Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.

Characterization of antifungal compounds produced by lactobacilli in cheese-mimicking matrix: Comparison between active and inactive strains.

Biopreservation of dairy products by acid lactic bacteria appears as a promising alternative to either replace or reduce the use of chemical preservatives. This study aimed at the identification of bacteria preventing fungal spoilers growth in dairy products, and, at the understanding of their antifungal activity. First, antifungal activity of eighteen Lactobacillus strains was tested against five molds and four yeasts leading to selection of L. casei 7006 which had an activity against seven fungal targets. Then, challenge tests against C. lusistaniae 3668 in a cheese-mimicking matrix have been performed demonstrating that this strain was able to reduce strongly this yeast growth after 14 and 21 days storages at 7 °C. Antifungal compounds produced in cheese-mimicking matrix containing L. casei 7006 strain were quantified, then compared to the one prepared with an inactive strain (L. casei 6960) or without Lactobacillus strain. Three compounds were differently produced between cheeses with or without Lactobacillus strain after 21 days at 7 °C: lactic acid, benzoic acid and diacetyl. However, lactic acid concentrations were similar between the three cheeses after 14 days at 7 °C, but an antifungal activity was only associated to L. casei 7006 presence. Benzoic acid concentrations between cheese with L. casei 7006 and negative control L. casei 6960 were also the same. Among the antifungal molecules retrieved from these analyses, diacetyl was the most significantly overproduced in cheese containing L. casei 7006, thus this volatile was associated to the antifungal activity of this strain.

Unearthing the Plant Growth-Promoting Traits of Bacillus megaterium RmBm31, an Endophytic Bacterium Isolated From Root Nodules of Retama monosperma.

Plants live in association with complex populations of microorganisms, including Plant Growth-Promoting Rhizobacteria (PGPR) that confer to plants an improved growth and enhanced stress tolerance. This large and diverse group includes endophytic bacteria that are able to colonize the internal tissues of plants. In the present study, we have isolated a nonrhizobial species from surface sterilized root nodules of Retama monosperma, a perennial leguminous species growing in poor and high salinity soils. Sequencing of its genome reveals this endophytic bacterium is a Bacillus megaterium strain (RmBm31) that possesses a wide range of genomic features linked to plant growth promotion. Furthermore, we show that RmBm31 is able to increase the biomass and positively modify the root architecture of seedlings of the model plant species Arabidopsis thaliana both in physical contact with its roots and via the production of volatile organic compounds. Lastly, we investigated the molecular mechanisms implicated in RmBm31 plant beneficial effects by carrying out a transcriptional analysis on a comprehensive set of phytohormone-responsive marker genes. Altogether, our results demonstrate that RmBm31 displays plant growth-promoting traits of potential interest for agricultural applications.

Identification of Atg8 from Acanthamoeba castellanii by genetic complementation in Saccharomyces cerevisiae.

Autophagy is a eukaryotic process responsible for the degradation of intracellular content such as damaged organelles. Several putative autophagy-related genes have been identified within the annotated genome of the free-living amoeba Acanthamoeba castellanii. However, the involvement of the corresponding proteins in the autophagy pathway had not been formerly established. Here, we report that AcAtg8 cDNA can complement ATG8-deficient Saccharomyces cerevisiae.

Increased fatty acid synthesis inhibits nitrogen starvation-induced autophagy in lipid droplet-deficient yeast.

Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets.

Canthin-6-one displays antiproliferative activity and causes accumulation of cancer cells in the G2/M phase.

Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle.

Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo.

Molecular evolution of the androgenic hormone in terrestrial isopods.

In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts.

Unsuspected pyocyanin effect in yeast under anaerobiosis.

