Antimicrobial capacity of the freshwater planarians against S. aureus is under the control of Timeless

Abstract

Planarians, which are non-parasitic flatworms, are highly resistant to bacterial infections. To better understand the mechanisms underlying this resistance, we investigated the role of the circadian machinery in the anti-bacterial response of the freshwater planarian Schmidtea mediterranea. We identified Smed-Tim from S. mediterranea as a homolog of the mammalian clock gene Tim. We showed via RNA interference that Smed-Tim is required for the anti-microbial activities of Schmidtea mediterranea against Staphylococcus aureus infection during the light/dark cycle. Indeed, S. aureus infection leads to the expression of Smed-Tim, which in turn promotes Smed-Traf6 and Smed-morn2, but not Smed-p38 MAPK expression, 2 master regulators of planarian anti-microbial responses.

Keywords: MORN2; S. aureus; Tim; anti-bacterial response; planarians;.

Figure 1.

Predicted amino acid sequence of Smed-ARNTl-1 and Smed-TIM. Alignment of (A) Smed-ARNTL-1 with Hs-ARNTL-1 (NP_001025443.1), and (B) Smed-TIM with Hs-TIM (NP_003911.2) were performed using the PRALINE multiple sequence alignment (Center for Integrative Bioinfomatics, http://www.ibi.vu.nl/programs/pralinewww/) setup with default parameters. The results are color-coded for amino acid conservation and the scoring scheme works from 0, for the least conserved alignment position, to 10, for the most conserved alignment position.

Figure 2.

Smed-TIM silencing leads to a delay in S. aureus elimination by the planarians. (A) S. mediterranea were silenced for eGFP (control), Smed-Arntl-1, and Smed-Tim. Silenced animals do not present a particular phenotype (representative micrographs of 2 experiments; 10 animals per experimental conditions; scale bars, 150 mm). The efficiency of the gene silencing by RNAi in the S. mediterranea was determined using RT-qPCR (Figure S2A). (B, C) The S. mediterranea eGFP (RNAi), the S. mediterranea Smed-Arntl-1 (RNAi), and S. mediterranea Smed-Tim (RNAi) under D/D cycle (B) or under L/D cycle (C) were fed with S. aureus (1 × 10e bacteria), and the CFUs per animal were counted over time. In the L/D condition, the silencing of Smed-Tim via RNAi delayed the clearance of S. aureus by S. mediterranea. The results are expressed as the mean ± SD (10 animals per time point, number of experiments = 3, *p < 0.05). (D) The amount of bacteria ingested by the S. mediterranea eGFP (RNAi) and the S. mediterranea Smed-Tim (RNAi) under L/D cycle, after feeding (Time 0 day) was determined by counting the CFUs. The invalidation of Smed-Tim does not affect significantly the capacity of worms to ingest bacteria compared with S. mediterranea eGFP (RNAi). The results are expressed as the mean ± SD (10 animals per time point, number of experiments = 3).

Figure 3.

Smed-Tim is a gene of the planarians circadian clock. (A) Planarians were submitted to the condition L/D and then Smed-Tim expression was evaluated using RT-qPCR. Planarians have a maximum of Tim expression in dark phase. The results are presented as the mean ± SD (3 worms per time point processed individually in triplicate, number of experiments = 3). (B) Planarians were submitted to D/D cycle and Smed-Tim expression was evaluated using RT-qPCR. Worms submitted to D/D condition have a desynchronized Smed-Tim expression cycle. The results are presented as the mean ± SD (3 worms per time point processed individually in triplicate, number of experiments = 3). (C) The expression of Smed-Tim was evaluated at CT20 in worms submitted to L/D condition and infected by S. aureus. (D) Whole-mount in situ hybridization analysis of the expression of Smed-Tim in control and S. aureus infected animals were fixed at CT 20 (representative micrographs of 2 experiments; 5 animals per experimental conditions; scale bars, 250 µm). See also Figure S3. (E) Melatonin level was determined between CT14 and CT22 in animals eGFP (RNAi) and Smed-Tim (RNAi) under the L/D cycle level was assessed in planarian controls or silenced for Smed-Tim via RNAi. The results are presented as the mean ± SD (30 worms per time point, number of experiments = 2, *p < 0.05).

Figure 4.

Smed-Tim controls the expression of Smed-morn2 and Smed-Traf6. (A, B, C) The level of expression of (A) Smed-morn2, (B) Smed-Traf6, and (C) Smed-p38 MAPK mRNA in planarians under L/D condition and challenged with S. aureus was assessed by RT-qPCR. The results are presented as the mean ± SD (3 worms per time point processed individually in triplicate, number of experiments = 3, *p < 0.05). (D, E, F) Planarians under L/D condition were silenced for Smed-Tim via RNAi, then infected with S. aureus, and the level of mRNA expression of Smed-morn2 (D), Smed-Traf6 (E), and Smed-p38 MAPK (F) was evaluated using RT-qPCR. The expression of Smed-Morn2 and Smed-Traf6 was significantly diminished in animals which have been silenced for Smed-Tim under L/D cycle. The results are presented as the mean ± SD (3 animals per time point processed individually in triplicate, number of experiments = 3, *p < 0.05).