Benzyl Farnesyl Amine Mimetics are Potent Inhibitors of the Sterol Biosynthesis Pathway in Leishmania Amazonensis Leading to Oxidative Stress, Growth Arrest, and Ultrastructural Alterations

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus spread around the world. Benzyl farnesyl amine mimetics are known class of compounds selectively designed to inhibit the squalene synthase (SQS) enzyme that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzyl farnesyl amine mimetics (SBC 37 - 43) against Leishmania amazonensis. After the first initial screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected for further studies. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating that they are highly selective. Analyses of free sterol showed that SBC 39 and SBC 40 inhibit two enzymes, sterol Δ8 → Δ7 isomerase and SQS, resulting in depletion of endogenous 24-methyl sterols. Physiological analysis and electron microscopy revealed three main alterations: 1) in the mitochondrion ultrastructure and function; 2) the presence of lipid bodies and autophagosomes; and 3) the appearance of projections in the plasma membrane and extracellular vesicles inside the flagellar pocket. In conclusion, our results support the notion that benzyl farnesyl amine mimics have a potent effect against Leishmania amazonensis and should be an interesting novel pharmaceutical lead for the development of new chemotherapeutic alternatives to treat leishmaniasis.

Maize Zmcyp710a8 Mutant as a Tool to Decipher the Function of Stigmasterol in Plant Metabolism

Sterols are integral components of membrane lipid bilayers in eukaryotic organisms and serve as precursors to steroid hormones in vertebrates and brassinosteroids (BR) in plants. In vertebrates, cholesterol is the terminal sterol serving both indirect and direct roles in cell signaling. Plants synthesize a mixture of sterols including cholesterol, sitosterol, campesterol, and stigmasterol but the signaling role for the free forms of individual plant sterols is unclear. Since stigmasterol is the terminal sterol in the sitosterol branch and produced from a single enzymatic step, modifying stigmasterol concentration may shed light on its role in plant metabolism. Although Arabidopsis has been the model of choice to study sterol function, the functional redundancy of AtCYP710A genes and the presence of brassicasterol may hinder our ability to test the biological function of stigmasterol. We report here the identification and characterization of ZmCYP710A8, the sole maize C-22 sterol desaturase involved in stigmasterol biosynthesis and the identification of a stigmasterol-free Zmcyp710a8 mutant. ZmCYP710A8 mRNA expression pattern correlated with transcripts for several sterol biosynthesis genes and loss of stigmasterol impacted sterol composition. Exogenous stigmasterol also had a stimulatory effect on mRNA for ZmHMGR and ZmSMT2. This demonstrates the potential of Zmcyp710a8 in understanding the role of stigmasterol in modulating sterol biosynthesis and global cellular metabolism. Several amino acids accumulate in the Zmcyp710a8 mutant, offering opportunity for genetic enhancement of nutritional quality of maize. Other cellular metabolites in roots and shoots of maize and Arabidopsis were also impacted by genetic modification of stigmasterol content. Yet lack of obvious developmental defects in Zmcyp710a8 suggest that stigmasterol might not be essential for plant growth under normal conditions. Nonetheless, the Zmcyp710a8 mutant reported here is of great utility to advance our understanding of the additional roles of stigmasterol in plant metabolism. A number of biological and agronomic questions can be interrogated using this tool such as gene expression studies, spatio-temporal localization of sterols, cellular metabolism, pathway regulation, physiological studies, and crop improvement.

Delineating the Upc2A Regulon in Candida glabrata

Candida glabrata is the second most common cause of invasive candidiasis. Intrinsic resistance has greatly limited the utility of the triazole antifungal, fluconazole, in the treatment of invasive fungal infection. The transcription factor Upc2 regulates the expression of sterol biosynthesis genes in yeast. Disrupting UPC2A in C. glabrata greatly increases its susceptibility to fluconazole (FLU) in both FLU-susceptible and -resistant clinical isolates. Therefore, the Upc2A and its target genes represent a potential pathway for overcoming FLU resistance in C. glabrata. We aimed to delineate the Upc2A regulon to determine its target genes involved in FLU resistance. Transcriptome sequencing (RNA-seq) analysis was used to compare gene expression profiles of: a) wild-type (WT) strains with and without UPC2A under non-stressed conditions; b) those same strains treated with three sterol biosynthesis inhibitors (SBIs; FLU, terbinafine, fenpropimorph); and c) strains with an activating mutation (GOF) in UPC2A. Global chromatin Immunoprecipitation (ChIP-seq) was used to identify genes whose promoters were bound by Upc2A in strains carrying WT and GOF alleles of Upc2A with and without fluconazole exposure. Only three genes (UPC2A, ERG25, and ERG3) were found to be downregulated in the absence of UPC2A. Sixteen genes were commonly upregulated in response to SBIs’ treatment in a Upc2A-dependent way, including ERG2, ERG3, and ERG11, the promoters of 10 of which were bound by Upc2A. A total of 15 genes were upregulated, including ERG2, ERG3, ERG25, and ERG11 in the strain containing GOF mutation and the promoters of 6 of these genes were bound by Upc2A. Based on our data, ERG3, ERG11, HEM13, and CAGL0H09592g (ScTIR1) could be potential targets of Upc2A in C. glabrata. This more comprehensive understanding of the Upc2A regulon in C. glabrata may eventually lead to strategies to overcome FLU resistance and enhance fluconazole activity against this important fungal pathogen.

Membrane Sterol Composition in Arabidopsis thaliana Affects Root Elongation via Auxin Biosynthesis

Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (β-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.

Clade-Specific Sterol Metabolites in Dinoflagellate Endosymbionts Are Associated with Coral Bleaching in Response to Environmental Cues

These results indicate that sterol metabolites in Symbiodiniaceae are clade specific, that their biosynthetic pathways share architectural and substrate specificity features with those of animals and plants, and that environmental stress-specific perturbation of sterol biosynthesis in dinoflagellates can impair a key mutualistic partnership for healthy reefs.