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secretory products from the gastrointestinal nematode Trichuris muris
Accepted Manuscript Excretory/secretory products from the gastrointestinal nematode Trichuris muris Lucienne Tritten, Mifong Tam, Mireille Vargas, Armando Jardim, Mary M. Stevenson, Jennifer Keiser, Timothy G. Geary PII:
S0014-4894(17)30079-6
DOI:
10.1016/j.exppara.2017.05.003
Reference:
YEXPR 7407
To appear in:
Experimental Parasitology
Received Date: 7 February 2017 Revised Date:
10 April 2017
Accepted Date: 18 May 2017
Please cite this article as: Tritten, L., Tam, M., Vargas, M., Jardim, A., Stevenson, M.M., Keiser, J., Geary, T.G., Excretory/secretory products from the gastrointestinal nematode Trichuris muris, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Excretory/secretory products from the gastrointestinal nematode Trichuris
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muris
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Lucienne Trittena,b,e *, Mifong Tamb,c, Mireille Vargasd, Armando Jardima,b, Mary M.
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Stevensonb,c, Jennifer Keiserd, Timothy G. Gearya,b
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Bellevue, Quebec H9X 3V9, Canada, b Centre for Host-Parasite Interactions, c Department
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of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, d
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Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health
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Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Sainte-Anne-de-
Institute, Socinstrasse 57, P.O. Box, CH-4002 Basel, Switzerland. e Current address:
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Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich,
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Switzerland
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* Corresponding author: Lucienne Tritten, Institute of Parasitology, University of Zurich,
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Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland. e-mail: [email protected],
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telephone: +41 44 635 85 24
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ACCEPTED MANUSCRIPT Abstract
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To better control gastrointestinal nematode infections in humans and animals, it is important
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to understand the strategies used by these parasites to modulate the host immune system.
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In this regard, molecules released by parasites have been attributed crucially important roles
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in host-parasite negotiations. We characterized the excretory/secretory (E/S) microRNA
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(miRNA) and protein profiles from the mouse gastrointestinal nematode parasite Trichuris
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muris. Released miRNAs were subjected to miRNA sequencing and E/S proteins were
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analysed by mass spectrometry. Fourteen miRNAs were identified in T. muris exosome-like
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vesicles, as well as 73 proteins of nematode origin, 11 of which were unique to this study.
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Comparison with published nematode protein secretomes revealed high conservation at the
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functional level.
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ACCEPTED MANUSCRIPT 1. Introduction
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Parasites are masterful modulators of their host’s immune system, diverting responses to
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their own advantage, a phenomenon in which microRNAs (miRNAs) and proteins have been
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suggested to participate. miRNAs are small noncoding RNA entities ubiquitously present in
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eukaryotes. They exert regulatory functions at the posttranscriptional level through
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messenger RNA (mRNA) degradation and suppression of translation 1. Exosomes are
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believed to be the main trafficking vehicle of extracellular miRNAs in biofluids 2 and display
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selective miRNA profiles that differ from the global miRNA contents of the parent cell or
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tissue 3. The miRBase registry contains a substantial number of parasitic helminth miRNAs,
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but few studies have focused on circulating miRNAs released by parasitic nematodes into
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their host or environment 4–9. Most efforts to characterize nematode miRNAs have been
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based on whole-worm extracts 10–12 and do not describe the miRNA populations released by
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parasites, which may serve in intercellular communication with their hosts. Awareness of the
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potential importance of miRNAs at the functional level in host-helminth interactions has
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begun to rise 4,13–15. For instance, a recent report revealed that exposure to exosomes from
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Heligmosomoides polygyrus bakeri impaired the development of an innate immune response
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to an allergen administered to mice 4.
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Extensively investigated across many nematode species, excretory/secretory (E/S) proteins
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are important players in immunomodulation, whose diverse roles have been (and are still
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being) progressively characterized 16,17. Several parasite-derived proteins have been shown
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to induce a Th2-skewed immune response, combined with regulatory and anti-inflammatory
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components 17. Anti-allergic and anti-inflammatory effects of helminth E/S proteins have
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been observed for many nematode species 18–22. E/S products fulfil a wide range of functions
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in worm migration and feeding, including disarmament of host responses by inactivating
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defence molecules (e.g., reactive oxygen species), and mimicry of host anti-inflammatory
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molecules, among others 23.
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ACCEPTED MANUSCRIPT In the present work, we describe the miRNA and protein profiles released by Trichuris muris
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in vesicles of the size of exosomes. T. muris is grouped in Clade I of phylum Nematoda and
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preferentially resides between the colon and the caecum 24,25. All parasitic stages stay in the
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lumen, their anterior end penetrating into gut epithelial cells from which they feed. The T.
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muris protein profile was compared to previously published secretomes of parasitic
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nematode species, including the clade V murine gastrointestinal nematode, H. p. bakeri. We
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also generated an E/S miRNA profile of H. p. bakeri, which was previously published
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elsewhere 4, to validate our workflow and cross-laboratory reproducibility of miRNA
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sequencing.
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2.1. T. muris miRNA profile in pelleted vesicles/particles
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13,684,186 raw reads were obtained from miRNA sequencing from T. muris isolated
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vesicles/particles of the size of exosomes using polymer precipitation (Exoquick TC, System
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Biosciences), 11,002,227 of which were mappable after removal of low-quality reads, junk
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and short sequences. Reads unmapped to nematode genomes were further mapped to
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mouse and mammalian genomes. In total, 130 unique nematode miRNA candidate
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sequences were obtained (Supplementary Table S1), and another 128 mapped to mouse
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miRNAs in miRBase (Supplementary Table S2). They were present at levels (copy numbers)
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comparable to the worm-derived candidates. Excluding other types of RNA, as well as low-
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confidence miRNAs (suboptimal mapping or folding properties), 14 high-confidence miRNA
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candidates were obtained (as defined by genome mapping, homology with other nematode
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miRNAs and secondary structure properties (see 5.4), Table 1 and Supplementary Table
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S1), only 7 of which were present in > 5 reads.
89 T. muris-secreted miRNA candidates
Name
Sequence
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Properties
#
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cel-let-7-5p
TGAGGTAGTAGGTTGTATAGTT
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asu-miR-252-5p_R-2
CTAAGTAGTAGTGCCGCAGGT
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hco-miR-9_R-2
TCTTTGGTTATCTAGCTGTAT
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TGGCAGTGTAGTTAGCTGGTTGT
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TAAGGCACGCGGTGAATGCC
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TGGAAGACTAGTGATTTTGTTGT
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TTGCATAGTCACAAAAGTGATC
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cel-miR-124-3p_R-1 asu-miR-7-5p_R1_1ss10GA bma-miR153_1ss22GC cel-miR-82-3p_R4_1ss10CA hco-mir-5972p5_2ss2GT21CA
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asu-miR-345p_1ss10GA
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AACCCGTAGATCCGATCTTGT
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Reads map to other nematode miRNAs/premiRNAs in miRBase. The reads map to the T. muris genome. The sequences may form hairpins.
asu-miR-100a-5p_R3_1ss16AT
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cel-miR-79-3p_R-2
TGAGATCATAGTGAAAGC
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TTAGGCTTAGGCTTAGGCTTAGGC
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ATAAAGCTAGGTTACCAAAG
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asu-mir-279aTGACTAGATCCACACTCATC 3p_2ss13TC19GT asu-miR-252-3p_RACCTGCTCCCTGCTACTTAAG 1_2ss15GA21CG bma-mir-236-1TAATACTGTCGGGTAAAGATGATT 3p_3ss11AG17TA20CT
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Table 1. T. muris miRNA high confidence candidates. miRNA candidates for which genome mapping, homology to known nematode miRNAs and secondary structure support characterization as high confidence candidates. Names in bold and italics denote sequences identical to mouse miRNAs.
