Differential expression of small RNA pathway genes associated with the <i>Biomphalaria glabrata/Schistosoma mansoni</i> interaction

<div><p>The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus <i>Schistosoma</i>. In Brazil, <i>Schistosoma mansoni</i> is the only species representative of the genus whose passage through an invertebrate host, snails of the genus <i>Biomphalaria</i>, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of <i>B</i>. <i>glabrata</i> makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of <i>B</i>. <i>glabrata</i> as well as its relationship to the parasite <i>S</i>. <i>mansoni</i>. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in <i>B</i>. <i>glabrata</i>. <i>In silico</i> analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with <i>S</i>. <i>mansoni</i>, suggesting that the machinery is required for miRNA and piRNA processing in <i>B</i>. <i>glabrata</i> at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the <i>B</i>. <i>glabrata/S</i>. <i>mansoni</i> interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.</p></div

Multiple alignment of the RIBOc domain of Drosha and Dicer proteins.

<p>The highlighted residues, D (aspartic acid) and E (glutamic acid), which are highly conserved in the RIBOc domain of <i>B</i>. <i>glabrata</i>, and their orthologues are responsible for its catalytic activity. The sequences used for the Dicer family were as follows: BGLB002125-PA (<i>Biomphalaria glabrata</i>), XP_005106232.1 (<i>Aplysia californica</i>), NP_524453.1 (<i>Drosophila melanogaster</i>), NP_683750.2 (<i>Mus musculus</i>), XP_006618601.1 (<i>Apis dorsata</i>), XP_004523314.1 (<i>Ceratitis capitata</i>), XP_003745061.1 (<i>Metaseiulus occidentalis</i>), NP_498761.2 (<i>Caenorhabditis elegans</i>), NP_001154925.1 (<i>Danio rerio</i>), XP_868526.3 (<i>Canis lupus familiaris</i>), XP_008774532.1 (<i>Rattus norvegicus</i>), NP_976235.1 (<i>Bos taurus</i>), XP_005179924.1 (<i>Musca domestica</i>) and Smp_169750.2 (<i>Schistosoma mansoni</i>). The sequences used for the Drosha family were as follows: BGLB003167-PA (<i>Biomphalaria glabrata</i>), XP_008199088.1 (<i>Tribolium castaneum</i>), XP_006618766.1 (<i>Apis dorsata</i>), XP_006558454.1 (<i>Apis mellifera</i>), XP_003394274.1 (<i>Bombus terrestris</i>), NP_477436.1 (<i>Drosophila melanogaster</i>), XP_005248351.1 (<i>Homo sapiens</i>), XP_006520084.1 (<i>Mus musculus</i>), EKC20603.1 (<i>Crassostrea gigas</i>), NP_001122460.2 (<i>Caenorhabditis elegans</i>), NP_001103942.1 (<i>Danio rerio</i>), NP_001101125.2 (<i>Rattus norvegicus</i>), XP_854135.2 (<i>Canis lupus familiaris</i>), XP_591998.4 (<i>Bos taurus</i>), XP_005186977.1 (<i>Musca domestica</i>), NP_001006379.1 (<i>Gallus gallus</i>), Smp_142510.2 (<i>Schistosoma mansoni</i>) and Sjp_0048900.1 (<i>Schistosoma japonicum</i>).</p

Domain structure of small RNA processing pathway proteins in <i>B</i>. <i>glabrata</i>.

<p><b>A—Bgl-Argonaute</b> has the following domains: ArgoN (PF16486—position 52 to 182 and E-value 8.5e^-31), ArgoL1 (PF08699—position 192 to 242 and E-value 9.8e^-24), PAZ (PF02170—position 256 to 364 and E-value 8.7e^-18), ArgoL2 (PF16488—position 373 to 419 and E-value 1.6e^-12), ArgoL2 (PF16488—position 424 to 460 and E-value 4.4e^-12), ArgoMid (PF16487—position 470 to 550 and E-value 2.4e^-34) and PIWI (PF02171—position 557 to 849 and E-value 4.7e^-104); <b>B—Bgl-Piwi</b> has the following domains: PAZ (PF02170 –position 274 to 404 and E-value 1.3e^-32) and PIWI (PF02171—position 547 to 839 and E-value 6.2e^-98); <b>C—Bgl-Drosha</b> has the following domains: Ribonucleas_3_3 (PF14622—position 923 to 1030 and E-value 1.3e^-21) and DSRM (PF00035—position 1081 to 1124 and E-value 2.00e^-06); <b>D—Bgl-Dicer</b> has the following domains: Helicase_C (PF00271—position 423 to 503 and E-value 5.4e^-12), Dicer_dimer (PF03368—position592 to 682 and E-value 1.3e^-24), PAZ (PF02170—position899 to 1050 and E-value 1.2e^-33), Ribonuclease_3 (PF00636 –position 1665 to 1842 and E-value 8.2e^-34) and Ribonuclease_3 (PF00636—position1951 to 2086 and E-value 9.7e^-22).</p

Phylogenetic tree of Bgl-Argonaute and Bgl-Piwi with their orthologues.

<p>The phylogenetic analysis was performed using Mega 5.2 with bootstrap analysis. Bootstrap percentages are indicated at each branch.</p

Relative gene expression of <i>B</i>. <i>glabrata</i> at several time points of infection by <i>S</i>. <i>mansoni</i> using uninfected snails at the same time points as a baseline.

