methods). Raw and processed sma-miRNA read data are found in Supplementary file 3.
Mentions: Among the 35 known sma-miRNAs identified in the schistosomula E/S products (Fig. 4), sma-bantam, sma-miR-10, sma-miR-3479 and sma-miR-n1 were all previously detected in sera obtained from individuals (mice, humans or rabbits) chronically infected with S. mansoni (49, 50). While none of the newly identified 170 sma-miRNAs have yet been detected in infected sera, their presence should be investigated due to their high abundance in schistosomula E/S products (Fig. 5 and Supplementary file 3). Nevertheless, our findings now indicate a potential mechanism, larval excretion/secretion of known and novel miRNAs, by which these sma-miRNAs are released from schistosomes and accumulate in host blood. The discovery that sma-bantam, sma-miR-10, sma-miR-3479 and sma-miR-n1 (among numerous novel sma-miRNAs) are found in similar abundance between EV-enriched and EV-depleted E/S fractions (Figs. 4a and 5a) would suggest that, in addition to EV encapsulation, serum-detectable sma-miRNAs could be stabilized by protein interactions. Alternatively, the comparable abundance of these (and other, Supplementary file 3) miRNAs found within EV-enriched and EV-depleted E/S fractions could also be indicative of vesicle lysing during the 72 h in vitro cultures. Until a time in which these 2 scenarios are resolved (using differential proteinase K and RNase A processing of EVs), quantitative reverse-transcription PCR detection of the most abundantly excreted/secreted 72 h schistosomula sma-miRNAs (sma-mir-new-215, sma-mir-new-271 and sma-mir-new-717; bold miRNAs highlighted in Fig. 5a and b) in infected mouse sera is currently being planned to confirm their presence in EV-enriched and EV-depleted host biofluids.

Bottom Line: Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences.Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5' tsRNAs and fourteen 3' tsRNAs).Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions.

View Article: PubMed Central - PubMed

Affiliation: IBERS, Aberystwyth University, Aberystwyth, UK.

ABSTRACT

Background: Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos.

Results: Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30-100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5' tsRNAs and fourteen 3' tsRNAs).

Conclusions: The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions.

No MeSH data available.


Related in: MedlinePlus