Impact of TR34/L98H, TR46/Y121F/T289A and TR53 Alterations in Azole-Resistant Aspergillus fumigatus on Sterol Composition and Modifications after In Vitro Exposure to Itraconazole and Voriconazole

Background: Sterols are the main components of fungal membranes. Inhibiting their biosynthesis is the mode of action of azole antifungal drugs that are widely used to treat fungal disease including aspergillosis. Azole resistance has emerged as a matter of concern but little is known about sterols biosynthesis in azole resistant Aspergillus fumigatus. Methods: We explored the sterol composition of 12 A. fumigatus isolates, including nine azole resistant isolates with TR34/L98H, TR46/Y121F/T289A or TR53 alterations in the cyp51A gene and its promoter conferring azole resistance. Modifications in sterol composition were also investigated after exposure to two azole drugs, itraconazole and voriconazole. Results: Overall, under basal conditions, sterol compositions were qualitatively equivalent, whatever the alterations in the target of azole drugs with ergosterol as the main sterol detected. Azole exposure reduced ergosterol composition and the qualitative composition of sterols was similar in both susceptible and resistant isolates. Interestingly TR53 strains behaved differently than other strains. Conclusions: Elucidating sterol composition in azole-susceptible and resistant isolates is of interest for a better understanding of the mechanism of action of these drugs and the mechanism of resistance of fungi.

Heat stress leads to rapid lipid remodelling and transcriptional adaptations in Nicotiana tabacum pollen tubes

After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, they likely evolved mechanisms to quickly adapt to temperature changes to sustain an elongation at the highest possible rate. We investigated these adaptions in Nicotiana tabacum pollen tubes grown in vitro under 22 °C and 37 °C by a multi-omic approach including lipidomic, metabolomic and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress while triacylglycerols changed less in respect to desaturation but showed higher levels. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acids increased during heat stress, including the non-codogenic amino acids γ-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially up- and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated, but also transcripts that have so far not been linked to heat stress. Transcripts involved in triacylglycerol synthesis were increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled indicating other means of regulation.

Spent Coffee Ground Oil as a Potential Alternative for Vegetable Oil Production: Evidence from Oil Content, Lipid Profiling, and Physicochemical Characterization

In this work, routinely measured physicochemical indices and lipid profiling of oil extracted from spent coffee grounds (SCG) were evaluated to assess the suitability of SCG as a new candidate for oil production. The obtained results reveal that the oil yield was 18.55±1.5 g/100g. Physicochemical indices were comparable to those of widely consumed vegetable oils in the range set in several studies. The main fatty acids of SCG oil were linoleic acid 43.20±2.19 g/100g, palmitic acid 31.78±2.02 g/100g, and oleic acid 12.68±1.15 g/100g dry basis. For sterol composition, β-sitosterol was the most abundant sterol (44.70±0.01%), followed by stigmasterol (27.57±0.01%) and campesterol (12.16±0.01%). In conclusion, this composition is typical for many other vegetable oils. Therefore, this oil may be considered a good alternative for vegetable oil production for new multi-purpose products such as cosmetic and industrial pharmaceutical uses.

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.

Trypanosoma cruzi Affects Rhodnius prolixus Lipid Metabolism During Acute Infection

The interaction between Rhodnius prolixus and Trypanosoma cruzi has huge medical importance because it responds to the transmission of Chagas disease, a neglected tropical disease that affects about eight million people worldwide. It is known that trypanosomatid pathogens depend on active lipid endocytosis from the insect host to meet growth and differentiation requirements. However, until now, knowledge on how the parasite affects the lipid physiology of individual insect organs was largely unknown. Herein, the biochemical and molecular dynamics of the triatomine R. prolixus lipid metabolism in response to T. cruzi acute infection were investigated. A qRT-PCR approach was used to determine the expression profile of 12 protein-coding genes involved in R. prolixus lipid physiology. In addition, microscopic and biochemical assays revealed the lipid droplet profile and the levels of the different identified lipid classes. Finally, spectrometry analyses were used to determine fatty acid and sterol composition and their modulation towards the infection. T. cruzi infection downregulated the transcript levels of protein-coding genes for lipid biosynthetic and degrading pathways in individual triatomine organs. On the other hand, upregulation of lipid receptor transcripts indicates an attempt to capture more lipids from hemolymphatic lipoproteins. Consequently, several lipid classes (such as monoacylglycerol, diacylglycerol, triacylglycerol, cholesteryl ester, phosphatidylcholine, and phosphatidylethanolamine) were involved in the response to the parasite challenge, although modulating only the insect fat body. T. cruzi never leaves the insect gut and yet it modulates non-infected tissues, suggesting that the association between the parasite and the vector organs is reached by cell signaling molecules. This hypothesis raises several intriguing issues to inspire future studies in the parasite-vector interaction field.

Sterol Composition Modulates the Response of Saccharomyces cerevisiae to Iron Deficiency

Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway.

Lactiplantibacillus plantarum Used as Single, Multiple, and Mixed Starter Combined with Candida boidinii for Table Olive Fermentations: Chemical, Textural, and Sensorial Characterization of Final Products

In this study, four different kinds of table olive fermentations belonging to Olea europaea L. Itrana cultivar were evaluated: A, spontaneous fermentation; B, fermentation with a single inoculum (Lactiplantibacillus plantarum B1); C, fermentation with multiple inoculum (L. plantarum B1 + L. plantarum B51 + L. plantarum B124, 1:1:1); and D, fermentation with mixed (bacterium + yeast) inoculum (L. plantarum B1 + Candida boidinii). This research focuses on the correlation between the different mixes of inoculations and their effect under the chemical, sensorial, and textural profiles in the final products (olives) for potential applications on table olive fermentation. During the fermentation, some specific parameters were monitored: chemical characterization of oil fraction (pigments, tocopherols, fatty acids, alkyl esters, and sterol composition), Texture Profile Analysis (TPA), determination of olive color, and sensory evaluation of the final products. The use of LAB starters (single and multiple inocula) compared to spontaneous process revealed a greater performance in preventing the spoilage process and in developing favorable physico-chemical conditions during the fermentation. In fact, the highest values of fatty acid alkyl esters were reached in spontaneous fermentation (~480 mg/kg in jar A). The presence of C. boidinii as inoculum in jar D was involved in table olive softening: the fermented olives showed the lowest values of the parameters related to consistence of fruit as hardness (~2300 g) and gumminess (~990 g) and high value of fatty acid methyl esters (~110 mg/kg).

Altering Sterol Composition Implied That Cholesterol Is Not Physiologically Associated With Diosgenin Biosynthesis in Trigonella foenum-graecum

Diosgenin serves as an important precursor of most steroidal drugs in market. Cholesterol was previously deemed as a sterol origin leading to diosgenin biosynthesis. This study reports that cholesterol is not in parallel with diosgenin biosynthesis in Trigonella foenum-graecum. We first perturbed its sterol composition using inhibitors specific for the upstream isoprenoid pathway enzymes, HMGR (3-hydroxy-3-methylgutaryl-CoA reductase) on the mevalonate (MVA) and DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) on the 2-C-methyl-D-erythritol-4-phophate (MEP) pathways, and have revealed that diosgenin and cholesterol reversely or differently accumulated in either the MVA or the MEP pathway-suppressed plants, challenging the previously proposed role of cholesterol in diosgenin biosynthesis. To further investigate this, we altered the sterol composition by suppressing and overexpressing the 24-sterol methyltransferase type 1 (SMT1) gene in T. foenum-graecum, as SMT1 acts in the first committed step of diverting the carbon flux of cholesterol toward biosynthesis of 24-alkyl sterols. Knockdown of TfSMT1 expression led to increased cholesterol level but caused a large reduction of diosgenin. Diosgenin was increased upon the TfSMT1-overexpressing, which, however, did not significantly affect cholesterol biosynthesis. These data consistently supported that diosgenin biosynthesis in T. foenum-graecum is not associated with cholesterol. Rather, campesterol, a 24-alkyl sterol, was indicative of being correlative to diosgenin biosynthesis in T. foenum-graecum.