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It should be noted that 6 of the 8 most abundant high-confidence sequences that map
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perfectly to the T. muris genome (asu-miR-100a-5p_R-3_1ss16AT, cel-let-7-5p, hco-miR5
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9_R-2, cel-miR-124-3p_R-1, asu-miR-7-5p_R-1_1ss10GA, and bma-miR-153_1ss22GC)
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present in the T. muris preparations are identical to known mouse miRNAs as well. Despite thorough washing of the parasites prior to culture, we cannot exclude the possibility that
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mouse material was still present, and so cannot prove that these sequences are parasite-
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derived.
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2.2. Validation of miRNA sequencing workflow using H. p. bakeri culture media
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The robustness of the sequencing method was confirmed by a general agreement between
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the previously published H. p. bakeri miRNA secretome 4, and our H. p. bakeri dataset
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(Supplementary File 1 and Supplementary Table S3), despite methodological differences.
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Following isolation of particles of the size of exosomes using polymer precipitation (Exoquick
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TC, System Biosciences), 18 high-confidence miRNA candidates were identified; all had
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been described by Buck and colleagues 4. Forty-four miRNA candidates mapped to the
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mouse genome (Supplementary Table S4). Nanoparticle tracking analysis of H. p. bakeri
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culture media revealed a main particle population of 109 ± 3.1 nm in diameter, at a total
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concentration of 6.77 x 1010 particles/ml (Supplementary Fig S1). We cannot exclude the
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presence of non-vesicular contaminants. Larger particles were also observed, but in much
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lower abundance.
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2.3. T. muris protein profile in pelleted vesicles/particles
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Proteomic analysis revealed the presence of 73 Trichuris proteins and 36 of mouse origin in
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the isolated material of the size of exosomes (Table 2 and Supplementary Table S5).
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Thirteen proteins were identified in only one of the two samples corresponding to two
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consecutive elutions of the protein-binding column. Good agreement was observed between
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both samples: the three most abundant proteins were the same, and 13 proteins appeared in
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the top 15 in both samples. Only 16 identified proteins (21.9 %) were predicted to contain a 6
ACCEPTED MANUSCRIPT signal peptide cleavage site and another 14 (19.2 %) were predicted to be non-classically
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secreted. An additional 19 proteins (26 %) in these samples have no known
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excretory/secretory sequence motifs, but have been repeatedly associated with exosomes
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and are listed in Exocarta (www.exocarta.org/). The remaining 24 proteins (33 %) may
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originate from damaged worms, a possibility we cannot exclude. However, we did not detect
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proteins that are typically the most abundant in cells, such as ribosomal or mitochondrial
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proteins (e.g., adenine nucleotide translocator proteins and voltage-dependent anion
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channel proteins 26), which would be expected to predominate in the sample if damaged
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worms were present, and so believe that the detected proteins do not indicate the presence
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of structurally compromised worms in the cultures.
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UniProt accession
Protein name Poly-cysteine and histidine tailed protein isoform 2
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Actin
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Glyceraldehyde 3 phosphate dehydrogenase
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Dynein light chain
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Histone domain containing protein
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Uncharacterized protein
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Uncharacterized protein
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Intermediate filament protein (ifa)
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Uncharacterized protein
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14-3-3 protein
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ATP synthase subunit beta
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Ubiquitin associated and SH3
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Triosephosphate isomerase
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Uncharacterized protein
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Fructose bisphosphate aldolase class I
T. trichiura, A0A077Z5Q5 T. trichiura, A0A077ZE37 T. trichiura, A0A077ZHV3 T. trichiura, A0A077ZJN3 T. trichiura, A0A077ZFV9 T. suis, A0A085MFD5 T. suis, A0A085MAM6 T. trichiura, A0A077Z6U0 T. suis, A0A085MIT4 T. trichiura, A0A077YXJ9 T. trichiura, A0A077Z3I0 T. trichiura, A0A077ZFQ2 T. trichiura, A0A077ZC84 T. trichiura, A0A077ZFC1 T. trichiura, A0A077Z6Y9
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Blast2GO description
Unique to this study
Poly-cysteine and histidine tailed protein isoform 2
yes
Actin-5c
no
Glyceraldehyde-3-phosphate dehydrogenase
no
Dynein light chain
no
Histone-like
no
Phosphoglycerate kinase
no
Galactoside-binding lectin
no
Intermediate filament protein ifa-1
no
Phosphoenolpyruvate carboxykinase
no
14-3-3 zeta
no
ATP synthase beta subunit
no
Ubiquitin associated and sh3 domain-containing protein b
no
Triosephosphate isomerase
no
Hypothetical protein TTRE_0000739101 Fructose-bisphosphate aldolase class-i
yes no
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Table 2. Fifteen most abundant T. muris proteins found in isolated vesicles/particles. Proteins were mapped to Trichuridae; the UniProt accession number and the closest 7
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Trichuris spp. used for identification are given, as well as the Blast2GO sequence description. Proteins are listed according to relative abundance.
140 We obtained gene ontology (GO) terms for 69 of the 73 protein names. Eleven proteins were
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unique to this study, and to our knowledge, have not been reported in other nematode
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secretomes. The most abundant predicted protein was mouse keratin, a common
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contaminant found in proteomic studies. In the isolated particles of the size of exosomes, the
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most abundant protein of nematode origin was poly-cysteine and histidine tailed protein
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isoform 2. More than 50 % (39/73) of the T. muris proteins had previously been associated
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with homologues found in mammalian exosomes, as noted in the Exocarta database.
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No secreted protein was common to all 12 nematode secretomes compared (Supplementary
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Table S5). Glutathione S-transferase was reported to be secreted by 9 of the compared
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species, and enolase in 8. Actin, triosephosphate isomerase, aldolase and galectins were
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found in the secreted particles of T. muris and 7 other parasitic nematodes. All are common
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exosomal proteins.
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In agreement with published H. p. bakeri data 27 (Fig 1) and other nematode protein
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secretomes reported from our laboratory 18,27–29, the GO terms “binding” (GO:0005488) and
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“catalytic activity” (GO:0003824) were the most frequent level 2 molecular function
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categories. Under “biological process” (level 2), the terms “cellular process” (GO:0009987),
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“metabolic process” (GO:0008152) and “single-organism process” were the major groups.
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Indeed, the biological processes in play presented some differences with H. p. bakeri, for
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which the three main categories were “multicellular organismal process”, “developmental
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process”, and “biological regulation”. Secretory protein profiles appeared relatively similar in
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T. muris (vesicular component), H. p. bakeri, and B. malayi; terms attributed to H. p. bakeri
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proteins resembled B. malayi more than T. muris.
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Figure 1. Gene ontology analysis of the identified T. muris proteins compared to other nematode secretomes. The distribution of level 2 GO terms for biological processes (A) and molecular functions (B), as assigned by Blast2GO. H. p. bakeri and B. malayi GO distribution data and the top 3 (level 2) GO terms for A. suum and D. immitis were retrieved from previous reports 18,27–29.
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The distribution of level 4 biological process and molecular function terms for T. muris
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closely follows the pattern observed for B. malayi and D. immitis secreted proteins 18,29
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(Supplementary Fig S2).
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ACCEPTED MANUSCRIPT 3. Discussion
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We present excretory/secretory miRNA and protein profiles of the murine gastrointestinal
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parasitic nematode T. muris. After subjecting concentrated culture media to a polymer-based
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vesicle precipitation, we identified 14 high confidence miRNA candidates and 73 proteins.
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Because worms were washed thoroughly before incubation in culture media, we did not
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expect a significant contribution from the host. However, since some miRNA candidate
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sequences and proteins were predicted to be of mouse origin, it is possible that some mouse
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intestinal tissue was removed with the worms and remained despite washes, releasing host
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material/vesicles into culture media. However, the possibility that host miRNAs and proteins
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are taken up by the worm and packaged into worm-derived vesicles cannot be dismissed.
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Similarly, it is possible that some worms may have been damaged upon removal from the
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host, releasing non-secretory material into the culture media.
