Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria.

The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.

Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline.

In the yeast Saccharomyces cerevisiae, the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p (scs2Δ) and a mutant, OPI1FFAT, lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis.

Activation of protein kinase C-mitogen-activated protein kinase signaling in response to inositol starvation triggers Sir2p-dependent telomeric silencing in yeast.

Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.

Coordination of storage lipid synthesis and membrane biogenesis: evidence for cross-talk between triacylglycerol metabolism and phosphatidylinositol synthesis.

Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino(-) phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino(-) phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino(-) phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells.

Interruption of inositol sphingolipid synthesis triggers Stt4p-dependent protein kinase C signaling.

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.

Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling.

Inositol auxotrophy (Ino(-) phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino(-) phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino(-) mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed.

Molecular Dialogues between Early Divergent Fungi and Bacteria in an Antagonism versus a Mutualism.

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

The emergence of yeast lipidomics.

The emerging field of lipidomics, driven by technological advances in lipid analysis, provides greatly enhanced opportunities to characterize, on a quantitative or semi-quantitative level, the entire spectrum of lipids, or lipidome, in specific cell types. When combined with advances in other high throughput technologies in genomics and proteomics, lipidomics offers the opportunity to analyze the unique roles of specific lipids in complex cellular processes such as signaling and membrane trafficking. The yeast system offers many advantages for such studies, including the relative simplicity of its lipidome as compared to mammalian cells, the relatively high proportion of structural and regulatory genes of lipid metabolism which have been assigned and the excellent tools for molecular genetic analysis that yeast affords. The current state of application of lipidomic approaches in yeast and the advantages and disadvantages of yeast for such studies are discussed in this report.

Biology of Fungi and Their Bacterial Endosymbionts.

Heritable symbioses, in which endosymbiotic bacteria (EB) are transmitted vertically between host generations, are an important source of evolutionary novelties. A primary example of such symbioses is the eukaryotic cell with its EB-derived organelles. Recent discoveries suggest that endosymbiosis-related innovations can be also found in associations formed by early divergent fungi in the phylum Mucoromycota with heritable EB from two classes, Betaproteobacteria and Mollicutes. These symbioses exemplify novel types of host-symbiont interactions. Studies of these partnerships fuel theoretical models describing mechanisms that stabilize heritable symbioses, control the rate of molecular evolution, and enable the establishment of mutualisms. Lastly, by altering host phenotypes and metabolism, these associations represent an important instrument for probing the basic biology of the Mucoromycota hosts, which remain one of the least explored filamentous fungi.

Bacterial endosymbionts influence host sexuality and reveal reproductive genes of early divergent fungi.

Many heritable mutualisms, in which beneficial symbionts are transmitted vertically between host generations, originate as antagonisms with parasite dispersal constrained by the host. Only after the parasite gains control over its transmission is the symbiosis expected to transition from antagonism to mutualism. Here, we explore this prediction in the mutualism between the fungus Rhizopus microsporus (Rm, Mucoromycotina) and a beta-proteobacterium Burkholderia, which controls host asexual reproduction. We show that reproductive addiction of Rm to endobacteria extends to mating, and is mediated by the symbiont gaining transcriptional control of the fungal ras2 gene, which encodes a GTPase central to fungal reproductive development. We also discover candidate G-protein-coupled receptors for the perception of trisporic acids, mating pheromones unique to Mucoromycotina. Our results demonstrate that regulating host asexual proliferation and modifying its sexual reproduction are sufficient for the symbiont's control of its own transmission, needed for antagonism-to-mutualism transition in heritable symbioses. These properties establish the Rm-Burkholderia symbiosis as a powerful system for identifying reproductive genes in Mucoromycotina.

Spatially restricted JAG1-Notch signaling in human thymus provides suitable DC developmental niches.

A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34+ CD123+ progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123+ monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1+ thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development.

Role of the unfolded protein response pathway in secretory stress and regulation of INO1 expression in Saccharomyces cerevisiae.

The unfolded protein response pathway (UPR) enables the cell to cope with the buildup of unfolded proteins in the endoplasmic reticulum (ER). UPR loss-of-function mutants, hac1Delta and ire1Delta, are also inositol auxotrophs, a phenotype associated with defects in expression of INO1, the most highly regulated of a set of genes encoding enzymes of phospholipid metabolism. We now demonstrate that the UPR plays a functional role in membrane trafficking under conditions of secretory stress in yeast. Mutations conferring a wide range of membrane trafficking defects exhibited negative genetic interaction when combined with ire1Delta and hac1Delta. At semipermissive temperatures, carboxypeptidase Y transit time to the vacuole was slower in Sec(-) cells containing an ire1Delta or hac1Delta mutation than in Sec(-) cells with an intact UPR. The UPR was induced in Sec(-) cells defective in subcellular membrane trafficking events ranging from ER vesicle trafficking to distal secretion and in erg6Delta cells challenged with brefeldin A. However, the high levels of UPR induction observed under these conditions were not correlated with elevated INO1 expression. Indeed, many of the Sec(-) mutants that had elevated UPR expression at semipermissive growth temperatures failed to achieve wild-type levels of INO1 expression under these same conditions.

