A multistrain approach to studying the mechanisms underlying compatibility in the interaction between Biomphalaria glabrata and Schistosoma mansoni

Abstract

In recent decades, numerous studies have sought to better understand the mechanisms underlying the compatibility between Biomphalaria glabrata and Schistosoma mansoni. The developments of comparative transcriptomics, comparative genomics, interactomics and more targeted approaches have enabled researchers to identify a series of candidate genes. However, no molecular comparative work has yet been performed on multiple populations displaying different levels of compatibility. Here, we seek to fill this gap in the literature. We focused on B. glabrata FREPs and S. mansoni SmPoMucs, which were previously demonstrated to be involved in snail/schistosome compatibility. We studied the expression and polymorphisms of these factors in combinations of snail and schistosome isolates that display different levels of compatibility. We found that the polymorphism and expression levels of FREPs and SmPoMucs could be linked to the compatibility level of S. mansoni. These data and our complementary results obtained by RNA-seq of samples from various snail strains indicate that the mechanism of compatibility is much more complex than previously thought, and that it is likely to be highly variable within and between populations. This complexity must be taken into account if we hope to identify the molecular pathways that are most likely to be good targets for strategies aimed at blocking transmission of the parasite through the snail intermediate host.

Fig 1. Compatibility trials between different strains of parasites and snails.

Each pairwise combination of the studied strains of schistosomes (SmLE, SmVEN, SmBRE, and SmGH2) and snails (BgBAR, BgVEN, BgBRE, and BgGUA) were tested for compatibility. The upper graphs present the prevalence (P in %) and intensity (I) of infection for each S. mansoni in the different B. glabrata host strains. The lower graphs present the prevalence and intensity of infection for each B. glabrata strain when confronted by the four S. mansoni strains. Prevalence values are represented by colored histograms, while intensity values are indicated by black squares. The sympatric snails and schistosomes bear the same color. The presented data represent the mean values obtained for three independent experiments, the data obtained by (Theron et al, 2014) and two other experiments performed in 2010 and 2012. Error bars represent the average absolute deviation of the mean.

Fig 2. Diverse SmPoMucs are expressed by the different strains of S. mansoni.

(A) SmPoMuc cDNA structure. The 5’ region is characterized by a variable number of tandem repeats. Two different types of repeats (r1 and r2) were mainly identified in previous studies [19,20]. SmPoMuc cDNA structure is shared by previously published SmBRE, and SmGH2 strains [19,20] and the new ones, SmLE and SmVEN. The 3’ region (exon 3 to exon 15) harbors sequence differences that enable the proteins to be classified into the only three groups (groups 1, 2, and 3.1) known to be expressed [19,20]. (B) Transcription patterns of SmPoMucs in 11 individuals of each schistosome strain. Nested RT-PCR amplicons were obtained from individual sporocysts (1 to 11) of SmLE, SmVEN, SmBRE, and SmGH2, and resolved by agarose gel electrophoresis. PCR was performed using consensus primers that amplified the complete coding sequence of all SmPoMuc transcripts (located in exon 1 and exon 15; vertical and dashed lines represent the positions of the primers used for PCR and nested PCR, respectively). “C-” denotes the negative control. SmBRE and SmGH2 nested PCR results were from [21] (C) Inter-strain polymorphism at the protein level. Protein extracts (8 μg) were obtained from sporocysts of each S. mansoni strain, and Western blot analysis was performed using anti-SmPoMuc polyclonal antibodies. Actin was detected as a control.

Fig 3. SmPoMuc variant numbers in the different S. mansoni strains.

Nested RT-PCR for SmPoMucs was performed on 11 individual sporocysts for each S. mansoni strain (SmLE, SmVEN, SmBRE, and SmGH2), and the obtained fragments were cloned and analyzed by Sanger sequencing (see S1 Table). The numbers of SmPoMuc cDNA variants obtained for group 1 (red), group 2 (blue), group 3.1 (purple), group 3.1(r1-r2) (yellow), and all groups (black) are indicated.

Fig 4. SmPoMuc expression between S. mansoni strains.

(A) Quantitative PCR was performed with primers that targeted all SmPoMuc transcripts or discriminated among groups 1, 2, 3.1, and 3.1(r1-r2). The primer positions and amplicon sizes are indicated. (B) RNA was extracted from pooled sporocysts of each S. mansoni strain (SmLE, SmVEN, SmBRE, and SmGH2). The results are presented as the mean Ct values normalized with respect to the α-tubulin gene, and were obtained from three biological replicates.

Fig 5. RNA-seq of the different B. glabrata strains, and enrichment analysis.

Venn diagram presenting the transcripts found to be over-represented (A) and under-represented (B) in the transcriptomes of BgBAR, BgVEN, and BgGUA versus BgBRE. Gene Ontology (GO) enrichment analysis of genes that were over-represented (C) and under-represented (D) in the transcriptome of BgBAR. Red arrows indicate biological processes belonging to the “response to biotic and abiotic stress” group, while the green arrow shows an immune-response-related biological process.

Fig 6. Analysis of immune-relevant genes differentially represented between BgBAR, BgVEN, BgGUA and BgBRE reference transcriptome.

(A) Histograms showing the proportion of genes bearing immune-relevant protein domains that were differentially represented (DESeq 2 analysis) in BgBAR (yellow), BgVEN (green) and BgGUA (blue) versus BgBRE. The monitored immune-relevant genes include immune recognition molecules, immune signaling proteins, and immune effectors. Within these categories, the genes were subdivided by shared protein domains (defined by Interproscan analysis of predicted protein sequences). Ig-fold: immunoglobulin-like fold, IPR013783. CTL: C-type lectin, IPR001304. FBG: fibrinogen-related domains, IPR002181. GLECT: galectin, IPR001079. PGRP: animal peptidoglycan protein homolog, IPR006619. LRR: leucine-rich repeat, IPR001611. SR: scavenger receptor cysteine-rich, IPR017448. BPI: lipopolysaccharide-binding protein, IPR001124 and IPR017942. TIR: toll-interleukin 1-receptor, IPR000157. C1q: complement component C1q domain, IPR001073. TNF: tumor necrosis factor family, IPR006052. CARD: caspase recruitment domain, IPR001315. MACPF: membrane-attack complex/perforin, IPR020864. DEATH: DEATH domain, found in proteins involved in cell death, IPR000488. (B) Genes previously described to have an immune function in B. glabrata species. The heat map represents the log2 fold change values for transcripts that are differentially represented between the snail strains. The table beside the heat map classifies the corresponding genes into three immune functions: effector, recognition, and cell migration.

Fig 7. Abundances of transcripts for the different FREP classes in the four B. glabrata strains.

(A) The histograms represent normalized count values (upper quartile method) obtained when Bowtie 2 was used to map the BgBAR, BgVEN, BgBRE, and BgGUA reads with respect to the sequences of the 24 classes of FREP transcripts. Asterisks indicate FREP classes that contain only one IgSF domain. (B) The diagram shows the sum of normalized count values for all the FREPs (total) or the FREPs containing one or two IgSF domains.

Fig 8. FREP variant polymorphism analysis.

Diversity was assessed by aligning the longest transcript from each FREP class to the transcriptomes of BgBAR, BgVEN, BgBRE, and BgGUA using Blastn (similarity, at least 95%). (A) Histograms showing the number of variants normalized by the total number of transcripts for each FREP class in all B. glabrata strains. (B) For all B. glabrata strains, diagrams showing the sum of normalized FREP variant numbers based on whether they have one or two IgSF domains.