Transcriptomic analysis of subarachnoid cysts of Taenia solium reveals mechanisms for uncontrolled proliferation and adaptations to the microenvironment

Abstract

Subarachnoid neurocysticercosis (SANCC) is caused by an abnormally transformed form of the metacestode or larval form of the tapeworm Taenia solium. In contrast to vesicular parenchymal and ventricular located cysts that contain a viable scolex and are anlage of the adult tapeworm, the subarachnoid cyst proliferates to form aberrant membranous cystic masses within the subarachnoid spaces that cause mass effects and acute and chronic arachnoiditis. How subarachnoid cyst proliferates and interacts with the human host is poorly understood, but parasite stem cells (germinative cells) likely participate. RNA-seq analysis of the subarachnoid cyst bladder wall compared to the bladder wall and scolex of the vesicular cyst revealed that the subarachnoid form exhibits activation of signaling pathways that promote proliferation and increased lipid metabolism. These adaptions allow growth in a nutrient-limited cerebral spinal fluid. In addition, we identified therapeutic drug targets that would inhibit growth of the parasite, potentially increase effectiveness of treatment, and shorten its duration.

Figure 1

Over-activation of signaling pathways that promotes cell proliferation in the bladder wall of subarachnoid cysts. (A) Quantitative gene expression for egf receptor, tgf-β receptor 1 isoforms, and transcription factor e2f4 in subarachnoid and vesicular cysts by qPCR. Significant overexpression in the evaluated genes was observed in the subarachnoid cysts. Statistically significant differences are indicated by asterisks (Mann–Whitney U test). Asterisks represent level of significance: ***p < 0.001; **p < 0.01. (B) Localization of phospho-AKT and (C) Localization of phospho-SMAD2 in the bladder wall of the subarachnoid (upper) and vesicular cysts (middle and lower) by IF. Confocal microscopy images of representative samples; positive cells were observed only in the bladder wall of subarachnoid cysts (red arrowheads). White scale bar: 20 µm, ×20, zoom4.

Figure 2

In vitro effects of EGF and TGF-β on germinative cells proliferation. (A) Representative images of germinative cells isolated from subarachnoid cysts and cultured for 15 days. (B) Characterization of cell cultures by FISH and IF. An LNA probe was designed to evaluate the expression of the germline marker argo2 together with SMAD2 phosphorylation. Representative images of confocal microscopy; white scale bar: 10 µm. (C) Cell growth curves of germinative cells isolated from subarachnoid cysts. EGF at 100 ng/mL produced a significant increase in cells after 72 h (black asterisks), TGF beta at 0.01 ng/mL after 24 h (green asterisks). Finally, the combined use of EGF and TGF at half concentration produced a significant increase in cell growth after 24 h (blue asterisks). Asterisks indicate statistically significant differences between groups (treated and control). One-way ANOVA test (***p < 0.001; ****p < 0.0001).

Figure 3

Increased expression and synthesis of extracellular matrix components in the bladder wall of subarachnoid cysts. (A) Localization of collagen in the bladder wall of subarachnoid and vesicular cysts by Masson's trichrome stain. Light microscopy images of representative samples at ×4 (upper) and ×40 (lower); blue-stain collagen fibers were observed in the bladder wall of subarachnoid cysts (red arrowheads). Red scale bar: 200 µm, black scale bar: 100 µm. BW: Bladder wall. (B) Quantitative gene expression of collagen IV in subarachnoid and vesicular cysts. Significant overexpression was observed in the subarachnoid cysts. Statistically significant differences are indicated by asterisks (Mann–Whitney U test). Asterisks represent level of significance: ***p < 0.001.

Figure 4

Increased lipid uptake and metabolism in the bladder wall of subarachnoid cysts. (A) Localization of TSCD36 in the bladder wall of the subarachnoid (upper) and vesicular cysts (middle and lower) by IF. Confocal microscopy images of representative samples; positive cells were observed only in the bladder wall of subarachnoid cysts (red arrowheads). White scale bar: 20 µm, ×20, zoom4. (B) Identification of lipid droplets in the bladder wall of the subarachnoid (upper) and vesicular cysts (middle and lower) by Nile red staining. Confocal microscopy images of representative samples; a high amount of lipid droplets were observed in the bladder wall of subarachnoid cysts (white arrowheads). White scale bar: 20 µm, ×20, zoom4. (C) Quantitative gene expression of tscd36, and long chain fatty acid CoA ligase 5 in subarachnoid and vesicular cysts. Significant overexpression in the evaluated genes was observed in the subarachnoid cysts. Statistically significant differences are indicated by asterisks (Mann–Whitney U test). Asterisks represent level of significance: ***p < 0.001; **p < 0.01.

Figure 5

Schematic representation of molecular changes associated with cell proliferation through the activation of signaling pathways, and the uptake/metabolism of lipids in subarachnoid cysts of T. solium.