Yeh1 constitutes the major steryl ester hydrolase under heme-deficient conditions in <i>Saccharomyces cerevisiae</i>

Steryl esters are stored in intracellular lipid droplets from which they are mobilized upon demand and hydrolyzed to yield free sterols and fatty acids. The mechanisms that control steryl ester mobilization are not well understood. We have previously identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester hydrolysis, Yeh1, Yeh2, and Tgl1 (R. Köffel, R. Tiwari, L. Falquet, and R. Schneiter, Mol. Cell. Biol. 25:1655-1668, 2005). Both Yeh1 and Tgl1 localize to lipid droplets, whereas Yeh2 is localized to the plasma membrane. To characterize the precise function of these three partially redundant lipases, we examined steryl ester mobilization under heme-deficient conditions. S. cerevisiae is a facultative anaerobic organism that becomes auxotrophic for sterols and unsaturated fatty acids in the absence of molecular oxygen. Anaerobic conditions can be mimicked in cells that are deficient for heme synthesis. We here report that Yeh1 is the sole active steryl ester hydrolase under such heme-deficient conditions, indicating that Yeh1 is activated whereas Yeh2 and Tgl1 are inactivated by the lack of heme. The heme-dependent activation of Yeh1 is mediated at least in part by an increase in steady-state levels of Yeh1 at the expense of Yeh2 and Tgl1 in exponentially growing cells. This increase in steady-state levels of Yeh1 requires Rox3, a component of the mediator complex that regulates transcription by RNA polymerase II. These data thus provide the first link between fat degradation and the transcriptional control of lipase activity in yeast

Engineered Liposomes Protect Immortalized Immune Cells from Cytolysins Secreted by Group A and Group G Streptococci.

The increasing antibiotic resistance of bacterial pathogens fosters the development of alternative, non-antibiotic treatments. Antivirulence therapy, which is neither bacteriostatic nor bactericidal, acts by depriving bacterial pathogens of their virulence factors. To establish a successful infection, many bacterial pathogens secrete exotoxins/cytolysins that perforate the host cell plasma membrane. Recently developed liposomal nanotraps, mimicking the outer layer of the targeted cell membranes, serve as decoys for exotoxins, thus diverting them from attacking host cells. In this study, we develop a liposomal nanotrap formulation that is capable of protecting immortalized immune cells from the whole palette of cytolysins secreted by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis-important human pathogens that can cause life-threatening bacteremia. We show that the mixture of cholesterol-containing liposomes with liposomes composed exclusively of phospholipids is protective against the combined action of all streptococcal exotoxins. Our findings pave the way for further development of liposomal antivirulence therapy in order to provide more efficient treatment of bacterial infections, including those caused by antibiotic resistant pathogens

Host-Derived Microvesicles Carrying Bacterial Pore-Forming Toxins Deliver Signals to Macrophages: A Novel Mechanism of Shaping Immune Responses

Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14+MHCIIlowCD86low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance

Eukaryotic sterol homeostasis: steryl ester hydrolases in "Saccharomyces cerevisiae"