The blue-green phenazine, Pyocyanin (PYO), is a well-known virulence factor produced by Pseudomonas aeruginosa, notably during cystic fibrosis lung infections. It is toxic to both eukaryotic and bacterial cells and several mechanisms, including the induction of oxidative stress, have been postulated. However, the mechanism of PYO toxicity under the physiological conditions of oxygen limitation that are encountered by P. aeruginosa and by target organisms in vivo remains unclear. In this study, wild-type and mutant strains of the yeast Saccharomyces cerevisiae were used as an effective eukaryotic model to determine the toxicity of PYO (100-500 µmol/L) under key growth conditions. Under respiro-fermentative conditions (with glucose as substrate), WT strains and certain H2 O2 -hypersensitive strains showed a low-toxic response to PYO. Under respiratory conditions (with glycerol as substrate) all the strains tested were significantly more sensitive to PYO. Four antioxidants were tested but only N-acetylcysteine was capable of partially counteracting PYO toxicity. PYO did not appear to affect short-term respiratory O2 uptake, but it did seem to interfere with cyanide-poisoned mitochondria through a complex III-dependent mechanism. Therefore, a combination of oxidative stress and respiration disturbance could partly explain aerobic PYO toxicity. Surprisingly, the toxic effects of PYO were more significant under anaerobic conditions. More pronounced effects were observed in several strains including a 'petite' strain lacking mitochondrial DNA, strains with increased or decreased levels of ABC transporters, and strains deficient in DNA damage repair. Therefore, even though PYO is toxic for actively respiring cells, O2 may indirectly protect the cells from the higher anaerobic-linked toxicity of PYO. The increased sensitivity to PYO under anaerobic conditions is not unique to S. cerevisiae and was also observed in another yeast, Candida albicans.

The MFS-type efflux pump Flr1 induced by Yap1 promotes canthin-6-one resistance in yeast.

Screening for suppressors of canthin-6-one toxicity in yeast identified Yap1, a transcription factor involved in cell response to a broad range of injuries. Although canthin-6-one did not promote a significant oxidative stress, overexpression of YAP1 gene clearly increased resistance to this drug. We demonstrated that Yap1-mediated resistance involves the plasma membrane major-facilitator-superfamily efflux pump Flr1 but not the vacuolar ATP-binding-cassette transporter Ycf1. FLR1 overexpression was sufficient to reduce sensitivity to the drug, but strictly dependent on a functional YAP1 gene.

A Saccharomyces cerevisiae strain unable to store neutral lipids is tolerant to oxidative stress induced by α-synuclein.

Parkinson disease is a neurodegenerative pathology that has been linked to several genetic mutations of the SNCA gene encoding the pro-oxidant α-synuclein protein. The budding yeast Saccharomyces cerevisiae is a valuable model for studying the cellular and molecular mechanisms of α-synuclein toxicity. Indeed heterologous expression of α-synuclein is toxic to wild-type yeast and exhibits the main features of damage caused to mammalian neurons, including an increase in neutral lipid storage (triglycerides and steryl esters, embedded into lipid droplets). To address the significance of this accumulation, we forced α-synuclein production in a strain unable to synthesize triglycerides and steryl esters. Surprisingly, the inability to store neutral lipids rendered the cells more tolerant to α-synuclein. Our results indicate that the level of α-synuclein toxicity is correlated with fatty acid synthase activity and intracellular redox status.

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae.

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis.

Hypersusceptibility to azole antifungals in a clinical isolate of Candida glabrata with reduced aerobic growth.

Petite mutations have been described in Saccharomyces cerevisiae and pathogenic yeasts. However, previous studies of the phenotypic traits of these petite mutants reported that they express azole resistance. We describe a clinical isolate of Candida glabrata with a striking association between increased susceptibility to azoles and respiratory deficiency. This isolate was obtained from a urine sample together with a respiration-competent C. glabrata isolate which exhibited azole resistance. The respiratory status of the two isolates was confirmed by cultivation on glycerol-containing agar and oxygraphy. Flow cytometry revealed the normal incorporation of rhodamine 123, and mitochondrial sections with typical cristae were seen by transmission electron microscopy for both isolates. Together, these results suggested a nuclear origin for the reduced respiratory capacity of the hypersusceptible isolate. The sterol contents of these isolates were similar to the sterol content of a reference strain. Sequencing of the ERG11 and PDR1 genes revealed that the sequences were identical in the two isolates, demonstrating their close relatedness. In addition to silent mutations, they carried a nonsense mutation in PDR1 that led to the truncation of transcription factor Pdr1p. They also overexpressed both PDR1 and one of its targets, CDR1, providing a possible explanation for the azole resistance of the respiration-competent isolate. In conclusion, in addition to azole resistance, which is a common feature of C. glabrata mitochondrial petite mutants, the mutation of a nuclear gene affecting aerobic growth may lead to azole hypersusceptibility; however, the mechanisms underlying this phenotype remain to be determined.

Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids.

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae. We show that SFA originating from either endogenous(preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.

Rsp5p ubiquitin ligase and the transcriptional activators Spt23p and Mga2p are involved in co-regulation of biosynthesis of end products of the mevalonate pathway and triacylglycerol in yeast Saccharomyces cerevisiae.

Rsp5p, a yeast S. cerevisiae ubiquitin ligase, is essential for regulation of unsaturated fatty acid synthesis via activation of the transcriptional activators Spt23p and Mga2p. Here we show that the conditional mutant rsp5-19 produces decreased levels of the end products of mevalonate pathway, such as ergosterol, ubiquinone and of dolichols, especially those with 19-24 isoprene units. The mechanism of Rsp5p involvement in the control of these lipid synthesis pathways was addressed by overproduction of Rsp5p-independent Spt23p or Mga2p. Expression of constitutively active forms of these transactivators resulted in excess production of ergosterol, but did not restore a wild-type level of dolichols. Moreover, synthesis of long chain dolichols was decreased in the wild-type and a rsp5-19 background. Finally, overproduction of active Spt23p or Mga2p was accompanied by the appearance of large lipid particles in the wild-type and rsp5-19 strains as observed by Nile Red staining, due to accumulation of unsaturated triacylglycerol. Thus, we conclude that Rsp5p, Spt23p and Mga2p may participate in the control of the homeostasis of lipids and lipid particles.

A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata.

Unlike the molecular mechanisms that lead to azole drug resistance, the molecular mechanisms that lead to polyene resistance are poorly documented, especially in pathogenic yeasts. We investigated the molecular mechanisms responsible for the reduced susceptibility to polyenes of a clinical isolate of Candida glabrata. Sterol content was analyzed by gas-phase chromatography, and we determined the sequences and levels of expression of several genes involved in ergosterol biosynthesis. We also investigated the effects of the mutation harbored by this isolate on the morphology and ultrastructure of the cell, cell viability, and vitality and susceptibility to cell wall-perturbing agents. The isolate had a lower ergosterol content in its membranes than the wild type, and the lower ergosterol content was found to be associated with a nonsense mutation in the ERG6 gene and induction of the ergosterol biosynthesis pathway. Modifications of the cell wall were also seen, accompanied by increased susceptibility to cell wall-perturbing agents. Finally, this mutation, which resulted in a marked fitness cost, was associated with a higher rate of cell mortality. Wild-type properties were restored by complementation of the isolate with a centromeric plasmid containing a wild-type copy of the ERG6 gene. In conclusion, we have identified the molecular event responsible for decreased susceptibility to polyenes in a clinical isolate of C. glabrata. The nonsense mutation detected in the ERG6 gene of this isolate led to a decrease in ergosterol content. This isolate may constitute a useful tool for analysis of the relevance of protein trafficking in the phenomena of azole resistance and pseudohyphal growth.

In vitro activity of terpenes against Candida biofilms.

The antibiofilm activity of 10 terpenes was tested in vitro against three Candida species by 24-h treatment of biofilms aged 1-5 days. Treatment of 24-h-old Candida albicans biofilms with carvacrol, geraniol or thymol (0.06%) resulted in >80% inhibition. Carvacrol (0.03%) inhibition was > or =75% independent of the age of the C. albicans biofilm. Carvacrol (0.125%) inhibition was >75% against Candida glabrata and Candida parapsilosis biofilms. Geraniol (> or =0.125%) and thymol (0.06% or 0.125%) inhibition was >75% against C. parapsilosis biofilms whatever their age. This study demonstrates the antibiofilm activity of terpenes and points out the exceptional efficiency of carvacrol, geraniol and thymol, which could represent candidates in the treatment of candidiasis associated with medical devices.

A lipid-mediated quality control process in the Golgi apparatus in yeast.

