Convergent evolution of a genotoxic stress response in a parasite-specific p53 homolog

Abstract

P53 is a widely studied tumor suppressor that plays important roles in cell-cycle regulation, cell death, and DNA damage repair. P53 is found throughout metazoans, even in invertebrates that do not develop malignancies. The prevailing theory for why these invertebrates possess a tumor suppressor is that P53 originally evolved to protect the germline of early metazoans from genotoxic stress such as ultraviolet radiation. This theory is largely based upon functional data from only three invertebrates, omitting important groups of animals including flatworms. Previous studies in the freshwater planarian flatworm Schmidtea mediterranea suggested that flatworm P53 plays an important role in stem cell maintenance and skin production, but these studies did not directly test for any tumor suppressor functions. To better understand the function of P53 homologs across diverse flatworms, we examined the function of two different P53 homologs in the parasitic flatworm Schistosoma mansoni. The first P53 homolog (p53-1) is orthologous to S. mediterranea P53(Smed-p53) and human TP53 and regulates flatworm stem cell maintenance and skin production. The second P53 homolog (p53-2) is a parasite-specific paralog that is conserved across parasitic flatworms and is required for the normal response to genotoxic stress in S. mansoni. We then found that Smed-p53 does not seem to play any role in the planarian response to genotoxic stress. The existence of this parasite-specific paralog that bears a tumor suppressor-like function in parasitic flatworms implies that the ability to respond to genotoxic stress in parasitic flatworms may have arisen from convergent evolution.

Keywords: flatworms; p53; parasitology; schistosomes.

Fig. 1.

Phylogenetic analysis of p53-1 and p53-2 orthologs. (A) Schematic of domain structure of human TP53/TP63/TP73, Amphimedon queenslandica (Aq) P53, S. mediterranea Smed-p53, and S. mansoni p53-1/p53-2. TAD, transactivation domain; DBD, DNA binding domain; TetD, tetramerization domain; SAM, sterile alpha motif. (B) Phylogenetic trees of p53-1 and p53-2 orthologs color coded by remarkable clades. Flatworm p53-1 and p53-2 orthologs are indicated with dashed lines. (C) Cartoon depicting model of p53-1 and p53-2 evolution. Dotted line with question mark indicates the possible loss of p53-2 orthologs in Monogenea.

Fig. 2.

Expression pattern of schistosome p53 homologs. (A) Colorimetric WISH showing expression patterns of p53-1 and p53-2. (B) Double FISH experiment showing expression of p53-1 relative to the tegument progenitor marker tsp-2 and the neoblast marker nanos2 as well as the expression of p53-2 relative to the proliferative cell marker h2b. (C) Uniform manifold approximation plots showing expression patterns of p53-1 and p53-2 in adult schistosomes. Red indicates high expression, orange indicates medium expression, and blue indicates low or no expression. Important cell populations are indicated. Percentage of p53-1–positive cells that are also marker positive is indicated in the upper right of appropriate panels in B. Counts were performed in three separate animals with more than 130 cells per gene comparison. (Scale bars, 100 µm [A], 10 µm [B]).

Fig. 3.

p53-1 RNAi perturbs neoblast maintenance and differentiation. (A) Double FISH of the gut neoblast marker eled and the tegument progenitor marker tsp-2 after p53-1 and p53-2 RNAi. Neoblasts are labeled with the thymidine analog EdU. (B and C) Quantification of tsp-2–positive cells and EdU-positive cells, respectively, after p53-1 and p53-2 RNAi (corresponds to 3A). (D) qPCR detection of expression of the neoblast marker nanos2 and the tegument progenitor marker tsp-2. (E) FISH of a mixture of genes that mark the tegument during p53-1 and p53-2 RNAi. Neoblast progeny are labeled with EdU via a pulse-chase experiment. (F) FISH of the gut marker ctsb after p53-1 and p53-2 RNAi. Neoblast progeny are labeled with EdU via a pulse-chase experiment. (G and H) Quantification of data from E and F, respectively. Fraction indicates number of worms that are similar to representative image. Data are from 10 animals per condition from one biological replicate (A) or >12 animals per condition from two biological replicates (E and F). *P < 0.05; ***P < 0.001; ns, not significant. Error bars indicate the 95% confidence interval. (Scale bars, 50 µm.)

Fig. 4.

p53-2 RNAi abrogates normal response to genotoxic stress. (A) EdU-labeled neoblasts following radiation after p53-1 or p53-2 RNAi. (B) Quantification of data from A. (C) EdU-labeled neoblasts following cisplatin treatment after p53-1 or p53-2 RNAi. Cis, cisplatin. (D) Quantification of data from C. Data are from >12 animals per condition from two biological replicates. Fraction indicates number of worms that are similar to representative image. Veh, vehicle. ***P < 0.001; ns, not significant. Horizontal bars indicate the conditions that are being compared with respect to statistical tests. (Scale bars, 50 µm.)

Fig. 5.

Smed-p53 RNAi does not protect planarian neoblasts from genotoxic stress. FACS plots of Smed-p53 RNAi annexin V flow cytometry experiment. Percentage of neoblasts remaining following irradiation (Upper) and percentage of apoptotic neoblasts (Lower) are indicated in the 2D flow plot and flow histogram, respectively. Graphs show representative data from one of two biological replicates. Each biological replicate used 30 RNAi-treated animals per condition.