Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability

Abstract

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.

Fig. 1.

Growth dependence on availability of heme and quantification of heme b in P. serpens and related flagellates. (A) Growth rate of P. serpens is the same without heme or when heme is supplied up to a concentration of 5 μM; 25 μM heme inhibits growth, likely attributable to toxic effects of free heme. Quite opposite dependence of growth on heme concentration is observed in the closely related C. fasciculata, which grows best when supplied with 25 μM heme and stops growing when heme concentration in the media is lowered to 1 μM. (B) Heme b extracted from equal numbers of cells from various kinetoplastids was separated by HPLC and detected by diode array detector. C. fasciculata was used as a related organism that possesses a complete set of respiratory complexes. It was grown in the same medium and supplemented with the same amount of heme (5 μM) as P. serpens. The bloodstream stage of T. brucei, which does not express its respiratory complexes III and IV in this life cycle stage, and thus functionally resembles P. serpens, was used as another control. The absence of respiratory complexes that normally consume most of heme is reflected in the much lower amount of extracted heme compared with C. fasciculata. The heme content in P. serpens is even lower than in T. brucei, which is in accordance with the lowest number of heme proteins found in the Phytomonas spp. genomes among all kinetoplastids (Table 1). Not even a trace amount of heme is detected in P. serpens grown without heme (dotted line). +H, with heme; −H, without heme.

Fig. 2.

Respiratory complex II (succinate dehydrogenase) is assembled and active in P. serpens grown with (+H) or without (−H) heme. (A) Clear native gel (3–12%) after in-gel staining for succinate dehydrogenase activity; C. fasciculata (Cf) served as a control. Ferritin (monomeric and dimeric forms) was used as a molecular weight marker (M). (B) Lysates from the same cells as in A were analyzed by SDS/PAGE and immunoblotted with specific antiserum against the T. brucei subunit of complex II, SDH1. (C) Activity of succinate dehydrogenase in P. serpens grown without heme. The decrease in absorbance (A₆₀₀) with time (curve 2) was caused by the addition of ubiquinone to the reaction, which mediated the electron transfer from succinate to 2,6-dichlorophenolindophenol. The activity was specifically inhibited using malonate (curve 3). Curve 1 represents the background without ubiquinone. (D) Activity did not significantly differ between P. serpens grown with (+H) or without (−H) heme. Medium values were calculated from three measurements.

Fig. 3.

Heme is not needed for desaturation of fatty acids but is required for ergosterol biosynthesis in P. serpens. (A) Schematic phylogenetic tree of Δ9-fatty acid desaturases (FADS). P. serpens is the only organism that secondarily lost the cytochrome b₅ domain. The full phylogenetic tree of Δ9-fatty acid desaturase is shown in Fig. S1. (B) Analyses of fatty acid composition by gas chromatography demonstrate that in P. serpens, the desaturation of fatty acids is not affected by the absence of heme. (C) Analysis of sterol composition by TLC. Ergosterol, which is the major membrane sterol of Trypanosomatida, and lanosterol, the precursor of heme-dependent demethylation, were used as standards (S). C. fasciculata (Cf) served as a control. P. serpens synthesized a sterol that corresponded to the ergosterol standard only when heme was added to the growth medium (+H). Cells grown without heme (−H) accumulated lanosterol.