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The overlap between miRNAs from both mouse gastrointestinal nematodes (T. muris and H.
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p. bakeri (both Buck and colleagues’ 4 and current datasets) was minor: of all T. muris
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miRNAs (high and low confidence), only four were 100% identical or varied in length only in
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the 3’ end compared to H. p. bakeri miRNAs (let-7-5p, miR-9, miR-124, and miR-79). miR-
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100a, miR-34, and miR-252 presented minor substitutions compared to H. p. bakeri miRNA
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sequences. miR-100a, the most abundant candidate in T. muris-derived particles of the size
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of exosomes, was also the second most abundant candidate in D. immitis-infected dog blood
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from our H. p. bakeri dataset, but was present in the previous work 4. miR-9 and miR-252
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were abundant in exosome-like vesicles from B. malayi L3 5 but remain functionally
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uncharacterized in nematodes. In mouse intestinal tissue, the most abundant miRNAs are
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miR-192, followed by miR-215, and members of the let-7 family 30. In both T. muris and H. p.
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bakeri culture media, neither miR-192 nor miR-215 ranks among the 30 most abundant
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sequences.
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. Among the 14 high confidence miRNAs present in T. muris media, miR-252 was absent
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ACCEPTED MANUSCRIPT Some miRNA sequences attributed to nematode secretions could be of mouse origin;
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sequence identity prevents the determination of the relative contribution by host vs. parasite.
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miR-124 was described in mouse intestine among other tissues 31. However, not all host-
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identical miRNA species found in T. muris exosome-like particles are reported to occur in
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murine intestinal tissue. For instance, miR-9 and miR-99a (identical to asu-miR-100a-5p_R-
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3_1ss16AT) are specifically brain-enriched miRNAs in the mouse 31. Others, like let-7a, are
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expressed in many mouse tissues 31. Besides well-described roles in developmental timing
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in C. elegans, let-7a acts as a regulator of the murine and human immune systems. For
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example, let-7a directly inhibits the expression of the pro-inflammatory cytokine IL-6 and has
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been linked to cancer 32.
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If most mammalian miRNA targets are highly conserved 33, it is also true for some miRNAs
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conserved across nematodes 14. Nearly 50 % (6/14) of the high-confidence miRNAs found in
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the T. muris culture media shared sequence identity with mouse miRNAs. In a context of
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host-pathogen crosstalk and of host manipulation by the parasite via miRNA transfer, as has
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been suggested 4,5, the extent of the overlap between host and parasite miRNAs (or seed
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sequences) may reflect host-parasite specificity. It is worth noting that several secreted
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parasite miRNAs (i.e., miR-9 and some members of the miR-100 family) are more conserved
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between B. malayi and its human host than with C. elegans 5. In line with this, a number of
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H. p. bakeri miRNAs released in exosomes were identical or homologous to mouse miRNAs
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and there is evidence that these are used to modulate host immune functions 4. Clearly,
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more investigations are needed to draw firm conclusions in this respect.
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mmu-miR-26a-5p was the most frequently detected mouse miRNA, whereas it is only the 8th
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most highly expressed murine gut miRNA 30. The intestinal epithelium expresses relatively
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high levels of miR-26a, which represses a number of proliferation-associated genes in this
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tissue, including genes involved in tumorigenesis 34.
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which may be due to different vesicle/particle isolation protocols between the studies, the
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number of worms used, or the duration of incubation in culture media (2 days versus 3
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weeks). Nonetheless, all high-confidence miRNA sequences identified in the current work
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were consistent. These results suggest that miRNA secretion is a robust phenomenon for
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nematodes in culture, and that high-confidence sequences obtained using different methods
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and equipment are comparable.
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Only 41 % of proteins released by T. muris in vesicles/particles were predicted to be
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secreted by SignalP or SecretomeP. However, many of the remaining protein sequences
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have been reported in E/S products from other nematodes, as described in Supplementary
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Table S5. Including hits from our search in Exocarta, 67 % of these proteins have been
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associated with secretion and/or exosomal transport, near the proportion reported for B.
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malayi larval exosome-like vesicles 5. We compared T. muris-derived proteins with 14
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published secretomes of nematodes classified in clades I, III, or V. Most proteins released by
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T. muris have been described in other nematode secretomes and overall, there was a high
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degree of conservation of secreted proteins at the functional level. Although we lack visual
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evidence for the presence of exosome-like vesicles in the T. muris sample, typical vesicular
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proteins were well represented in our sample, dominated by cytoskeletal proteins, metabolic
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enzymes, zeta polypeptide, heat shock proteins, etc.35 . This is in line with recent evidence
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that different species and stages of nematodes release microvesicles 4,5,36. Heat shock
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protein 70, GAPDH, ezrin, moesin, actin and tubulin are common cargo of many different
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types of vesicles 37. A few known immunomodulators were recognized in this study, among
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them a galectin and a glutathione-S-transferase 38. We identified ten enzymes involved in
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energy metabolism (glycolysis, tricarboxylic acid cycle, and gluconeogenesis). Among them,
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the well-known exosomal proteins aldolase, enolase and triosephosphate isomerase are
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commonly observed in nematode E/S products 18,28,29,39–41. Trypsins were described in the
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secretome of T. spiralis, another clade I nematode 42, and H. p. bakeri 40. Hasnain and
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ACCEPTED MANUSCRIPT colleagues reported serine protease activity in T. muris secretions 43, and we found five
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proteins with trypsin domains that could have a similar biological function. A Von Willebrand
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factor-like protein has also been described to be secreted proteins by the liver fluke Fasciola
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hepatica 44; its role remains to be explored. Thirty-nine % of the level 2 GO terms describing
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molecular functions indicated catalytic activity. Level 4 GO terms revealed peptidase activity
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and serine hydrolase activity, among others. The importance of helminth proteases at all
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stages of infection has been widely acknowledged 45, and in T. muris chronic infections, they
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degrade mucins which may confer protection from parasite expulsion 43.
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Because experimental differences (vesicle/particle precipitation, technical aspects in LC-MS
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analysis and amount of protein analysed for these studies) impact on the outcome, a direct
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cross-study comparison is both qualitatively and quantitatively difficult. However, available
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data obtained by gene ontology predict that more T. muris proteins are functionally similar to
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the B. malayi (up to 26 5,18,19), N. brasiliensis (19 46), and D. immitis (16 29) secretomes than
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to the H.p. bakeri (14 27) secretome and that of the phylogenetically closer species T. spiralis
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muscle larvae 42.
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A major difference between the secretions from the two gastrointestinal nematodes H. p.
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bakeri and T. muris is the absence of venom allergen-like proteins in the T. muris sample
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compared to their predominance in the material from H. p. bakeri 27,40, and other gut
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parasites such as A. caninum and H. contortus 41,47, all of which are clade V nematodes.
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Similarly, these proteins are absent in secretions from A. suum 28. If venom allergens are
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present in the genus Trichuris, they are only distant homologues of H. polygyrus, B. malayi,
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or Ancylostoma venom allergen proteins, as revealed by a BLAST analysis yielding poor E-
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values (not shown). The role of nematode venom allergen-like proteins is unclear, and the
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functional significance of their apparent absence from T. muris remains to be resolved.
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4. Conclusion 13
ACCEPTED MANUSCRIPT We report 14 high-confidence T. muris miRNA candidates released in culture media, four of
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which are identical to miRNAs released by another mouse gastrointestinal parasitic
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nematode, H. p. bakeri. Some features in the ES protein profiles of the vesicular component
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we present here are common to other parasitic species, and may represent a general
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secretomic signature of parasitic nematodes. Differences in the composition of secreted
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material may reflect both methodological differences and/or the evolutionary distance
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between T. muris and the compared nematode species.
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5. Methods
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5.1. Ethics statement
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All experimental protocols were approved, either by the Animal Ethics Committee of McGill
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University and the Canadian Council on Animal Care or by the veterinary authorities of the
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Canton Basel-Stadt, Switzerland (licence no. 2070), based on Swiss regulations.