Sterol-Homeostase in Eukaryonten beruht auf der reziproken Umwandlung von freien Sterolen und Sterol-Estern (STE). Die Mechanismen des Aufbaus von STE sind bekannt, diejenigen der Mobilisierung von STE sind jedoch noch weitgehend unbekannt. STE stellen eine wichtige Speicherform für Fettsäuren und Sterole dar und werden intrazellulär in Lipidpartikeln gespeichert. Um die Enzyme, welche für die Mobilisierung von STE notwendig sind, zu identifizieren, führten wir eine in silico-Analyse des Hefegenoms zur Auffindung von für STE-Hydrolasen kodierenden Genen durch. Die Genomanalyse zeigte acht potentielle STE-Hydrolase kodierende Gene. Um zu testen, ob eine oder mehrere dieser Lipasen für die Mobilisierung von STE notwendig sind, entwickelten wir einen in vivo-Assay, mit welchem wir die Hydrolyse von radioaktiv markierten STE in Saccharomyces cerevisiae beobachten konnten. In Mutanten, welche für Lipase-Kandidaten Gene deletiert waren, wurde durch Blockierung der endogenen Sterol-Biosynthese mit Terbinafine die STE Mobilisierung initiiert. Dies ermöglichte uns die Rate der Hydrolyse von [3H]Palmitinsäure oder [14C]Cholesterol-markierten STE in diesen Mutanten zu bestimmen. Diese Experimente zeigten, dass die Rate der STE-Mobilisierung in Zellen, die für YLL012/YEH1 (Steryl-Ester- Hydrolase 1), YLR020/YEH2 oder TGL1 deletiert waren, sehr gering war. Dies traf jedoch nicht auf die Hydrolyse von Triacylglycerol (TAG) zu. Diese drei Lipasen sind Paraloge der menschlichen sauren Lipasefamilie, welche aus der lysosomalen sauren Lipase, der gastrischen Lipase und vier neuen, bisher nicht charakterisierten menschlichen ORFs besteht. Lipasemutanten mobilisieren STE in verschiedenem Ausmaß, was durch eine teilweise funktionelle Überlappung der Aktivität der drei Genprodukte bedingt ist. Eine yeh1Δ yeh2Δ tgl1Δ-Triple-Mutante hingegen zeigt keine STE-Mobilisierung mehr, was darauf schließen lässt, dass die drei Genprodukte zusammen für sämtliche STE-Hydrolase-Aktivitäten in Hefe verantwortlich sind. Die STE-Hydrolase-Aktivität der drei Lipasen wurde durch in vitro- Assays, in denen radioaktiv markierte Cholesteryl-Ester als Substrat verwendet wurden, bestätigt. Funktionelle GFP-markierte STE-Hydrolasen Yeh1p und Tgl1p kolokalisieren mit Erg6p, einem Markerprotein für Lipidpartikel, und sind in isolierten Lipidpartikeln angereichert. Interessanterweise lokalisiert die dritte Lipase, Yeh2p, an der Zellperipherie und kann nicht in Lipidpartikeln gefunden werden. Laut Sequenzanalysen kodieren YEH1, YEH2 und TGL1 für Membranproteine. Die drei Lipasen können nur in Gegenwart von Detergenz solubilisiert werden, was darauf schließen lässt, dass sie sich tatsächlich wie integrale Membranproteine verhalten. Dadurch sind diese die ersten Membran-verankerten Lipasen, die bisher beschrieben wurden. Proteinase-Verdau-Experimente, in denen GFP-markierte Lipasen benutzt wurden, ließen uns auch die Membrantopologie der drei Lipasen feststellen. Die Mechanismen, welche die STE-Mobilisierung kontrollieren, sind noch weitgehend unbekannt. Beispielweise wird die menschliche hormonsensitive Lipase (HSL) nach lipolytischer Stimulation phosphoriliert, was in einer hundertfachen Aktivitätssteigerung resultiert. Eine detaillierte Analyse der drei STE-Hydrolasen zeigte, dass zwar Yeh2p durch Phosphorilierung modifiziert wird, nicht aber Yeh1p oder Tgl1p. Diese Phosphorilierung hängt von der Wachstumsphase der Zelle ab. Ein Mutantenscreen nach Proteinkinasen, welche für die Phosphorilierung von Yeh2p nötig sind, zeigte, dass jede der vier Proteinkinasen, SWE1, VHS1, KCC4 und YNR047 für die Phosphorilierung von Yeh2p notwendig ist. Die biologische Signifikanz dieser posttranslationalen Modifikation ist bisher noch nicht bekannt. Die Hefe ist ein fakultativ anaerober Organismus, der in Abwesenheit von Sauerstoff auxotroph für Sterole und ungesättigte Fettsäuren wird. Es wird in dieser Arbeit auch gezeigt, dass Yeh1p die einzige aktive STE-Hydrolase unter Häm-depletierten Konditionen ist. Die “steady-state-levels“ von Yeh1p sind in Häm-depletierten Zellen signifikant erhöht, was mit der Beobachtung kongruiert, dass Yeh1p wichtig für STEMobilisierung unter diesen Bedingungen ist. Zusammengefasst bedeutet dies, dass die hier präsentierte Arbeit eine neue Klasse von Membran-verankerten Lipasen, die für die Mobilisierung von STE in Hefe notwendig sind, beschreibt. Diese Lipasen unterscheiden sich in ihrer subzellulären Lokalisation und der Membrantopologie. Zukünftige Studien, die sich auf das hier erarbeitete Material stützen, könnten detaillierte Einblicke in die Regulation der drei Hefelipasen geben und dazu beitragen, die physiologischen Aspekte von STE in Hefe zu klären. Da man erst beginnt, die regulatorischen Aspekte von STE-Lagerung und -Mobilisierung im Menschen zu verstehen, besteht ein großes Interesse an der Regulierung und vor allem an den Konsequenzen von Defekten im Neutral-Lipid-Metabolismus. Heutzutage steigt die Anzahl schwerer Krankheiten, verursacht durch Lipid-Akkumulation, wie z.B. Arteriosklerose, Adipositas und Typ-2-Diabetes, stetig an. Die drei neu entdeckten und hier beschriebenen STE-Hydrolasen sind Paraloge der menschlichen sauren Lipasefamilie. Somit stellt die Hefe einen exzellenten Modellorganismus dar, der wichtige Fragen betreffend die Regulation von STE-Mobilisierung klären kann. Die Ergebnisse sollten zu einem besseren Verständnis der grundsätzlichen Mechanismen der Cholesterol-Homeostase in Menschen führen.Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters (STE). The formation of STE is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. STE constitute an important storage form for fatty acids and sterols that are deposited in intracellular lipid particles. To identify genes that are required for the mobilization of STE, we performed an in silico analysis of the yeast genome to identify putative STE hydrolase encoding genes. This candidate gene approach revealed eight putative lipase encoding genes. To test whether one or more of these putative lipases are required for STE hydrolysis we developed in vivo assays to monitor hydrolysis of radiolabeled steryl esters in Saccharomyces cerevisiae. Upon depletion of endogenous sterol biosynthesis by terbinafine, hydrolysis of [3H]palmitic acid- or [14C]cholesterol-labeled steryl esters was monitored in cells bearing deletion of candidate hydrolase genes. This analysis revealed that the rate of STE mobilization, but not that of triacylglyerol (TAG), is strongly decreased in cells lacking YLL012/YEH1 (steryl ester hydrolase 1), YLR020/YEH2, or TGL1. These lipases are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase single mutants mobilize STE to various degrees, indicating partial functional redundancy of the three gene products. A triple yeh1Δ yeh2Δ tgl1Δ mutant shows no STE mobilization, indicating that these three genes together encode for all the STE hydrolyase activity present in yeast. STE hydrolase activity of the three lipases was confirmed by in vitro assays with radiolabeled cholesteryl ester as substrate. Functional GFP-tagged Yeh1p and Tgl1p co-localize with Erg6p, a marker protein for lipid particles, and are enriched in isolated lipid particles. Interestingly, the third lipase, Yeh2p, is localized to the cell periphery and is absent from lipid particles. YEH1, YEH2, and TGL1 encode predicted membrane proteins and were shown to be solubilized by detergent treatment only, indicating that they indeed behave as integral membrane proteins. This makes them the first membrane-anchored lipases described so far. Proteinase protection experiments, using GFP-tagged lipases, enabled us to also determine their membrane topology. The mechanisms that control STE mobilization are not well understood. The mammalian hormone sensitive lipase (HSL) has been shown to be phosphorylated upon lipolytic stimulation, which resulted in a 100-fold increase of its activity. Detailed analysis of the three STE hydrolases revealed that Yeh2p, but not Yeh1p or Tgl1p, is modified by phosphorylation and this phosphorylation depends on the growth phase of the cell. A deletion mutant screen for protein kinases that are required for phosphorylation of Yeh2p revealed that every one of the four protein kinases, SWE1, VHS1, KCC4, and YNR047 is required for phosphorylation of Yeh2p. The biological significance of this posttranslational modification, however, is not yet known. Yeast is a facultative anaerobic organism that becomes auxotrophic for sterols and unsaturated fatty acids in the absence of oxygen. It is also reported here that Yeh1p is the only active STE hydrolase in heme-deficient conditions. The steady-state levels of Yeh1p are significantly increased in heme-deficient cells, which is in line with the observation that Yeh1p is important for STE mobilization under these conditions. Taken together, the work presented here describes a novel class of membrane-anchored lipases required for STE mobilization in yeast. These lipases differ in their subcellular localization and membrane topology. Future studies based on the data acquired here will allow providing detailed insight into regulation of the three lipases and also help to clarify the physiological importance of STE in yeast. The question how STE storage and mobilization is regulated in humans is important for the understanding of an increasing number of human disorders in lipid metabolism, such as atherosclerosis, obesity and type 2 diabetes. The three STE hydrolases described in this work are paralogues of the mammalian acid lipase family and yeast will thus provide an excellent model organism to address the question how STE mobilization is regulated which in turn should be valuable for the understanding of basic principles of STE homeostasis in mammals