When heme biosynthesis is disrupted, the yeast Saccharomyces cerevisiae becomes unable to synthesize its major sterol, ergosterol, and desaturate fatty acids. We took advantage of this physiological peculiarity to evaluate the consequences of ergosterol and/or unsaturated fatty acid (UFA) depletions on the biogenesis of a model polytopic plasma membrane protein, the uracil permease Fur4p. We show that under UFA shortage, which results in low amounts of diunsaturated phospholipid species, and under ergosterol depletion, Fur4p is prematurely routed from the Golgi apparatus to the vacuolar lumen in a process that requires the ubiquitin ligase Rsp5p. Interestingly, this diversion is not correlated to Fur4p exclusion from detergent-resistant membranes. In an independent set of experiments, we show that Fur4p targeting to the plasma membrane depends on phosphatidylethanolamine amounts and more specifically on the propensity of this phospholipid to form a hexagonal phase. In light of recent literature, we propose a model in which ergosterol and diunsaturated phospholipid species maintain optimal membrane curvature for Fur4p to evade the Golgi quality control process and to be properly delivered to its normal destination.

SFH2 regulates fatty acid synthase activity in the yeast Saccharomyces cerevisiae and is critical to prevent saturated fatty acid accumulation in response to haem and oleic acid depletion.

The yeast Saccharomyces cerevisiae is a facultative anaerobic organism. Under anaerobiosis, sustained growth relies on the presence of exogenously supplied unsaturated fatty acids and ergosterol that yeast is unable to synthesize in the absence of oxygen or upon haem depletion. In the absence of exogenous supplementation with unsaturated fatty acid, a net accumulation of SFA (saturated fatty acid) is observed that induces significant modification of phospholipid profile [Ferreira, Régnacq, Alimardani, Moreau-Vauzelle and Bergès (2004) Biochem. J. 378, 899-908]. In the present paper, we focus on the role of SFH2/CSR1, a hypoxic gene related to SEC14 and its involvement in lipid metabolism upon haem depletion in the absence of oleic acid supplementation. We observed that inactivation of SFH2 results in enhanced accumulation of SFA and phospholipid metabolism alterations. It results in premature growth arrest and leads to an exacerbated sensitivity to exogenous SFA. This phenotype is suppressed in the presence of exogenous oleic acid, or by a controlled expression of FAS1, one of the two genes encoding FAS. We present several lines of evidence to suggest that Sfh2p and oleic acid regulate SFA synthase in yeast at different levels: whereas oleic acid acts on FAS2 at the transcriptional level, we show that Sfh2p inhibits fatty acid synthase activity in response to haem depletion.

Reduced susceptibility to polyenes associated with a missense mutation in the ERG6 gene in a clinical isolate of Candida glabrata with pseudohyphal growth.

Little information is available about the molecular mechanisms responsible for polyene resistance in pathogenic yeasts. A clinical isolate of Candida glabrata with a poor susceptibility to polyenes, as determined by disk diffusion method and confirmed by determination of MIC, was recovered from a patient treated with amphotericin B. Quantitative analysis of sterols revealed a lack of ergosterol and an accumulation of late sterol intermediates, suggesting a defect in the final steps of the ergosterol pathway. Sequencing of CgERG11, CgERG6, CgERG5, and CgERG4 genes revealed exclusively a unique missense mutation in CgERG6 leading to the substitution of a cysteine by a phenylalanine in the corresponding protein. In addition, real-time reverse transcription-PCR demonstrated an overexpression of genes encoding enzymes involved in late steps of the ergosterol pathway. Moreover, this isolate exhibited a pseudohyphal growth whatever the culture medium used, and ultrastructural changes of the cell wall of blastoconidia were seen consisting in a thinner inner layer. Cell wall alterations were also suggested by the higher susceptibility of growing cells to Calcofluor white. Additionally, complementation of this isolate with a wild-type copy of the CgERG6 gene restored susceptibility to polyenes and a classical morphology. Together, these results demonstrated that mutation in the CgERG6 gene may lead to a reduced susceptibility to polyenes and to a pseudohyphal growth due to the subsequent changes in sterol content of the plasma membrane.