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Experiments were conducted in accordance with the approved guidelines.
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5.2. Parasite maintenance and culture
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The T. muris life cycle was maintained as described 48. Dexamethasone-treated (1 mg/l
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drinking water, Sigma-Aldrich) C57BL/10 female mice (Harlan, Blackthorn, UK) were
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infected orally with 200 embryonated eggs and maintained for 6 weeks until sacrifice (CO2)
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and dissection. Approximately 3000 adult T. muris were washed and cultured for 18-48 h at
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37°C, in filter-sterilized RPMI 1640 supplemented with 100 U/ml penicillin G, 100 µg/ml
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streptomycin and 2.5 µg/ml amphotericin B at a concentration of 10-20 worms/ml. Media
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were pooled and centrifuged at 5000 x g for 10 min prior to concentration on 3000 Da
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centrifugal filter units (Amicon Ultracel, Millipore), centrifuged at 8000 x g for 10 min and
304
stored at -20°C.
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5.3. Vesicle isolation, RNA and protein purification
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Biosciences). Total RNA and protein were subsequently isolated with a kit (Norgen Biotek,
308
#24100, “blood” protocol), yielding 6.5 µg RNA. For proteins, two consecutive column
309
elutions were performed, resulting in 565 and 661 µg of protein, respectively.
310
5.4. RNA sequencing
311
RNA quality was assessed by LC Sciences, and Illumina deep-sequencing of small RNAs
312
was performed (50 base pair single-end reads) following a proprietary pipeline using 1 µg
313
RNA. Low-quality sequences and other RNA populations (Rfam, Repbase; 49,50 were
314
removed and the remaining sequences of 18-24 nucleotides in length were mapped to the T.
315
muris genome
316
(ftp://ftp.sanger.ac.uk/pub/project/pathogens/Trichuris/muris/genome/version_2b/Tmuris_v2b
317
.genome_scaffolds.fa), mRNA
318
(ftp://ftp.sanger.ac.uk/pub/project/pathogens/Trichuris/muris/genes/geneSet2.1_Feb2013.nu
319
cl.fasta), and nematode miRNAs/pre-miRNAs in miRBase v.21.0 51. Next, sequences were
320
mapped to the mouse genome (M. musculus). Since no entry exists in miRBase for T. muris,
321
high confidence candidates are defined by successful mapping to the worm genome,
322
homology with other nematode miRNAs and the capacity of the extended genome region to
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form a hairpin. Raw data are available from the NCBI GEO database at
324
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93667.
325
5.5. Proteomic analysis
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T. muris proteins from the vesicular component were analysed by liquid chromatography-
327
tandem mass spectrometry (LC-MS/MS) at the Quebec Genomics Centre proteomics
328
platform, University Laval, for proteomics analysis.
329
An overnight tryptic digestion was performed on 1.0 µg protein in solution and analysed as
330
described 52. Reverse-phase nanoscale capillary liquid chromatography (Agilent 12000 nano
331
pump) was employed to separate peptides before analysis by electrospray tandem MS/MS
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ACCEPTED MANUSCRIPT (5600 Mass Spectrometer, AB Sciex, Farmingham, MA, USA). PicoFrit columns (New
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Objective, Woburn, MA, USA) packed with Jupiter (Phenomonex) 5u, 300A C18 served for
334
separation and were eluted with a linear gradient of acetonitrile (2-30 %) solvent, 0.1 %
335
formic acid. Spectra were acquired using Analyst v.1.6 and analysed with Mascot (Matrix
336
Science, London, UK; v. 2.4.1) and X! Tandem (The GPM, thegpm.org; version CYCLONE
337
(2010.12.01.1)). The TAX_MusMusculus_10090_20141118 database (unknown version,
338
116602 entries) and the TAX_Trichuris_36086_20141217 database were used. Scaffold (v.
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4.4.1.1, Proteome Software Inc., Portland, OR, USA) enabled the validation of MS/MS-
340
based peptide and protein identities. For peptide identification, >16.0 % probability to
341
achieve a false discovery rate (FDR) <1.0 % was set. Protein identities were validated when
342
>99.0 % probability to achieve a FDR <1.0 % and a minimum of 2 identified peptides were
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achieved. Protein probabilities were assigned by the Protein Prophet algorithm 53. Proteins
344
containing undistinguishable peptides were grouped to satisfy the principles of parsimony.
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Protein relative abundance (within the sample only) was assessed using the normalized
346
spectral abundance factor in Scaffold (NSAF). Protein descriptions for UniProt accession
347
numbers were obtained in Blast2GO (v. 3.0.8) 54. Both protein names from Scaffold and
348
sequence descriptions obtained from Blast2GO were used for comparison in published
349
nematode secretomes 5,18,19,27–29,40–42,46,47 and the Litomosoides sigmodontis predicted
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secretome 39. The protein names or Blast2GO descriptions were submitted to Exocarta 35
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(accessed on 28th of December 2016), in search of reported associations with exosomes in
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mammals. Secretory properties were further evaluated using SignalP (v. 4.1) and
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SecretomeP (v. 2.0) 55,56.
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Acknowledgments
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We are grateful to Daniel Defoy from the plate-forme protéomique du Centre Génomique de
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Québec, Université Laval for assistance with the proteomics analyses.
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We thank the Canadian Institutes for Health Research (fellowship # 320382 to LT, and grant
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# MOP130369 to MMS), the Canada Research Chairs, Canadian Natural Sciences and
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Engineering Research Council (to TGG), the Centre for Host-Parasite Interactions (Fonds de
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Recherche du Québec – Nature et Technologies), and the European Research Council
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(ERC-2013-CoG 614739-A_HERO to JK) for funding and support.
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504 Authors’ contributions
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LT, AJ, and TGG designed the study. LT, MT, and MV performed the experiments. MMS and
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JK provided the parasite material. LT analysed the data and wrote the first draft of the
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manuscript, AJ, MMS, JK and TGG revisited the manuscript critically. All authors read and
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approved the final manuscript.
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Additional information
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Competing financial interests
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The authors declare that they have no competing interests.
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Supplementary Figure S1. NanoSight measurement of microvesicles in H. p. bakeri culture media. Size distribution of observed microvesicles obtained by dividing the average size by the concentration. Red bars indicate ±1x standard error of the mean
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Supplementary Figure S2. Level 4 GO terms distribution for T. muris secreted proteins. A) biological process, B) molecular functions.
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11. Legends to supplementary files submitted separately:
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Supplementary File 1. Methods and Results to H. p. bakeri miRNA sequencing.
538 Supplementary Table S1. T. muris miRNA candidates. miRNA candidates were grouped
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into clusters according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S2. Mouse miRNAs found in T. muris exosomes. miRNA
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candidates were also mapped to the host genome and similarly grouped into clusters
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according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S3. H. p. bakeri miRNA candidates from isolated exosomes.
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miRNA candidates were grouped into clusters according to genome mapping and homology
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to known nematode miRNAs.
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Supplementary Table S4. Mouse miRNAs found in H. p. bakeri exosomes. miRNA
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candidates were also mapped to the host genome and similarly grouped into clusters
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according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S5. T. muris excretory/secretory proteins. Detailed information on
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T. muris and mouse proteins identified by mass spectrometry in culture media in two
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samples. For predicted T. muris proteins, a Blast2GO description is given, as well as
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predictions for secretion and presence in other nematode secretomes.
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Highlights We characterized the microRNAs and proteins in vesicles released by Trichuris muris.
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Fourteen miRNAs were identified in T. muris exosome-like vesicles.
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We found 73 proteins of nematode origin, 11 of which were unique to this study.
