The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase.

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCE: Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a Ki value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.

Pps1, phosphatidylserine synthase, regulates the salt stress response in Schizosaccharomyces pombe.

Phosphatidylserine (PS) is important for maintaining growth, cytoskeleton, and various functions in yeast; however, its role in stress responses is poorly understood. In Schizosaccharomyces pombe, the PS synthase deletion (pps1∆) mutant shows defects in growth, morphology, cytokinesis, actin cytoskeleton, and cell wall integrity, and these phenotypes are rescued by ethanolamine supplementation. Here, we evaluated the role of Pps1 in the salt stress response in S. pombe. We found that pps1∆ cells are sensitive to salt stresses such as KCl and CaCl2 even in the presence of ethanolamine. Loss of the functional cAMP-dependent protein kinase (git3∆ or pka1∆) or phospholipase B Plb1 (plb1∆) enhanced the salt stress-sensitive phenotype in pps1∆ cells. Green fluorescent protein (GFP)-Pps1 was localized at the plasma membrane and endoplasmic reticulum regardless of the stress conditions. In pka1∆ cells, GFP-Pps1 was accumulated around the nucleus under the KCl stress. Pka1 was localized in the nucleus and the cytoplasm under normal conditions and transferred from the nucleus to the cytoplasm under salt-stress conditions. Pka1 translocated from the nucleus to the cytoplasm during CaCl2 stress in the wild-type cells, while it remained localized in the nucleus in pps1∆ cells. Expression and phosphorylation of Pka1-GFP were not changed in pps1∆ cells. Our results demonstrate that Pps1 plays an important role in the salt stress response in S. pombe.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

Defining ER-mitochondria contact dynamics in Plasmodium falciparum by targeting component of phospholipid synthesis pathway, phosphatidylserine synthase (PfPSS).

The malaria parasite completes the asexual cycle inside the host erythrocyte, which requires extensive membrane biogenesis for its development and multiplication. Metabolic pathways for the synthesis of membrane phospholipids (PL), including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), are crucial for parasite survival. Here, we have studied the P. falciparum enzyme responsible for PS synthesis, Phosphatidylserine synthase (PfPSS), GFP targeting approach confirmed it to be localized in the parasite ER as well as in ER-protrusions. Detailed high resolution microscopy, using these transgenic parasites expressing PfPSS-GFP, redefined the dynamics of ER during the intraerythrocytic life cycle and its association with the mitochondria. We report for the first time presence of ER-mitochondria contact (ERMC) in Plasmodium; ERMC is formed by PfPSS containing ER-protrusions, which associate with the mitochondria surface throughout the parasite growth cycle. Further, ERMC is found to be stable and refractory to ER and mitochondrial stresses, suggesting that it is formed through strong tethering complexes. PfPSS was found to interact with other major key enzyme involved in PL synthesis, choline/Etn-phosphotransferase (CEPT), which suggest that ER is the major site for PL biosynthesis. Overall, this study defines the morphological organisation of ERMC which mediates PL synthesis/transport in the Plasmodium.

Phosphatidylserine Synthase PTDSS1 Shapes the Tumor Lipidome to Maintain Tumor-Promoting Inflammation.

An altered lipidome in tumors may affect not only tumor cells themselves but also their microenvironment. In this study, a lipidomics screen reveals increased amounts of phosphatidylserine (PS), particularly ether-PS (ePS), in murine mammary tumors compared with normal tissue. PS was produced by phosphatidylserine synthase 1 (PTDSS1), and depletion of Ptdss1 from tumor cells in mice reduced ePS levels accompanied by stunted tumor growth and decreased tumor-associated macrophage (TAM) abundance. Ptdss1-deficient tumor cells exposed less PS during apoptosis, which was recognized by the PS receptor MERTK. Mammary tumors in macrophage-specific Mertk-/- mice showed similarly suppressed growth and reduced TAM infiltration. Transcriptomic profiles of TAMs from Ptdss1-knockdown tumors and Mertk-/- TAMs revealed that macrophage proliferation was reduced when the Ptdss1/Mertk pathway was targeted. Moreover, PTDSS1 expression correlated positively with TAM abundance but negatively with breast carcinoma patient survival. PTDSS1 thus may be a target to modify tumor-promoting inflammation. SIGNIFICANCE: This study shows that inhibiting the production of ether-phosphatidylserine by targeting phosphatidylserine synthase PTDSS1 limits tumor-associated macrophage expansion and breast tumor growth.

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases.

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

Mapping the Substrate-Binding Sites in the Phosphatidylserine Synthase in Candida albicans.

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.

