The response of Caenorhabditis elegans to hydrogen sulfide and hydrogen cyanide

Abstract

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia-reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.

Figure 1

Wild-type nematodes exposed to 50 ppm RA/H2S exhibit a brief increase in movement initially and then continue to move (A, black line). When either wild-type or hif-1 mutant animals are exposed to 150 ppm RA/H2S or 50 ppm RA/H2S, respectively, they stop moving and when the RA/H2S is removed, they resume movement (blue and red lines, A and B). Examples of Hif-1 moving, stopped, and reanimated worms are shown in C.

Figure 2

H₂S sensitivity screen method and results. (A) Schematic of initial paralysis screen and lethality rescreen. (B) Histogram of the number of strains isolated for each penetrance bin. The strains with zero survivors after sulfide exposure are highlighted in red. These strains were subsequently characterized.

Figure 3

Enrichment of H₂S-sensitive phenotype after crossing mutant lines with the Hawaiian strain CB4856. SNP mapping data show five apparent complementation groups. For suls-1 and suls-2 the apparent location of mutation is indicated by an arrow. The known locations of sqrd-1, cysl-1, and hif-1 are also indicated by arrows. Black indicates an N2/HI ratio >5.

Figure 4

Complementation data showing that each genetic locus consists of only one complementation group (blue, green, yellow, and pink shades). suls-1 is not shown because it is dominant. sqrd-1, cysl-1 double heterozygotes are slightly sensitive to RA/H₂S.

Figure 5

(A) Cysteine synthase (CYS) catalyzes the formation of cysteine and acetate from OAS and H₂S. (B) Cyanoalanine synthase (CAS) catalyzes the formation of β-cyanoalanine and H₂S from cysteine and HCN. (C) Amino acid alignment of a selected region of CYS and CBS homologs. C. elegans Cysl genes are homologous to CYS, whereas C. elegans Cbs genes are homologous to cystathionine β-synthase (CBS).

Figure 6

(A) Both sqrd-1and cysl-2 mRNA levels increase relative to untreated animals as measured by quantitative reverse transciptase PCR. Y axis is relative mRNA concentration compared to room air-treated control animals. *P < 0.05 as compared to animals grown in room air. (B) hif-1 is required for sqrd-1 and cysl-2 mRNA induction when exposed to RA/H₂S or RA/HCN. Arrowheads indicate that mRNA levels were observed at very low levels. *P < 0.05 when comparing wild-type and hif-1 mutant animals. (C) Western blot shows SQRD-1 protein induction in RA/H₂S. This induction requires hif-1. No immunoreactivity is observed in sqrd-1 mutant animals. SQRD-1 protein is abundant in egl-9 mutant animals. Circles indicate protein size markers of 116, 82, 62, and 49 kDa from top to bottom. The calculated molecular weight of 53 kDa corresponds well with the observed relative migration.

Figure 7

SQRD-1::GFP expression is induced by exposure to RA/H₂S. (A) SQRD-1::GFP is not expressed in room air. (B) RA/H₂S at 50 ppm induces expression of SQRD-1::GFP. (C) Confocal microscopy at ×40 magnification shows that the expression is brightest in the muscle dense bodies (tailed arrows) and the hypodermis (tail-less arrow). (D) Cross-section (Z-stack) shows expression is most intense in the hypodermis.

Figure 8

Proposed mechanism. Both H₂S and HCN can inhibit electron transport. Electron transport inhibition causes induction of HIF-1 activity resulting in high expression of SQRD-1 and CYSL-2. SQRD-1 catalyzes the first step in the H₂S-oxidation pathway, ultimately resulting in production of sulfate and thiosulfate. CYSL-2 catalyzes the first step in the HCN assimilation pathway, producing H₂S. The resultant H₂S is detoxified by CYSL-1.