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S0014-4894(17)30079-6
DOI:
10.1016/j.exppara.2017.05.003
Reference:
YEXPR 7407
To appear in:
Experimental Parasitology
Received Date: 7 February 2017 Revised Date:
10 April 2017
Accepted Date: 18 May 2017
Please cite this article as: Tritten, L., Tam, M., Vargas, M., Jardim, A., Stevenson, M.M., Keiser, J., Geary, T.G., Excretory/secretory products from the gastrointestinal nematode Trichuris muris, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Excretory/secretory products from the gastrointestinal nematode Trichuris
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muris
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Lucienne Trittena,b,e *, Mifong Tamb,c, Mireille Vargasd, Armando Jardima,b, Mary M.
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Stevensonb,c, Jennifer Keiserd, Timothy G. Gearya,b
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a
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Bellevue, Quebec H9X 3V9, Canada, b Centre for Host-Parasite Interactions, c Department
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of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada, d
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Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health
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Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Sainte-Anne-de-
Institute, Socinstrasse 57, P.O. Box, CH-4002 Basel, Switzerland. e Current address:
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Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich,
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Switzerland
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* Corresponding author: Lucienne Tritten, Institute of Parasitology, University of Zurich,
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Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland. e-mail: [email protected],
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telephone: +41 44 635 85 24
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ACCEPTED MANUSCRIPT Abstract
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To better control gastrointestinal nematode infections in humans and animals, it is important
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to understand the strategies used by these parasites to modulate the host immune system.
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In this regard, molecules released by parasites have been attributed crucially important roles
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in host-parasite negotiations. We characterized the excretory/secretory (E/S) microRNA
29
(miRNA) and protein profiles from the mouse gastrointestinal nematode parasite Trichuris
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muris. Released miRNAs were subjected to miRNA sequencing and E/S proteins were
31
analysed by mass spectrometry. Fourteen miRNAs were identified in T. muris exosome-like
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vesicles, as well as 73 proteins of nematode origin, 11 of which were unique to this study.
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Comparison with published nematode protein secretomes revealed high conservation at the
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functional level.
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ACCEPTED MANUSCRIPT 1. Introduction
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Parasites are masterful modulators of their host’s immune system, diverting responses to
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their own advantage, a phenomenon in which microRNAs (miRNAs) and proteins have been
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suggested to participate. miRNAs are small noncoding RNA entities ubiquitously present in
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eukaryotes. They exert regulatory functions at the posttranscriptional level through
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messenger RNA (mRNA) degradation and suppression of translation 1. Exosomes are
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believed to be the main trafficking vehicle of extracellular miRNAs in biofluids 2 and display
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selective miRNA profiles that differ from the global miRNA contents of the parent cell or
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tissue 3. The miRBase registry contains a substantial number of parasitic helminth miRNAs,
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but few studies have focused on circulating miRNAs released by parasitic nematodes into
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their host or environment 4–9. Most efforts to characterize nematode miRNAs have been
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based on whole-worm extracts 10–12 and do not describe the miRNA populations released by
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parasites, which may serve in intercellular communication with their hosts. Awareness of the
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potential importance of miRNAs at the functional level in host-helminth interactions has
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begun to rise 4,13–15. For instance, a recent report revealed that exposure to exosomes from
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Heligmosomoides polygyrus bakeri impaired the development of an innate immune response
53
to an allergen administered to mice 4.
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Extensively investigated across many nematode species, excretory/secretory (E/S) proteins
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are important players in immunomodulation, whose diverse roles have been (and are still
56
being) progressively characterized 16,17. Several parasite-derived proteins have been shown
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to induce a Th2-skewed immune response, combined with regulatory and anti-inflammatory
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components 17. Anti-allergic and anti-inflammatory effects of helminth E/S proteins have
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been observed for many nematode species 18–22. E/S products fulfil a wide range of functions
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in worm migration and feeding, including disarmament of host responses by inactivating
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defence molecules (e.g., reactive oxygen species), and mimicry of host anti-inflammatory
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molecules, among others 23.
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ACCEPTED MANUSCRIPT In the present work, we describe the miRNA and protein profiles released by Trichuris muris
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in vesicles of the size of exosomes. T. muris is grouped in Clade I of phylum Nematoda and
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preferentially resides between the colon and the caecum 24,25. All parasitic stages stay in the
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lumen, their anterior end penetrating into gut epithelial cells from which they feed. The T.
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muris protein profile was compared to previously published secretomes of parasitic
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nematode species, including the clade V murine gastrointestinal nematode, H. p. bakeri. We
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also generated an E/S miRNA profile of H. p. bakeri, which was previously published
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elsewhere 4, to validate our workflow and cross-laboratory reproducibility of miRNA
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sequencing.
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2.1. T. muris miRNA profile in pelleted vesicles/particles
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13,684,186 raw reads were obtained from miRNA sequencing from T. muris isolated
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vesicles/particles of the size of exosomes using polymer precipitation (Exoquick TC, System
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Biosciences), 11,002,227 of which were mappable after removal of low-quality reads, junk
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and short sequences. Reads unmapped to nematode genomes were further mapped to
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mouse and mammalian genomes. In total, 130 unique nematode miRNA candidate
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sequences were obtained (Supplementary Table S1), and another 128 mapped to mouse
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miRNAs in miRBase (Supplementary Table S2). They were present at levels (copy numbers)
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comparable to the worm-derived candidates. Excluding other types of RNA, as well as low-
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confidence miRNAs (suboptimal mapping or folding properties), 14 high-confidence miRNA
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candidates were obtained (as defined by genome mapping, homology with other nematode
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miRNAs and secondary structure properties (see 5.4), Table 1 and Supplementary Table
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S1), only 7 of which were present in > 5 reads.
89 T. muris-secreted miRNA candidates
Name
Sequence
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Copy
Properties
#
359
cel-let-7-5p
TGAGGTAGTAGGTTGTATAGTT
126
asu-miR-252-5p_R-2
CTAAGTAGTAGTGCCGCAGGT
40
hco-miR-9_R-2
TCTTTGGTTATCTAGCTGTAT
18
TGGCAGTGTAGTTAGCTGGTTGT
12
TAAGGCACGCGGTGAATGCC
9
TGGAAGACTAGTGATTTTGTTGT
7
TTGCATAGTCACAAAAGTGATC
4
cel-miR-124-3p_R-1 asu-miR-7-5p_R1_1ss10GA bma-miR153_1ss22GC cel-miR-82-3p_R4_1ss10CA hco-mir-5972p5_2ss2GT21CA
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asu-miR-345p_1ss10GA
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AACCCGTAGATCCGATCTTGT
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Reads map to other nematode miRNAs/premiRNAs in miRBase. The reads map to the T. muris genome. The sequences may form hairpins.
asu-miR-100a-5p_R3_1ss16AT
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cel-miR-79-3p_R-2
TGAGATCATAGTGAAAGC
2
TTAGGCTTAGGCTTAGGCTTAGGC
2
ATAAAGCTAGGTTACCAAAG
1
90 91 92 93 94
AC C
asu-mir-279aTGACTAGATCCACACTCATC 3p_2ss13TC19GT asu-miR-252-3p_RACCTGCTCCCTGCTACTTAAG 1_2ss15GA21CG bma-mir-236-1TAATACTGTCGGGTAAAGATGATT 3p_3ss11AG17TA20CT
1 1 1
Table 1. T. muris miRNA high confidence candidates. miRNA candidates for which genome mapping, homology to known nematode miRNAs and secondary structure support characterization as high confidence candidates. Names in bold and italics denote sequences identical to mouse miRNAs.
95
96
It should be noted that 6 of the 8 most abundant high-confidence sequences that map
97
perfectly to the T. muris genome (asu-miR-100a-5p_R-3_1ss16AT, cel-let-7-5p, hco-miR5
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9_R-2, cel-miR-124-3p_R-1, asu-miR-7-5p_R-1_1ss10GA, and bma-miR-153_1ss22GC)
99
present in the T. muris preparations are identical to known mouse miRNAs as well. Despite thorough washing of the parasites prior to culture, we cannot exclude the possibility that
101
mouse material was still present, and so cannot prove that these sequences are parasite-
102
derived.
