Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Qo site). We identified the G37V change within the cytochrome b Qi site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Qo site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.

Artemisinin and its derivatives target mitochondrial c-type cytochromes in yeast and human cells.

Artemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detect cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells.

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms.

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt-cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt-cyb encodes the core subunit (MT-CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt-cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human-associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt-cyb variations in key catalytic domains of MT-CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt-cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt-cyb-biochemical relationships in yeast provides a resource for future investigations in humans.

The antimalarial drug primaquine targets Fe-S cluster proteins and yeast respiratory growth.

Malaria is a major health burden in tropical and subtropical countries. The antimalarial drug primaquine is extremely useful for killing the transmissible gametocyte forms of Plasmodium falciparum and the hepatic quiescent forms of P. vivax. Yet its mechanism of action is still poorly understood. In this study, we used the yeast Saccharomyces cerevisiae model to help uncover the mode of action of primaquine. We found that the growth inhibitory effect of primaquine was restricted to cells that relied on respiratory function to proliferate and that deletion of SOD2 encoding the mitochondrial superoxide dismutase severely increased its effect, which can be countered by the overexpression of AIM32 and MCR1 encoding mitochondrial enzymes involved in the response to oxidative stress. This indicated that ROS produced by respiratory activity had a key role in primaquine-induced growth defect. We observed that Δsod2 cells treated with primaquine displayed a severely decreased activity of aconitase that contains a Fe-S cluster notoriously sensitive to oxidative damage. We also showed that in vitro exposure to primaquine impaired the activity of purified aconitase and accelerated the turnover of the Fe-S cluster of the essential protein Rli1. It is suggested that ROS-labile Fe-S groups are the primary targets of primaquine. Aconitase activity is known to be essential at certain life-cycle stages of the malaria parasite. Thus primaquine-induced damage of its labile Fe-S cluster - and of other ROS-sensitive enzymes - could inhibit parasite development.

Interplay between the hinge region of iron sulphur protein and the Qo site in the bc1 complex - Analysis of Plasmodium-like mutations in the yeast enzyme.

The respiratory chain bc1 complex is central to mitochondrial bioenergetics and the target of antiprotozoals. We characterized a modified yeast bc1 complex that more closely resemble Plasmodium falciparum enzyme. The mutant version was generated by replacing ten cytochrome b Qo site residues by P. falciparum equivalents. The Plasmodium-like changes caused a major dysfunction of the catalytic mechanism of the bc1 complex resulting in superoxide overproduction and respiratory growth defect. The defect was corrected by substitution of the conserved residue Y279 by a phenylalanine, or by mutations in or in the vicinity of the hinge domain of the iron-sulphur protein. It thus appears that side-reactions can be prevented by the substitution Y279F or the modification of the iron-sulphur protein hinge region. Interestingly, P. falciparum - and all the apicomplexan - contains an unusual hinge region. We replaced the yeast hinge region by the Plasmodium version and combined it with the Plasmodium-like version of the Qo site. This combination restored the respiratory growth competence. It could be suggested that, in the apicomplexan, the hinge region and the cytochrome b Qo site have co-evolved to maintain catalytic efficiency of the bc1 complex Qo site.

Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.