Cysteine synthases CYSL-1 and CYSL-2 mediate C. elegans heritable adaptation to P. vranovensis infection

Abstract

Parental exposure to pathogens can prime offspring immunity in diverse organisms. The mechanisms by which this heritable priming occurs are largely unknown. Here we report that the soil bacteria Pseudomonas vranovensis is a natural pathogen of the nematode Caenorhabditis elegans and that parental exposure of animals to P. vranovensis promotes offspring resistance to infection. Furthermore, we demonstrate a multigenerational enhancement of progeny survival when three consecutive generations of animals are exposed to P. vranovensis. By investigating the mechanisms by which animals heritably adapt to P. vranovensis infection, we found that parental infection by P. vranovensis results in increased expression of the cysteine synthases cysl-1 and cysl-2 and the regulator of hypoxia inducible factor rhy-1 in progeny, and that these three genes are required for adaptation to P. vranovensis. These observations establish a CYSL-1, CYSL-2, and RHY-1 dependent mechanism by which animals heritably adapt to infection.

Fig. 1. C. elegans heritably adapts to infection by Pseudomonas vranovensis.

a Graphical representation of experimental set up. Embryos collected by bleaching were placed onto fresh plates seeded with BIGb446 immediately after egg prep and the percent of surviving animals was counted after 24 h. b Percent of wild-type animals surviving on plates seeded with either E. coli HB101 or bacterial isolates BIGb446 and BIGb468 after 24 h. Data presented as mean values ± s.d. n = 3–4 experiments of >100 animals. c Percent of wild-type animals surviving on plates seeded with bacterial isolate BIGb446. Data presented as mean values surviving at each time point from three separate experiments ± s.d. n = 3 experiments of 500 animals. Log-rank test of individual Kaplan–Meier survival curves p < 0.001. d Images of wild-type animals surviving after 120 h of feeding on bacterial isolate BIGb446. 1000 animals were used at t = 0 in each condition and surviving animals were resuspended in 20 μl M9 and imaged. Scale bars 1 mm. Experiment repeated three times with similar results. e Percent of wild-type animals surviving on E. coli HB101 or bacterial isolate BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. *p < 0.05, **p < 0.01, ****p < 0.0001. Source data are provided as a Source Data file. See statistics and reproducibility section for statistical tests run.

Fig. 2. C. elegans exposure to P. vranovensis can have multigenerational effects.

a Percent of wild-type animals surviving on plates seeded with bacterial isolate BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. b Percent of wild-type animals surviving on plates seeded with bacterial isolate BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3–6 experiments of 500 animals each. Wild-type animals carried the integrated transgene nIs470. c Graphical representation of multigenerational experimental set up. See Fig. 1a for legend. Embryos were collected by bleaching (E.C.B.) in each generation and were placed onto fresh plates seeded with E. coli HB101 until the L4 developmental stage at each generation until the final generation, where animals were placed onto fresh plates seeded with P. vranovensis BIGb446 and the percent of surviving animals was counted after 24 h. Source data are provided as a Source Data file. ***p = 0.003, ****p < 0.0001. See section “statistics and reproducibility” for statistical tests run.

Fig. 3. Parental infection by P. vranovensis alters gene expression in offspring.

a Gene expression changes in embryos from parents fed P. vranovensis BIGb446 when compared to embryos from parents fed E. coli HB101. Values represent averages from three replicates. b Venn diagram of genes exhibiting a greater than two-fold change in RNA expression in adults fed P. vranovensis BIGb446 and embryos from adults fed P. vranovensis BIGb446. p-value represents normal approximation to the hypergeometric probability (see section “Statistics and reproducibility”). c Percent of F1 wild-type animals surviving on plates seeded with bacterial isolate BIGb446 after 24 h. P0 animals were fed their normal laboratory diet of E. coli HB101 or were exposed to the bacterial pathogens P. vranovensis BIGb446, P. aeruginosa PA14, or P. luminescens Hb for 24 h. Data presented as mean values ± s.d. n = 3 experiments of 500 animals. d Venn diagram of genes exhibiting a greater than two-fold change in RNA expression in embryos from parents fed P. vranovensis BIGb446, P. aeruginosa PA14, and P. luminescens Hb. p-value represents normal approximation to the hypergeometric probability (see section “Statistics and reproducibility”). e Venn diagram of number of genes exhibiting a greater than two-fold change in RNA expression in embryos from parents exposed to P. vranovensis BIGb446 and wdr-23(mac32) mutant embryos. p-value represents normal approximation to the hypergeometric probability (see section “Statistics and reproducibility”). f Percent of wild-type and skn-1(zj15) mutants surviving on plates seeded with P. vranovensis BIGb446 after 72 h. Error bars, s.d. n = 3 experiments of >100 animals. Source data are provided as a Source Data file. *p = 0.0057, ****p < 0.0001. See section “Statistics and reproducibility” for statistical tests run.

Fig. 4. CYSL-1 and CYSL-2 are required for C. elegans to adapt to P. vranovensis.

a Fold Change of cysl-1 mRNA in wild-type embryos from parents fed E. coli HB101 or P. vranovensis BIGb446. Data presented as mean values ± s.d. n = 3–6 replicates. b Fold Change of cysl-2 mRNA in wild-type embryos from parents fed E. coli HB101 or P. vranovensis BIGb446. Data presented as mean values ± s.d. n = 3–6 replicates. Data is the same as found in Supplementary Data 5 and  7. c Representative images of cysl-2::GFP in embryos from parents fed E. coli HB101 or P. vranovensis  BIGb446. Scale bars 100 μm. d Percent of wild-type and cysl-1(ok762) mutants surviving on plates seeded with bacterial isolates BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. e Percent of wild-type and cysl-2(ok3516) mutants surviving on plates seeded with bacterial isolates BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. ****p < 0.0001. Source data are provided as a Source Data file. See section “Statistics and reproducibility” for statistical tests run.

Fig. 5. RHY-1 is required for C. elegans adaptation to P. vranovensis.

a Transcripts per million (TPM) of rhy-1 in wild-type embryos from parents fed E. coli HB101 or P. vraonvensis BIGb446. Data presented as mean values ± s.d. n = 3–6 replicates. b Percent of wild-type and rhy-1 mutants surviving on plates seeded with bacterial isolates BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. c Percent of wild-type, egl-9(n586), hif-1(ia4), and rhy-1(n5500) mutants surviving on plates seeded with bacterial isolates BIGb446 after 24 h. Data presented as mean values ± s.d. n = 3 experiments of >100 animals. ****p < 0.0001. Source data are provided as a Source Data file. See section “Statistics and reproducibility” for statistical tests run.

Fig. 6. Model of C. elegans adaptation to P. vranovensis.

C. elegans heritably adapts to P. vranovensis exposure via a mechanism that requires CYSL-1, CYSL-2, and RHY-1. Briefly, our data indicates that parental exposure of C. elegans to P. vranovensis results in the activation of the SKN-1 transcription factor and increased expression of the cysteine synthases cysl-1 and cysl-2 and the predicted O-acyltransferase rhy-1 in offspring. These three genes are required for the observed increase in offspring survival in response to P. vranovensis infection.