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2.2. Validation of miRNA sequencing workflow using H. p. bakeri culture media
105
The robustness of the sequencing method was confirmed by a general agreement between
106
the previously published H. p. bakeri miRNA secretome 4, and our H. p. bakeri dataset
107
(Supplementary File 1 and Supplementary Table S3), despite methodological differences.
108
Following isolation of particles of the size of exosomes using polymer precipitation (Exoquick
109
TC, System Biosciences), 18 high-confidence miRNA candidates were identified; all had
110
been described by Buck and colleagues 4. Forty-four miRNA candidates mapped to the
111
mouse genome (Supplementary Table S4). Nanoparticle tracking analysis of H. p. bakeri
112
culture media revealed a main particle population of 109 ± 3.1 nm in diameter, at a total
113
concentration of 6.77 x 1010 particles/ml (Supplementary Fig S1). We cannot exclude the
114
presence of non-vesicular contaminants. Larger particles were also observed, but in much
115
lower abundance.
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2.3. T. muris protein profile in pelleted vesicles/particles
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Proteomic analysis revealed the presence of 73 Trichuris proteins and 36 of mouse origin in
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the isolated material of the size of exosomes (Table 2 and Supplementary Table S5).
120
Thirteen proteins were identified in only one of the two samples corresponding to two
121
consecutive elutions of the protein-binding column. Good agreement was observed between
122
both samples: the three most abundant proteins were the same, and 13 proteins appeared in
123
the top 15 in both samples. Only 16 identified proteins (21.9 %) were predicted to contain a 6
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125
secreted. An additional 19 proteins (26 %) in these samples have no known
126
excretory/secretory sequence motifs, but have been repeatedly associated with exosomes
127
and are listed in Exocarta (www.exocarta.org/). The remaining 24 proteins (33 %) may
128
originate from damaged worms, a possibility we cannot exclude. However, we did not detect
129
proteins that are typically the most abundant in cells, such as ribosomal or mitochondrial
130
proteins (e.g., adenine nucleotide translocator proteins and voltage-dependent anion
131
channel proteins 26), which would be expected to predominate in the sample if damaged
132
worms were present, and so believe that the detected proteins do not indicate the presence
133
of structurally compromised worms in the cultures.
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UniProt accession
Protein name Poly-cysteine and histidine tailed protein isoform 2
2
Actin
3
Glyceraldehyde 3 phosphate dehydrogenase
4
Dynein light chain
5
Histone domain containing protein
6
Uncharacterized protein
7
Uncharacterized protein
8
Intermediate filament protein (ifa)
9
Uncharacterized protein
10
14-3-3 protein
11
ATP synthase subunit beta
12
Ubiquitin associated and SH3
13
Triosephosphate isomerase
14
Uncharacterized protein
15
Fructose bisphosphate aldolase class I
T. trichiura, A0A077Z5Q5 T. trichiura, A0A077ZE37 T. trichiura, A0A077ZHV3 T. trichiura, A0A077ZJN3 T. trichiura, A0A077ZFV9 T. suis, A0A085MFD5 T. suis, A0A085MAM6 T. trichiura, A0A077Z6U0 T. suis, A0A085MIT4 T. trichiura, A0A077YXJ9 T. trichiura, A0A077Z3I0 T. trichiura, A0A077ZFQ2 T. trichiura, A0A077ZC84 T. trichiura, A0A077ZFC1 T. trichiura, A0A077Z6Y9
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Blast2GO description
Unique to this study
Poly-cysteine and histidine tailed protein isoform 2
yes
Actin-5c
no
Glyceraldehyde-3-phosphate dehydrogenase
no
Dynein light chain
no
Histone-like
no
Phosphoglycerate kinase
no
Galactoside-binding lectin
no
Intermediate filament protein ifa-1
no
Phosphoenolpyruvate carboxykinase
no
14-3-3 zeta
no
ATP synthase beta subunit
no
Ubiquitin associated and sh3 domain-containing protein b
no
Triosephosphate isomerase
no
Hypothetical protein TTRE_0000739101 Fructose-bisphosphate aldolase class-i
yes no
135 136 137
Table 2. Fifteen most abundant T. muris proteins found in isolated vesicles/particles. Proteins were mapped to Trichuridae; the UniProt accession number and the closest 7
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Trichuris spp. used for identification are given, as well as the Blast2GO sequence description. Proteins are listed according to relative abundance.
140 We obtained gene ontology (GO) terms for 69 of the 73 protein names. Eleven proteins were
142
unique to this study, and to our knowledge, have not been reported in other nematode
143
secretomes. The most abundant predicted protein was mouse keratin, a common
144
contaminant found in proteomic studies. In the isolated particles of the size of exosomes, the
145
most abundant protein of nematode origin was poly-cysteine and histidine tailed protein
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isoform 2. More than 50 % (39/73) of the T. muris proteins had previously been associated
147
with homologues found in mammalian exosomes, as noted in the Exocarta database.
148
No secreted protein was common to all 12 nematode secretomes compared (Supplementary
149
Table S5). Glutathione S-transferase was reported to be secreted by 9 of the compared
150
species, and enolase in 8. Actin, triosephosphate isomerase, aldolase and galectins were
151
found in the secreted particles of T. muris and 7 other parasitic nematodes. All are common
152
exosomal proteins.
153
In agreement with published H. p. bakeri data 27 (Fig 1) and other nematode protein
154
secretomes reported from our laboratory 18,27–29, the GO terms “binding” (GO:0005488) and
155
“catalytic activity” (GO:0003824) were the most frequent level 2 molecular function
156
categories. Under “biological process” (level 2), the terms “cellular process” (GO:0009987),
157
“metabolic process” (GO:0008152) and “single-organism process” were the major groups.
158
Indeed, the biological processes in play presented some differences with H. p. bakeri, for
159
which the three main categories were “multicellular organismal process”, “developmental
160
process”, and “biological regulation”. Secretory protein profiles appeared relatively similar in
161
T. muris (vesicular component), H. p. bakeri, and B. malayi; terms attributed to H. p. bakeri
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proteins resembled B. malayi more than T. muris.
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Figure 1. Gene ontology analysis of the identified T. muris proteins compared to other nematode secretomes. The distribution of level 2 GO terms for biological processes (A) and molecular functions (B), as assigned by Blast2GO. H. p. bakeri and B. malayi GO distribution data and the top 3 (level 2) GO terms for A. suum and D. immitis were retrieved from previous reports 18,27–29.
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The distribution of level 4 biological process and molecular function terms for T. muris
171
closely follows the pattern observed for B. malayi and D. immitis secreted proteins 18,29
172
(Supplementary Fig S2).
173 174 175 176
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ACCEPTED MANUSCRIPT 3. Discussion
178
We present excretory/secretory miRNA and protein profiles of the murine gastrointestinal
179
parasitic nematode T. muris. After subjecting concentrated culture media to a polymer-based
180
vesicle precipitation, we identified 14 high confidence miRNA candidates and 73 proteins.
181
Because worms were washed thoroughly before incubation in culture media, we did not
182
expect a significant contribution from the host. However, since some miRNA candidate
183
sequences and proteins were predicted to be of mouse origin, it is possible that some mouse
184
intestinal tissue was removed with the worms and remained despite washes, releasing host
185
material/vesicles into culture media. However, the possibility that host miRNAs and proteins
186
are taken up by the worm and packaged into worm-derived vesicles cannot be dismissed.
187
Similarly, it is possible that some worms may have been damaged upon removal from the
188
host, releasing non-secretory material into the culture media.
189
The overlap between miRNAs from both mouse gastrointestinal nematodes (T. muris and H.