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix.

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

Expression of the VRK (vaccinia-related kinase) gene family of p53 regulators in murine hematopoietic development.

The vaccinia-related kinase (VRK) proteins are a new group of three Ser-Thr kinases in the human kinome. VRK proteins are upstream regulators of several transcription factors. VRK1 phosphorylates p53 in Thr-18 within the region of binding to mdm2 preventing their interaction. The tissue distribution of three genes is still largely unknown. In the present report the expression of these genes was analyzed during murine hematopoietic development. The three genes are expressed in fetal liver and peripheral blood, with higher levels between days 11.5 and 13.5, a time when there is a massive expansion of liver cells, and thereafter their expression falls significantly. VRK genes are expressed, particularly at mid-gestation, in embryo thymus and spleen, but in adult thymus and spleen their levels are very low. VRK2 is expressed at lower levels than VRK1 and VRK3 in the mouse embryo. VRK genes play a role during embryonic development of hematopoiesis.

The response to inositol: regulation of glycerolipid metabolism and stress response signaling in yeast.

This article focuses on discoveries of the mechanisms governing the regulation of glycerolipid metabolism and stress response signaling in response to the phospholipid precursor, inositol. The regulation of glycerolipid lipid metabolism in yeast in response to inositol is highly complex, but increasingly well understood, and the roles of individual lipids in stress response are also increasingly well characterized. Discoveries that have emerged over several decades of genetic, molecular and biochemical analyses of metabolic, regulatory and signaling responses of yeast cells, both mutant and wild type, to the availability of the phospholipid precursor, inositol are discussed.

Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

Cell wall integrity MAPK pathway is essential for lipid homeostasis.

The highly conserved yeast cell wall integrity mitogen-activated protein kinase pathway regulates cellular responses to cell wall and membrane stress. We report that this pathway is activated and essential for viability under growth conditions that alter both the abundance and pattern of synthesis and turnover of membrane phospholipids, particularly phosphatidylinositol and phosphatidylcholine. Mutants defective in this pathway exhibit a choline-sensitive inositol auxotrophy, yet fully derepress INO1 and other Opi1p-regulated genes when grown in the absence of inositol. Under these growth conditions, Mpk1p is transiently activated by phosphorylation and stimulates the transcription of known targets of Mpk1p signaling, including genes regulated by the Rlm1p transcription factor. mpk1Delta cells also exhibit severe defects in lipid metabolism, including an abnormal accumulation of phosphatidylcholine, diacylglycerol, triacylglycerol, and free sterols, as well as aberrant turnover of phosphatidylcholine. Overexpression of the NTE1 phospholipase B gene suppresses the choline-sensitive inositol auxotrophy of mpk1Delta cells, whereas overexpression of other phospholipase genes has no effect on this phenotype. These results indicate that an intact cell wall integrity pathway is required for maintaining proper lipid homeostasis in yeast, especially when cells are grown in the absence of inositol.

A block in endoplasmic reticulum-to-Golgi trafficking inhibits phospholipid synthesis and induces neutral lipid accumulation.

Seeking to better understand how membrane trafficking is coordinated with phospholipid synthesis in yeast, we investigated lipid synthesis in several Sec(-) temperature-sensitive mutants, including sec13-1. Upon shift of sec13-1 cells to the restrictive temperature of 37 degrees C, phospholipid synthesis decreased dramatically relative to the wild type control, whereas synthesis of neutral lipids, especially triacylglycerol (TAG), increased. When examined by fluorescence microscopy, the number of lipid droplets appeared to increase and formed aggregates in sec13-1 cells shifted to 37 degrees C. Electron microscopy confirmed the increase in lipid droplet number and revealed that many were associated with the vacuole. Analysis of lipid metabolism in strains lacking TAG synthase genes demonstrated that the activities of the products of these genes contribute to accumulation of TAG in sec13-1 cells after the shift to 37 degrees C. Furthermore, the permissive temperature for growth of the sec13-1 strain lacking TAG synthase genes was 3 degrees C lower than sec13-1 on several different growth media, indicating that the synthesis of TAG has physiological significance under conditions of secretory stress. Together these results suggest that following a block in membrane trafficking, yeast cells channel lipid metabolism from phospholipid synthesis into synthesis of TAG and other neutral lipids to form lipid droplets. We conclude that this metabolic switch provides a degree of protection to cells during secretory stress.

Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae.

The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from approximately 40% of total phosphatidylcholine to a basal level of less than 5%. nte1Delta cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast.