Identification of miRNAs of Strongyloides stercoralis L1 and iL3 larvae isolated from human stool

Abstract

Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics.

Figure 1

miRNAs identification for S. stercoralis. (A) Known and novel miRNAs were identified in S. stercoralis compared to S. ratti, S. papillosus and C. elegans. (B) RT-qPCR was used to verify the expression and modulation of miRNAs between L1 and iL3. Data are expressed as NRQ. For the comparison analysis, L1 expression value of each miRNA was set equal to 1. P value < 0.05*; < 0.01**.

Figure 2

Scatter plot of mRNA log2 fold change of iL3-vs-L1 expression values (on the y-axis) versus the mean of normalized counts (on the x-axis). Dots represent individual transcripts. Blue dots indicate the 81 modulated genes (p-adjusted ≤ 0.05).

Figure 3

Flowchart of the study.

Figure 4

Flowchart of mRNASeq and miRNASeq bioinformatic analysis.