Lack of heme synthesis in a free-living eukaryote

Abstract

In most free-living eukaryotes studied thus far, heme is synthesized from a series of intermediates through a well defined evolutionarily conserved pathway. We found that free-living worms, including the model genetic organism Caenorhabditis elegans, and parasitic helminths are unable to synthesize heme de novo, even though these animals contain hemoproteins that function in key biological processes. Radioisotope, fluorescence labeling, and heme analog studies suggest that C. elegans acquires heme from exogenous sources. Iron-deprived worms were unable to grow in the presence of adequate heme unless rescued by increasing heme levels in the growth medium. These data indicate that although worms use dietary heme for incorporation into hemoproteins, ingested heme is also used as an iron source when iron is limiting. Our results provide a biochemical basis for the dependence of worm growth and development on heme, and they suggest that pharmacologic targeting of heme transport pathways in worms could be an important control measure for helminthic infections.

Fig. 1.

Heme auxotrophy of worms. (A) Dithionite-reduced minus ferricyanide-oxidized absorption spectra of pyridine hemochromes from total homogenate and membrane- and cytosolic-enriched fractions of C. elegans grown in axenic mCeHR medium supplemented with 20 μM hemin chloride. A peak at 557 nm and a trough at 541 nm indicate pyridine protohemochrome. All samples were reduced with 5 mM sodium dithionite or oxidized with 1 mM potassium ferricyanide. The vertical bar represents a ΔA of 0.005 for total homogenate, 0.012 for membrane fraction, and 0.02 for cytosolic fraction. (Inset) Immunoblot of the same samples (50 μg) that were separated by SDS/4–20% PAGE and probed with ATP2p antisera followed by chemiluminescent detection. This immunoblot was stripped to remove ATP2p antibodies and reprobed with α-tubulin antibody. (B) Ultralow-temperature spectrum of whole homogenate from C. elegans grown in mCeHR medium supplemented with 20 μM hemin. Only α bands are indicated for cytochromes c and b and cytochrome oxidase (a+a3). The vertical bar represents a ΔA of 1.0. (C) Aerobic growth of C. elegans in mCeHR medium supplemented with 0 or 20 μM hemin chloride or 20 μM protoporphyrin IX (disodium salt). Equal numbers of synchronized L1 larvae were used as primary inoculum in 24-well plates in triplicate and the cultures were analyzed quantitatively for growth at days 1, 3, and 7. (D) Biphasic response of C. elegans cultured in the presence of increasing amounts of hemin chloride (μM). Equal numbers of synchronized L1 larvae were grown in 24-well plates in mCeHR medium for 9 days and quantified (worms perμl) by microscopy. Each data point represents the mean ± SD from three separate experiments performed in triplicate. (E) Metabolic labeling in C. elegans cultured in the presence of heme. Synchronized L1 larvae were grown in mCeHR medium containing either ⁵⁹FeCl₃ or [⁵⁹Fe]heme (9.4 × 10⁶ dpm), and the worms were harvested as gravid adults. Heme was extracted and concentrated, and then resolved by TLC followed by detection with a PhosphorImager (Upper). Lane 5, [⁵⁹Fe]heme control. Radiolabeled bands were quantified in a γ counter and cpm was normalized to total protein (Lower). To correct for nonspecific binding of the radiolabeled Fe and heme, parallel experiments were conducted in the presence of 1 mM sodium azide (samples 1 and 3).

Fig. 2.

Characterization of heme uptake in C. elegans. (A) Aerobic growth of C. elegans in mCeHR medium with 20 μM hemin supplemented with either gallium protoporphyrin IX (GaPP) or gallium salts. Synchronized L1 larvae were grown for 9 days in 24-well plates and quantified (worms per μl) by microscopy. Each data point represents the mean from a single experiment and each experiment was performed in triplicate. Inset depicts the GaPP analysis at lower concentrations for clarity. (B) Effect of heme on the cytotoxicity of GaPP. Synchronized L1 larvae were inoculated in 24-well plates containing mCeHR medium with 0, 2, 4, or 6 μM GaPP and increasing hemin (μM). The number of worms per μl was measured on day 9, and the data are presented as mean ± SD. (C) Fluorescent metabolic labeling of worms with either 40 μM hemin (images 1 and 4) or 40 μM ZnMP/4 μM hemin (images 2, 3, 5, and 6) for 3 h followed by confocal microscopy with a 546 laser (images 1–3) and differential interference contrast optics (images 4–6). Arrowheads indicate ZnMP fluorescence accumulation within intestinal cells and developing embryos. For clarity, the boxed portion of image 2 is magnified in images 3 and 6. (Bar, 100 μm.) (D) Worms were incubated with 40μM ZnMP/4μM hemin for 16 h followed by a chase with 40 μM hemin. Worms were analyzed by epifluorescence microscopy (TRITC channel) and differential interference contrast optics. Experiments were performed in the absence (images 1–4) or presence (images 5–8) of NaN₃ during the chase periods to test for the nonspecific loss of ZnMP fluorescence. Photomicrograph 4 is shown at a lower power to depict the complete loss of ZnMP fluorescence. (Bar, 100 μm.) For C and D, four separate experiments were performed with a minimum of 50 worms per data point per experiment. The data are representative for >90% of worms analyzed.

Fig. 3.

Worms use heme-iron under iron deprivation. Equal numbers of synchronized L1 larvae were grown in the presence of 4, 20, and 100 μM hemin, in basal mCeHR medium (set 1), or basal medium lacking exogenous iron (set 2), or as set 2 with 1 mM iron chelator ferrozine (set 3), or as set 3 with 486 μM ferrous ammonium sulfate (set 4). These values of ferrozine and iron were empirically determined by performing dose–response experiments and analyzing worm growth. The number of worms per μl was measured on day 9 and the data are presented as mean ± SD performed in triplicate. P < 0.001 between sets 1 and 3. Within each set, values with different letters are significantly different. * denotes significant differences with the corresponding heme concentrations in set 1.