Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes

Figarella, Katherine; Marsiccobetre, Sabrina; Arocha, Irina; Colina, W; Hasegawa, Maya; Rodríguez, María Cecilia; Rodrı́guez-Acosta, Alexis; Duszenko, Michael; Benaím, Gustavo; Nl, Uzcategui

doi:10.15698/mic2016.01.471

Cited by 8 publications

(8 citation statements)

References 39 publications

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“…We also detected a modest swelling of mitochondria in c14dm� mutants (Fig 1B and S1A Fig). These findings are consistent with the previously reported hyperpolarization of mitochondrial membrane in Leishmania mexicana following treatment with triazole compounds [59].…”

Section: Discussionsupporting

confidence: 93%

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

Mukherjee

Moitra

et al. 2020

PLoS Pathog

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Sterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm�). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm�mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm� mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm�. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.

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Section: Discussionsupporting

confidence: 93%

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

Mukherjee

Moitra

et al. 2020

PLoS Pathog

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“…We also detected a modest swelling of mitochondria in c14dm - mutants (Fig 1B and Fig S1A). These findings are consistent with the previously reported hyperpolarization of mitochondrial membrane in Leishmania mexicana following treatment with triazole compounds [59].…”

Section: Discussionsupporting

confidence: 93%

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance inLeishmania major

Mukherjee¹,

Moitra

Xu³

et al. 2020

Preprint

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ABSTRACTSterol 14-α-demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm-). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm- mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm- mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm-. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions.AUTHOR SUMMARYSterols are well recognized for their stabilizing effects on the plasma membrane, but their functions in intracellular organelles are under explored, which hampers the development of sterol synthesis inhibitors as drugs. Our previous studies have demonstrated significant plasma membrane instability in the sterol biosynthetic mutant c14dm- in Leishmania major, a pathogenic protozoan responsible for cutaneous leishmaniasis causing 1-1.5 million infections a year. While the plasma membrane defects have undoubtedly contributed to the reduced virulence exhibited by c14dm- mutants, it was not clear whether other cellular processes were also affected. In this study, we revealed profound mitochondrial dysfunctions and elevated level of reactive oxygen species in c14dm- mutants. These sterol mutants rely heavily on glycolysis to generate energy and are extremely sensitive to glucose restriction. In addition, the accumulation of oxidants appears to be responsible (at least in part) for the previously observed heat sensitivity in c14dm- mutants. Thus, genetic or chemical inactivation of C14DM can influence the functions of cellular organelles beyond the plasma membrane. These findings shed light on the mechanism of action for azole compounds and provide new insight into the roles of sterol biosynthesis in Leishmania parasites.

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“… Figarella et al (2015) observed mitochondrial abnormalities using the same principles used in this study, with similar techniques. Ergosterone-coupled triazol molecules caused an increase of ROS production, leading to DNA damage, augmentation of autophagy and phosphatidylserine exposure, conforming reported by scientific literature for Trypanosomatidae family members ( Figarella et al, 2006 , Chowdhury et al, 2014 ).…”

Section: Discussionmentioning

confidence: 60%

Induction of Early Autophagic Process on Leishmania amazonensis by Synergistic Effect of Miltefosine and Innovative Semi-synthetic Thiosemicarbazone

et al. 2017

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Drug combination therapy is a current trend to treat complex diseases. Many benefits are expected from this strategy, such as cytotoxicity decrease, retardation of resistant strains development, and activity increment. This study evaluated in vitro combination between an innovative thiosemicarbazone molecule – BZTS with miltefosine, a drug already consolidated in the leishmaniasis treatment, against Leishmania amazonensis. Cytotoxicity effects were also evaluated on macrophages and erythrocytes. Synergistic antileishmania effect and antagonist cytotoxicity were revealed from this combination therapy. Mechanisms of action assays were performed in order to investigate the main cell pathways induced by this treatment. Mitochondrial dysfunction generated a significant increase of reactive oxygen and nitrogen species production, causing severe cell injuries and promoting intense autophagy process and consequent apoptosis cell death. However, this phenomenon was not strong enough to promote dead in mammalian cell, providing the potential selective effect of the tested combination for the protozoa. Thus, the results confirmed that drugs involved in distinct metabolic routes are promising agents for drug combination therapy, promoting a synergistic effect.

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Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes

Cited by 8 publications

References 39 publications

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance inLeishmania major

Induction of Early Autophagic Process on Leishmania amazonensis by Synergistic Effect of Miltefosine and Innovative Semi-synthetic Thiosemicarbazone

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