Comparative Study of Transcriptome Profiles of Mechanical- and Skin-Transformed <em>Schistosoma mansoni</em> Schistosomula

<div><p>Schistosome infection begins with the penetration of cercariae through healthy unbroken host skin. This process leads to the transformation of the free-living larvae into obligate parasites called schistosomula. This irreversible transformation, which occurs in as little as two hours, involves casting the cercaria tail and complete remodelling of the surface membrane. At this stage, parasites are vulnerable to host immune attack and oxidative stress. Consequently, the mechanisms by which the parasite recognises and swiftly adapts to the human host are still the subject of many studies, especially in the context of development of intervention strategies against schistosomiasis infection. Because obtaining enough material from <i>in vivo</i> infections is not always feasible for such studies, the transformation process is often mimicked in the laboratory by application of shear pressure to a cercarial sample resulting in mechanically transformed (MT) schistosomula. These parasites share remarkable morphological and biochemical similarity to the naturally transformed counterparts and have been considered a good proxy for parasites undergoing natural infection. Relying on this equivalency, MT schistosomula have been used almost exclusively in high-throughput studies of gene expression, identification of drug targets and identification of effective drugs against schistosomes. However, the transcriptional equivalency between skin-transformed (ST) and MT schistosomula has never been proven. In our approach to compare these two types of schistosomula preparations and to explore differences in gene expression triggered by the presence of a skin barrier, we performed RNA-seq transcriptome profiling of ST and MT schistosomula at 24 hours post transformation. We report that these two very distinct schistosomula preparations differ only in the expression of 38 genes (out of ∼11,000), providing convincing evidence to resolve the skin vs. mechanical long-lasting controversy.</p> </div

Mitochondrial activity is higher in ST schistosomula.

<p>AlamarBlue reactivity of 24-hour old MT and ST schistosomula incubated for either 3 hours (blue) or 24 hours (green). Longer incubation time in AlamarBlue provided the necessary resolution to establish a significant difference (<i>t-</i>test, p-value<0.01, indicated with * ) in metabolic activity between the mechanically (M) and skin (S) transformed schistosomula; (B) Blank wells.</p

RT-qPCR and RNA-seq fold change values are highly correlated.

<p>Bar plots and inset scatter plot show a comparison of RNA-seq and RT-qPCR fold change values obtained for 33 differentially expressed genes. Fold change values are represented in the log₂ scale and error bars represent 95% confidence interval (fold change ±1.96xSD). The inset scatter plot highlights the high correlation found between both methods (Pearson's correlation = 0.89, p-value = 3.85E⁻¹²). The solid lane represents the linear regression of this correlation; as a guide the x = y correlation is also shown as a dashed line.</p

The microexon gene Smp_124000 is expressed as different isoforms in the ST and MT schistosomula.

<p>The exons or lack of them that contribute to the new isoform are marked in dashed boxes while exons that are differentially expressed between the ST and MT schistosomula are marked with a star (*).</p

Diagram and photographs of assemblies used in skin-transformation of schistosomula.

<p>A – Graphical representation of a transformation assembly. B – Photograph of one of the transformation assembly prior to use. C – Three transformation assemblies in use during an experiment. The lower compartment of the assembly is placed in a water bath with a constant temperature of 37°C while the upper compartment is left at a room temperature (28°C).</p

Genes with higher expression in skin-transformed schistosomula.

<p>Differentially expressed genes with adjusted p-value<0.05 are shown. Gene identification numbers with a prefix “Smp_90” are those encoded in the mitochondrial genome.</p

Summary of sequenced samples and alignment to the <i>S. mansoni</i> reference genome.

<p>MT: mechanically transformed; ST: skin-transformed.</p

Improving the mechanical transformation protocol.

<p>A – Parasites were transformed according to a standard protocol <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002091#pntd.0002091-Brink1" target="_blank">[10]</a> where parasites are subjected to 23 passages through a syringe needle and separation of heads and tails is performed with 60% Percoll solution at room temperature. B – Transformation of parasites was performed using the optimized protocol described in Material and Methods; which involved shaking of the cercariae suspension in a vortex mixer followed by only 12–13 passages through a syringe needle. Separation of schistosomula/heads from tails was performed using 70% ice-cold Percoll solution. Arrows indicate damaged schistosomula, arrowheads point to contaminating cercariae or tails. Both panels (A and B) show light microscope images of 3-hours post-transformation schistosomula.</p

Skin-transformed (ST) and mechanically-transformed (MT) schistosomula are transcriptionally very similar.

<p>A – Correlation of gene expression values between MT (x-axis) and ST (y-axis) schistosomula. Both Pearson and Spearman's correlations are high (0.98 and 0.99 respectively) indicating very low variability between these two samples. B – Differential gene expression (MA plot) between MT and ST parasites at 24 hours after transformation (adjusted p-value<0.05). Relative concentration (x-axis) is plotted against fold change values (y-axis) in the log₂ scale. Positive log₂ fold changes represent transcripts more expressed in ST schistosomula while negative log₂ fold changes represent transcripts more expressed in MT schistosomula.</p

Genes with higher expression in mechanically transformed schistosomula.

<p>Differentially expressed genes with adjusted p-value<0.05 are shown.</p