<p><b>A—Argonaute</b> was significantly down-regulated 4 hours after infection compared to 24 hours, 7 days and 21 days after infection; <b>B—Piwi</b> was significantly down-regulated at 4 hours compared to 24 hours and 7 days but was significantly up-regulated at 7 days compared to 15 days; <b>C—Drosha</b> was significantly down-regulated at 4 hours compared to 12 hours and 7 days but was significantly up-regulated at 7 days compared to 30 days; <b>D—Exportin-5</b> was significantly down-regulated at 4 hours compared to 7 days and 21 days; and significantly up-regulated at 21 days compared to 15 and 30 days; <b>E—Tudor</b> was significantly down-regulated at 4 hours compared to 24 hours and significantly down-regulated at 12 hours compared to 24 hours and 30 days.</p

Phylogenetic tree of Bgl-Drosha and Bgl-Dicer with their orthologues.

<p>The phylogenetic analysis was performed using Mega 5.2 with bootstrap analysis. Bootstrap percentages are indicated at each branch.</p

Multiple alignment of the PIWI domain of Ago-like and Piwi-like proteins.

<p>The highlighted residues, D (aspartic acid) and H (Histidine), which are highly conserved in the PIWI domains of <i>B</i>. <i>glabrata</i> and their orthologues, are responsible for the catalytic activity. The sequences used for the Ago-like family were as follows: BGLB002396-PA (<i>Biomphalaria glabrata</i>), XP_005107587.1 (<i>Aplysia californica</i>), XP_009064000.1 (<i>Lottia gigantea</i>), EKC19600.1 (<i>Crassostrea gigas</i>), XP_003747659.1 (<i>Metaseiulus occidentalis</i>), XP_004529840.1 (<i>Ceratitis capitata</i>), NP_725341.1 (<i>Drosophila melanogaster</i>), XP_006622112.1 (<i>Apis dorsata</i>), XP_005175308.1 (<i>Musca domestica</i>), XP_003400152.1 (<i>Bombus terrestris</i>), NP_001257239.1 (<i>Caenorhabditis elegans</i>), Sjp_0044720.1 (<i>Schistosoma japonicum</i>), Smp_198380.1 (<i>Schistosoma mansoni</i>), NP_067608.1 (<i>Rattus norvegicus</i>), NP_001289151.1 (<i>Danio rerio</i>), XP_005629013.1 (<i>Canis lupus familiaris</i>), XP_004940123.1 (<i>Gallus gallus</i>), NP_991363.1 (<i>Bos taurus</i>), NP_036286.2 (<i>Homo sapiens</i>) and NP_700451.2 (<i>Mus musculus</i>). The sequences used for the Piwi-like family were as follows: BGLB010170-PA (<i>Biomphalaria glabrata</i>), XP_005096149.1 (<i>Aplysia californica</i>), XP_009064630.1 (<i>Lottia gigantea</i>), EKC35279.1 (<i>Crassostrea gigas</i>), XP_001641994.1 (<i>Nematostella vectensis</i>), NP_001274302.1 (<i>Hydra vulgaris</i>), NP_476875.1 (<i>Drosophila melanogaster</i>), XP_001652945.1 (<i>Aedes aegypti</i>), XP_003400353.1 (<i>Bombus terrestris</i>), XP_005183556.1 (<i>Musca domestica</i>), NP_004755.2 (<i>Homo sapiens</i>), NP_067286.1 (<i>Mus musculus</i>), NP_899181.1 (<i>Danio rerio</i>), XP_534638.2 (<i>Canis lupus familiaris</i>), XP_008764202.1 (<i>Rattus norvegicus</i>), NP_001092322.1 (<i>Gallus gallus</i>) and XP_618020.4 (<i>Bos taurus</i>).</p

Relative gene expression for several development times in <i>B</i>. <i>glabrata</i> using the time of mass eggs as the baseline of assay for all genes.

<p><b>A—Argonaute</b> was significantly up-regulated at 20 days of development compared to 5 days; <b>B—Piwi</b> was significantly up-regulated at 10 and 40 days compared to 5 days; <b>C—Drosha</b> levels were not significantly different among the groups; <b>D—Exportin-5</b> was not significantly different among the groups; <b>E—Tudor</b> was significantly down-regulated at 10 days compared to all other times.</p

Silver-stained 6% polyacrylamide gel showing RFLP profiles.

<p>Lane 1: molecular size markers (PhiX 174); Lane 3: negative control (i.e., no template DNA); Lane 13: pool of <i>S. mansoni</i> cercariae; Lane 14: <i>B. tenagophila</i> Taim; Lanes 6 and 9: profile of the local strain of <i>B. tenagophila</i>; Lanes 2, 4–5, 7–8 and 10–12: profiles of <i>B. tenagophila</i> collected 14 months after Taim introduction (i.e., containing the 350 bp marker).</p

Susceptibility of <i>B. tenagophila</i> collected before and after the introduction of the resistant lineage to infection after exposure to the SJ strain of <i>S. mansoni</i>.

<p>PI: post-introduction.</p><p>*Significant differences between <i>B. tenagophila</i> collected before and after the introduction of the resistant lineage (<i>p</i><0.05). Pearson’s chi-square test which is suitable for comparisons between proportions, and Fisher’s exact test were used at frequencies lower than 5.</p