Green Microalgae Strain Improvement for the Production of Sterols and Squalene

Sterols and squalene are essential biomolecules required for the homeostasis of eukaryotic membrane permeability and fluidity. Both compounds have beneficial effects on human health. As the current sources of sterols and squalene are plant and shark oils, microalgae are suggested as more sustainable sources. Nonetheless, the high costs of production and processing still hinder the commercialization of algal cultivation. Strain improvement for higher product yield and tolerance to harsh environments is an attractive way to reduce costs. Being an intermediate in sterol synthesis, squalene is converted to squalene epoxide by squalene epoxidase. This step is inhibited by terbinafine, a commonly used antifungal drug. In yeasts, some terbinafine-resistant strains overproduced sterols, but similar microalgae strains have not been reported. Mutants that exhibit greater tolerance to terbinafine might accumulate increased sterols and squalene content, along with the ability to tolerate the drug and other stresses, which are beneficial for outdoor cultivation. To explore this possibility, terbinafine-resistant mutants were isolated in the model green microalga Chlamydomonas reinhardtii using UV mutagenesis. Three mutants were identified and all of them exhibited approximately 50 percent overproduction of sterols. Under terbinafine treatment, one of the mutants also accumulated around 50 percent higher levels of squalene. The higher accumulation of pigments and triacylglycerol were also observed. Along with resistance to terbinafine, this mutant also exhibited higher resistance to oxidative stress. Altogether, resistance to terbinafine can be used to screen for strains with increased levels of sterols or squalene in green microalgae without growth compromise.

Coupled sterol synthesis and transport machineries at ER–endocytic contact sites

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike animals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14-demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target. In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis in Leishmania plays an unexpected role in global gene regulation.

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Leishmania species are parasitic protozoans which cause leishmaniasis affecting millions of people worldwide. Sterols are important components of plasma and organellar membranes and serve as precursors for the synthesis of signaling molecules. In contrast to animals, Leishmania do not produce cholesterol but instead synthesize ergostane-based sterols. C14-demethylase is a key enzyme involved in the biosynthesis of sterols in Leishmania parasites and an important drug target. Its inactivation leads to multiple defects including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis plays an unexpected role in global gene regulation in Leishmania parasites.

Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes

The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the role of metabolic heterogeneity. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Incorporation of 13C-glutamine into endogenously synthesized sterols is increased with BPTES treatment, an inhibitor of host glutamine metabolism that sensitizes amastigotes to azoles. Similarly, amastigotes are re-sensitized to azoles following addition of metabolites upstream of CYP51, raising the possibility that flux through the sterol synthesis pathway is a determinant of sensitivity to azoles and highlighting the potential role for metabolic heterogeneity in recalcitrant T. cruzi infection.

Cholesterol homeostasis in the vertebrate retina: Biology and pathobiology

Cholesterol is a quantitatively and biologically significant constituent of all mammalian cell membrane, including those that comprise the retina. Retinal cholesterol homeostasis entails the interplay between de novo synthesis, uptake, intra-retinal sterol transport, metabolism and efflux. Defects in these complex processes are associated with several congenital and age-related disorders of the visual system. Herein, we provide an overview of the following topics: a) cholesterol synthesis in the neural retina; b) lipoprotein uptake and intraretinal sterol transport in the neural retina and the retinal pigment epithelium (RPE); c) cholesterol efflux from the neural retina and the RPE; and d) biology and pathobiology of defects in sterol synthesis and sterol oxidation in the neural retina and the RPE. We focus, in particular, on studies involving animal models of monogenic disorders pertinent to the above topics, as well as in vitro models using biochemical, metabolic, and omic approaches. We also identify current knowledge gaps as well as opportunities in the field that beg further research in this topic area.

Characterization of the transcriptional response of Candida parapsilosis to the antifungal peptide MAF-1A

Candida parapsilosis is a major fungal pathogen that leads to sepsis. New and more effective antifungal agents are required due to the emergence of resistant fungal strains. MAF-1A is a cationic antifungal peptide isolated from Musca domestica that is effective against a variety of Candida species. However, the mechanism(s) of its antifungal activity remains undefined. Here, we used RNA-seq to identify differentially expressed genes (DEGs) in Candida parapsilosis following MAF-1A exposure. The early (6 h) response included 1,122 upregulated and 1,065 downregulated genes. Late (18 h) responses were associated with the increased expression of 101 genes and the decreased expression of 151 genes. Upon MAF-1A treatment for 18 h, 42 genes were upregulated and 25 genes were downregulated. KEGG enrichment showed that the DEGs in response to MAF-1A were mainly involved in amino acid synthesis and metabolism, oxidative phosphorylation, sterol synthesis, and apoptosis. These results indicate that MAF-1A exerts antifungal activity through interference with Candida parapsilosis cell membrane integrity and organelle function. This provides new insight into the interaction between Candida parapsilosis and this antimicrobial peptide and serves as a reference for future Candida parapsilosis therapies.