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization

An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved

Tailored liposomal nanotraps for the treatment of Streptococcal infections.

BACKGROUND Streptococcal infections are associated with life-threatening pneumonia and sepsis. The rise in antibiotic resistance calls for novel approaches to treat bacterial diseases. Anti-virulence strategies promote a natural way of pathogen clearance by eliminating the advantage provided to bacteria by their virulence factors. In contrast to antibiotics, anti-virulence agents are less likely to exert selective evolutionary pressure, which is a prerequisite for the development of drug resistance. As part of their virulence mechanism, many bacterial pathogens secrete cytolytic exotoxins (hemolysins) that destroy the host cell by destabilizing their plasma membrane. Liposomal nanotraps, mimicking plasmalemmal structures of host cells that are specifically targeted by bacterial toxins are being developed in order to neutralize-by competitive sequestration-numerous exotoxins. RESULTS In this study, the liposomal nanotrap technology is further developed to simultaneously neutralize the whole palette of cytolysins produced by Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis-pathogens that can cause life-threatening streptococcal toxic shock syndrome. We show that the mixture of liposomes containing high amounts of cholesterol and liposomes composed exclusively of choline-containing phospholipids is fully protective against the combined action of exotoxins secreted by these pathogens. CONCLUSIONS Unravelling the universal mechanisms that define targeting of host cells by streptococcal cytolysins paves the way for a broad-spectrum anti-toxin therapy that can be applied without a diagnostic delay for the treatment of bacterial infections including those caused by antibiotic-resistant pathogens

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of immune cell types

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore‐forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans‐membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T‐cell line Jurkat, and myeloid cell lines U937 and THP‐1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP‐1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first‐line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin