Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin

Abstract

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery in species with high anhydrobiosis capacities. However, it was difficult to distinguish whether transcriptomic changes were specific to DNA damage or mutual with anhydrobiosis. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families, including a previously identified novel anti-oxidative stress family, which may be a core component of the cross-tolerance mechanism. We also identified enrichment of the tryptophan metabolism pathway, for which metabolomic analysis suggested engagement of this pathway in stress tolerance. These results provide several candidates for the core component of cross-tolerance, as well as possible anhydrobiosis machinery.

Keywords: bleomycin; metabolome; tardigrade; transcriptome; tryptophan.

Fig. 1

(Color online) Transcriptome analysis of bleomycin-exposed H. exemplaris. [A] Experimental setup of the transcriptome analysis. H. exemplaris specimens were exposed to 100 μM bleomycin (BLM) for 24 h and were sampled at the corresponding time points. A control sample without bleomycin was also prepared. [B] PCA of transcriptome profiles. Transcripts with average expression > 1 were subjected to PCA. [C] Clustering of expression profiles. Transcripts with average expression > 1 were clustered based on Spearman’s correlation and split into eight groups based on total within sum of squares. A dynamic change was observed between 0-1 h, 9-12 h, and 12-24 h. [D] An illustration of the broader clustering of the eight groups. E, M, and L indicate the Early, Middle, and Late responses.

Fig. 2

(Color online) Comparison with anhydrobiosis entry. [A] UpSet plot showing the number of genes shared between each cluster and anhydrobiosis entry. [B] Number of GO BP terms significantly enriched between each response cluster and anhydrobiosis. [C] Features of the 102 NLS predicted transcripts without annotations, induced in anhydrobiosis and either one of clusters A, B, C. Transcripts are ordered based on the clustering performed at Supplement Fig. S1C. Z-scaled expression profiles (Expression profile), DEG profiles (DEG), averaged IUPRED3 score for protein sequence (IUPRED, estimates disordered regions within protein sequences), conservation patterns across Tardigrada and relative lineages are shown as “Conservation” (species names omitted for visualization). Specific species names can be found in the Supplementary Table S6. The red line for IUPRED indicates the average of the group. A protein sequence IUPRED3 score > 0.5 typically infer strong disordered nature of the protein.

Fig. 3

(Color online) AlphaFold structures of NLS-containing proteins. AlphaFold2 structures modeled from NLS-predicted proteins that had no SwissProt BLASTP hits (E-value > 1e-15). [AB] Structures for those that had a FoldSeek hit against the AlphaFold2 SwissProt database (E-value > 1E-05). The yellow and blue structures indicate the reference and the predicted H. exemplaris protein structures, respectively. [A] BV898_05254.p01 (5-hydroxytryptamine receptor 1E (AF-Q6VB83-F1-model_v4.pdb E-value = 6.22E-12, RMSD between 163 pruned atom pairs is 1.124 angstroms; across all 353 pairs: 10.861). [B] Ecdysone-induced protein 78C (AF-P45447-F1-model_v4 E-value = 4.48E-08, RMSD between 84 pruned atom pairs is 1.044 angstroms; across all 391 pairs: 32.283). [C-F] Transcripts that had an IUPRED3 score of 1.0. [C] BV898_03773.p01. [D] BV898_07132.p01. [E] BV898_12866.p01. [F] BV898_18010.p01.

Fig. 4

(Color online) Metabolomic profiles of active, anhydrobiotic, and bleomycin-exposed H. exemplaris. Z-scaled concentration profiles for 113 metabolites with significant differences between Control-Anhydrobiosis, Control-Bleomycin, Anhydrobiosis-Bleomycin.

Fig. 5

(Color online) KEGG pathway map of tryptophan metabolism. Data for two comparisons are indicated in a single circle (metabolite) or square (transcript); For metabolites, Left: Control vs Bleomycin; Right : Control vs Anhydrobiosis; For transcripts; Left: Control vs Bleomycin-0 h (24-h exposure, no recovery time), Right: Active vs Tun. Colors correspond to the following conditions: Gray: not detectable or not detected (0 μg/μL for each condition), Blue: decrease in expression/concentration (lower in Bleomycin/Anhydrobiosis); White: detected, but no significant difference; Red: increase in expression/concentration (higher in Bleomycin/Anhydrobiosis).