Phosphatidylserine flux into mitochondria unveiled by organelle-targeted Escherichia coli phosphatidylserine synthase PssA.

Most phospholipids are synthesised in the endoplasmic reticulum and distributed to other cellular membranes. Although the vesicle transport contributes to the phospholipid distribution among the endomembrane system, exactly how phospholipids are transported to, from and between mitochondrial membranes remains unclear. To gain insights into phospholipid transport routes into mitochondria, we expressed the Escherichia coli phosphatidylserine (PS) synthase PssA in various membrane compartments with distinct membrane topologies in yeast cells lacking a sole PS synthase (Cho1). Interestingly, PssA could complement loss of Cho1 when targeted to the endoplasmic reticulum (ER), peroxisome, or lipid droplet membranes. Synthesised PS could be converted to phosphatidylethanolamine (PE) by Psd1, the mitochondrial PS decarboxylase, suggesting that phospholipids synthesised in the peroxisomes and low doses (LDs) can efficiently reach mitochondria. Furthermore, we found that PssA which has been integrated into the mitochondrial inner membrane (MIM) from the matrix side could partially complement the loss of Cho1. The PS synthesised in the MIM was also converted to PE, indicating that PS flops across the MIM to become PE. These findings expand our understanding of the intracellular phospholipid transport routes via mitochondria.

Phosphatidylserine Synthase from Salicornia europaea Is Involved in Plant Salt Tolerance by Regulating Plasma Membrane Stability.

Salinity-induced lipid alterations have been reported in many plant species; however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here, a constitutively much higher phosphatidylserine (PS) content in the plasma membrane (PM) was found in the euhalophyte Salicornia europaea than in Arabidopsis. A gene encoding PS synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested that SePSS belongs to a base exchange-type PSS, which localises to the endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, and increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, the upregulation of SePSS leads to increased PS and phosphatidylethanolamine levels and enhanced salt tolerance in Arabidopsis, along with a lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than in wild-type (WT). These results suggest a positive correlation between PS levels and plant salt tolerance, and that SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea.

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination.IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

Characterization of a Phosphatidylserine Synthase of Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.

Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae.

To increase the growth of industrial strains under environmental stress, the Saccharomyces cerevisiae BY4741 salt-tolerant strain Y00 that tolerates 1.2 M NaCl was cultured through nitroguanidine mutagenesis. The metabolomics and transcription data of Y00 were compared with those of the wild-type strain BY4741. The comparison identified two genes related to salt stress tolerance, cds1 and cho1. Modular assembly of cds1 and cho1 redistributed the membrane phospholipid component and decreased the ratio of anionic-to-zwitterionic phospholipid in strain Y03 that showed the highest salt tolerance. Therefore, significantly increased membrane potential and membrane integrity helped strain Y03 to resist salt stress (1.2 M NaCl). This study provides an effective membrane engineering strategy to enhance salt stress tolerance.

Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production.

Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.

Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms.

Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. Here, we report that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIα partially rescues the mitochondrial defects of pss RNAi. Together, our results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms.

Molecular and functional evidence of phosphatidylserine synthase in Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Exposure of Candida albicans ß (1,3)-glucan is promoted by activation of the Cek1 pathway.

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of ß (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of ß (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in ß (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of ß (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased ß (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.

Plasma membrane architecture protects Candida albicans from killing by copper.

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

Contribution of phosphatidylserine to Rho1- and Pkc1-related repolarization of the actin cytoskeleton under stressed conditions in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae undergoes polarized cell growth, which is established in association with actin polarization. Rho1, one of the Rho-type GTPases in S. cerevisiae, is crucial for maintaining polarized cell growth and actin polarization and controlling the downstream signaling pathway, the Pkc1-Mpk1 MAP kinase cascade, through a physical interaction with Pkc1, the sole protein kinase C in this yeast. The Pkc1-Mpk1 MAP kinase cascade is important for the repolarization of actin under heat shock-stressed conditions. We recently reported that phosphatidylserine (PS), a membrane phospholipid component, played a pivotal role in the physical interaction between Rho1 and Pkc1 as well as the activation of the Pkc1-Mpk1 MAP kinase cascade. However, it currently remains unclear whether PS is involved in actin polarization by regulating the physical interaction between Rho1 and Pkc1. We herein demonstrated that the C1 domain of Pkc1, which is responsible for the interaction with Rho1, was crucial for Rho1-regulated actin polarization. We also found that actin repolarization under heat shock-stressed conditions was impaired in a mutant defective in CHO1 encoding PS synthase. These results suggest that PS contributes to actin polarization in which Rho1 and Pkc1 play a crucial role.