190
p. bakeri (both Buck and colleagues’ 4 and current datasets) was minor: of all T. muris
191
miRNAs (high and low confidence), only four were 100% identical or varied in length only in
192
the 3’ end compared to H. p. bakeri miRNAs (let-7-5p, miR-9, miR-124, and miR-79). miR-
193
100a, miR-34, and miR-252 presented minor substitutions compared to H. p. bakeri miRNA
194
sequences. miR-100a, the most abundant candidate in T. muris-derived particles of the size
195
of exosomes, was also the second most abundant candidate in D. immitis-infected dog blood
196
8
197
from our H. p. bakeri dataset, but was present in the previous work 4. miR-9 and miR-252
198
were abundant in exosome-like vesicles from B. malayi L3 5 but remain functionally
199
uncharacterized in nematodes. In mouse intestinal tissue, the most abundant miRNAs are
200
miR-192, followed by miR-215, and members of the let-7 family 30. In both T. muris and H. p.
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bakeri culture media, neither miR-192 nor miR-215 ranks among the 30 most abundant
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sequences.
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. Among the 14 high confidence miRNAs present in T. muris media, miR-252 was absent
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sequence identity prevents the determination of the relative contribution by host vs. parasite.
205
miR-124 was described in mouse intestine among other tissues 31. However, not all host-
206
identical miRNA species found in T. muris exosome-like particles are reported to occur in
207
murine intestinal tissue. For instance, miR-9 and miR-99a (identical to asu-miR-100a-5p_R-
208
3_1ss16AT) are specifically brain-enriched miRNAs in the mouse 31. Others, like let-7a, are
209
expressed in many mouse tissues 31. Besides well-described roles in developmental timing
210
in C. elegans, let-7a acts as a regulator of the murine and human immune systems. For
211
example, let-7a directly inhibits the expression of the pro-inflammatory cytokine IL-6 and has
212
been linked to cancer 32.
213
If most mammalian miRNA targets are highly conserved 33, it is also true for some miRNAs
214
conserved across nematodes 14. Nearly 50 % (6/14) of the high-confidence miRNAs found in
215
the T. muris culture media shared sequence identity with mouse miRNAs. In a context of
216
host-pathogen crosstalk and of host manipulation by the parasite via miRNA transfer, as has
217
been suggested 4,5, the extent of the overlap between host and parasite miRNAs (or seed
218
sequences) may reflect host-parasite specificity. It is worth noting that several secreted
219
parasite miRNAs (i.e., miR-9 and some members of the miR-100 family) are more conserved
220
between B. malayi and its human host than with C. elegans 5. In line with this, a number of
221
H. p. bakeri miRNAs released in exosomes were identical or homologous to mouse miRNAs
222
and there is evidence that these are used to modulate host immune functions 4. Clearly,
223
more investigations are needed to draw firm conclusions in this respect.
224
mmu-miR-26a-5p was the most frequently detected mouse miRNA, whereas it is only the 8th
225
most highly expressed murine gut miRNA 30. The intestinal epithelium expresses relatively
226
high levels of miR-26a, which represses a number of proliferation-associated genes in this
227
tissue, including genes involved in tumorigenesis 34.
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ACCEPTED MANUSCRIPT We detected only a fraction of the released miRNAs previously reported for H. p. bakeri 4,
229
which may be due to different vesicle/particle isolation protocols between the studies, the
230
number of worms used, or the duration of incubation in culture media (2 days versus 3
231
weeks). Nonetheless, all high-confidence miRNA sequences identified in the current work
232
were consistent. These results suggest that miRNA secretion is a robust phenomenon for
233
nematodes in culture, and that high-confidence sequences obtained using different methods
234
and equipment are comparable.
235
Only 41 % of proteins released by T. muris in vesicles/particles were predicted to be
236
secreted by SignalP or SecretomeP. However, many of the remaining protein sequences
237
have been reported in E/S products from other nematodes, as described in Supplementary
238
Table S5. Including hits from our search in Exocarta, 67 % of these proteins have been
239
associated with secretion and/or exosomal transport, near the proportion reported for B.
240
malayi larval exosome-like vesicles 5. We compared T. muris-derived proteins with 14
241
published secretomes of nematodes classified in clades I, III, or V. Most proteins released by
242
T. muris have been described in other nematode secretomes and overall, there was a high
243
degree of conservation of secreted proteins at the functional level. Although we lack visual
244
evidence for the presence of exosome-like vesicles in the T. muris sample, typical vesicular
245
proteins were well represented in our sample, dominated by cytoskeletal proteins, metabolic
246
enzymes, zeta polypeptide, heat shock proteins, etc.35 . This is in line with recent evidence
247
that different species and stages of nematodes release microvesicles 4,5,36. Heat shock
248
protein 70, GAPDH, ezrin, moesin, actin and tubulin are common cargo of many different
249
types of vesicles 37. A few known immunomodulators were recognized in this study, among
250
them a galectin and a glutathione-S-transferase 38. We identified ten enzymes involved in
251
energy metabolism (glycolysis, tricarboxylic acid cycle, and gluconeogenesis). Among them,
252
the well-known exosomal proteins aldolase, enolase and triosephosphate isomerase are
253
commonly observed in nematode E/S products 18,28,29,39–41. Trypsins were described in the
254
secretome of T. spiralis, another clade I nematode 42, and H. p. bakeri 40. Hasnain and
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256
proteins with trypsin domains that could have a similar biological function. A Von Willebrand
257
factor-like protein has also been described to be secreted proteins by the liver fluke Fasciola
258
hepatica 44; its role remains to be explored. Thirty-nine % of the level 2 GO terms describing
259
molecular functions indicated catalytic activity. Level 4 GO terms revealed peptidase activity
260
and serine hydrolase activity, among others. The importance of helminth proteases at all
261
stages of infection has been widely acknowledged 45, and in T. muris chronic infections, they
262
degrade mucins which may confer protection from parasite expulsion 43.
263
Because experimental differences (vesicle/particle precipitation, technical aspects in LC-MS
264
analysis and amount of protein analysed for these studies) impact on the outcome, a direct
265
cross-study comparison is both qualitatively and quantitatively difficult. However, available
266
data obtained by gene ontology predict that more T. muris proteins are functionally similar to
267
the B. malayi (up to 26 5,18,19), N. brasiliensis (19 46), and D. immitis (16 29) secretomes than
268
to the H.p. bakeri (14 27) secretome and that of the phylogenetically closer species T. spiralis
269
muscle larvae 42.
270
A major difference between the secretions from the two gastrointestinal nematodes H. p.
271
bakeri and T. muris is the absence of venom allergen-like proteins in the T. muris sample
272
compared to their predominance in the material from H. p. bakeri 27,40, and other gut
273
parasites such as A. caninum and H. contortus 41,47, all of which are clade V nematodes.
274
Similarly, these proteins are absent in secretions from A. suum 28. If venom allergens are
275
present in the genus Trichuris, they are only distant homologues of H. polygyrus, B. malayi,
276
or Ancylostoma venom allergen proteins, as revealed by a BLAST analysis yielding poor E-
277
values (not shown). The role of nematode venom allergen-like proteins is unclear, and the
278
functional significance of their apparent absence from T. muris remains to be resolved.
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4. Conclusion 13
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282
which are identical to miRNAs released by another mouse gastrointestinal parasitic
283
nematode, H. p. bakeri. Some features in the ES protein profiles of the vesicular component
284
we present here are common to other parasitic species, and may represent a general
285
secretomic signature of parasitic nematodes. Differences in the composition of secreted
286
material may reflect both methodological differences and/or the evolutionary distance
287
between T. muris and the compared nematode species.
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5. Methods
290
5.1. Ethics statement
291
All experimental protocols were approved, either by the Animal Ethics Committee of McGill
292
University and the Canadian Council on Animal Care or by the veterinary authorities of the
293
Canton Basel-Stadt, Switzerland (licence no. 2070), based on Swiss regulations.
294
Experiments were conducted in accordance with the approved guidelines.
295
5.2. Parasite maintenance and culture
296
The T. muris life cycle was maintained as described 48. Dexamethasone-treated (1 mg/l
297
drinking water, Sigma-Aldrich) C57BL/10 female mice (Harlan, Blackthorn, UK) were
298
infected orally with 200 embryonated eggs and maintained for 6 weeks until sacrifice (CO2)
299
and dissection. Approximately 3000 adult T. muris were washed and cultured for 18-48 h at
300
37°C, in filter-sterilized RPMI 1640 supplemented with 100 U/ml penicillin G, 100 µg/ml
301
streptomycin and 2.5 µg/ml amphotericin B at a concentration of 10-20 worms/ml. Media
302
were pooled and centrifuged at 5000 x g for 10 min prior to concentration on 3000 Da
303
centrifugal filter units (Amicon Ultracel, Millipore), centrifuged at 8000 x g for 10 min and
304
stored at -20°C.
305
5.3. Vesicle isolation, RNA and protein purification
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307
Biosciences). Total RNA and protein were subsequently isolated with a kit (Norgen Biotek,
308
#24100, “blood” protocol), yielding 6.5 µg RNA. For proteins, two consecutive column
309
elutions were performed, resulting in 565 and 661 µg of protein, respectively.
310
5.4. RNA sequencing
311
RNA quality was assessed by LC Sciences, and Illumina deep-sequencing of small RNAs
312
was performed (50 base pair single-end reads) following a proprietary pipeline using 1 µg
313
RNA. Low-quality sequences and other RNA populations (Rfam, Repbase; 49,50 were
314
removed and the remaining sequences of 18-24 nucleotides in length were mapped to the T.
315
muris genome
316
(ftp://ftp.sanger.ac.uk/pub/project/pathogens/Trichuris/muris/genome/version_2b/Tmuris_v2b
317
.genome_scaffolds.fa), mRNA
318
(ftp://ftp.sanger.ac.uk/pub/project/pathogens/Trichuris/muris/genes/geneSet2.1_Feb2013.nu
319
cl.fasta), and nematode miRNAs/pre-miRNAs in miRBase v.21.0 51. Next, sequences were
320
mapped to the mouse genome (M. musculus). Since no entry exists in miRBase for T. muris,
321
high confidence candidates are defined by successful mapping to the worm genome,
322
homology with other nematode miRNAs and the capacity of the extended genome region to
323
form a hairpin. Raw data are available from the NCBI GEO database at
324
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93667.
325
5.5. Proteomic analysis
326
T. muris proteins from the vesicular component were analysed by liquid chromatography-
327
tandem mass spectrometry (LC-MS/MS) at the Quebec Genomics Centre proteomics
328
platform, University Laval, for proteomics analysis.
329
An overnight tryptic digestion was performed on 1.0 µg protein in solution and analysed as
330
described 52. Reverse-phase nanoscale capillary liquid chromatography (Agilent 12000 nano
331
pump) was employed to separate peptides before analysis by electrospray tandem MS/MS
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333
Objective, Woburn, MA, USA) packed with Jupiter (Phenomonex) 5u, 300A C18 served for
334
separation and were eluted with a linear gradient of acetonitrile (2-30 %) solvent, 0.1 %
335
formic acid. Spectra were acquired using Analyst v.1.6 and analysed with Mascot (Matrix
336
Science, London, UK; v. 2.4.1) and X! Tandem (The GPM, thegpm.org; version CYCLONE
337
(2010.12.01.1)). The TAX_MusMusculus_10090_20141118 database (unknown version,
338
116602 entries) and the TAX_Trichuris_36086_20141217 database were used. Scaffold (v.
339
4.4.1.1, Proteome Software Inc., Portland, OR, USA) enabled the validation of MS/MS-
340
based peptide and protein identities. For peptide identification, >16.0 % probability to
341
achieve a false discovery rate (FDR) <1.0 % was set. Protein identities were validated when
342
>99.0 % probability to achieve a FDR <1.0 % and a minimum of 2 identified peptides were
343
achieved. Protein probabilities were assigned by the Protein Prophet algorithm 53. Proteins
344
containing undistinguishable peptides were grouped to satisfy the principles of parsimony.
345
Protein relative abundance (within the sample only) was assessed using the normalized
346
spectral abundance factor in Scaffold (NSAF). Protein descriptions for UniProt accession
347
numbers were obtained in Blast2GO (v. 3.0.8) 54. Both protein names from Scaffold and
348
sequence descriptions obtained from Blast2GO were used for comparison in published
349
nematode secretomes 5,18,19,27–29,40–42,46,47 and the Litomosoides sigmodontis predicted
350
secretome 39. The protein names or Blast2GO descriptions were submitted to Exocarta 35
351
(accessed on 28th of December 2016), in search of reported associations with exosomes in
352
mammals. Secretory properties were further evaluated using SignalP (v. 4.1) and
353
SecretomeP (v. 2.0) 55,56.
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6. References
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1. Rana, T. M. Illuminating the silence: understanding the structure and function of small
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3. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel
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4. Buck, A. H. et al. Exosomes secreted by nematode parasites transfer small RNAs to
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mammalian cells and modulate innate immunity. Nat. Commun. 5, 5488 (2014).
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13. Coakley, G., Maizels, R. M. & Buck, A. H. Exosomes and other extracellular vesicles: the new communicators in parasite infections. Trends Parasitol. 31, 477–489 (2015).
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14. Tritten, L. & Geary, T. G. MicroRNAs of filarial nematodes: a new frontier in host-
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Acknowledgments
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We are grateful to Daniel Defoy from the plate-forme protéomique du Centre Génomique de
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Québec, Université Laval for assistance with the proteomics analyses.
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We thank the Canadian Institutes for Health Research (fellowship # 320382 to LT, and grant
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# MOP130369 to MMS), the Canada Research Chairs, Canadian Natural Sciences and
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Engineering Research Council (to TGG), the Centre for Host-Parasite Interactions (Fonds de
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Recherche du Québec – Nature et Technologies), and the European Research Council
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(ERC-2013-CoG 614739-A_HERO to JK) for funding and support.
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504 Authors’ contributions
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LT, AJ, and TGG designed the study. LT, MT, and MV performed the experiments. MMS and
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JK provided the parasite material. LT analysed the data and wrote the first draft of the
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manuscript, AJ, MMS, JK and TGG revisited the manuscript critically. All authors read and
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approved the final manuscript.
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Additional information
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Competing financial interests
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The authors declare that they have no competing interests.
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Supplementary Figure S1. NanoSight measurement of microvesicles in H. p. bakeri culture media. Size distribution of observed microvesicles obtained by dividing the average size by the concentration. Red bars indicate ±1x standard error of the mean
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Supplementary Figure S2. Level 4 GO terms distribution for T. muris secreted proteins. A) biological process, B) molecular functions.
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11. Legends to supplementary files submitted separately:
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Supplementary File 1. Methods and Results to H. p. bakeri miRNA sequencing.
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into clusters according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S2. Mouse miRNAs found in T. muris exosomes. miRNA
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candidates were also mapped to the host genome and similarly grouped into clusters
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according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S3. H. p. bakeri miRNA candidates from isolated exosomes.
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miRNA candidates were grouped into clusters according to genome mapping and homology
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Supplementary Table S4. Mouse miRNAs found in H. p. bakeri exosomes. miRNA
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candidates were also mapped to the host genome and similarly grouped into clusters
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according to genome mapping and homology to known nematode miRNAs.
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Supplementary Table S5. T. muris excretory/secretory proteins. Detailed information on
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T. muris and mouse proteins identified by mass spectrometry in culture media in two
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samples. For predicted T. muris proteins, a Blast2GO description is given, as well as
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predictions for secretion and presence in other nematode secretomes.
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Highlights We characterized the microRNAs and proteins in vesicles released by Trichuris muris.
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Fourteen miRNAs were identified in T. muris exosome-like vesicles.
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We found 73 proteins of nematode origin, 11 of which were unique to this study.
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