Site-directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus.

Reactive intermediate deaminase A (RidA) is a highly conserved enzyme that catalyzes the hydrolysis of 2-imino acids to the corresponding 2-keto acids and ammonia. RidA thus prevents the accumulation of such potentially harmful compounds in the cell, as exemplified by its role in the degradation of 2-aminoacrylate, formed during the metabolism of cysteine and serine, catalyzing the conversion of its stable 2-iminopyruvate tautomer into pyruvate. Capra hircus (goat) RidA (ChRidA) was the first mammalian RidA to be isolated and described. It has the typical homotrimeric fold of the Rid superfamily, characterized by remarkably high thermal stability, with three active sites located at the interface between adjacent subunits. ChRidA exhibits a broad substrate specificity with a preference for 2-iminopyruvate and other 2-imino acids derived from amino acids with non-polar non-bulky side chains. Here we report a biophysical and biochemical characterization of eight ChRidA variants obtained by site-directed mutagenesis to gain insight into the role of specific residues in protein stability and catalytic activity. Each mutant was produced in Escherichia coli cells, purified and characterized in terms of quaternary structure, thermal stability and substrate specificity. The results are rationalized in the context of the high-resolution structures obtained by x-ray crystallography.

Apis mellifera RidA, a novel member of the canonical YigF/YER057c/UK114 imine deiminase superfamily of enzymes pre-empting metabolic damage.

The Reactive intermediate deiminase (Rid) protein family is a group of enzymes widely distributed in all Kingdoms of Life. RidA is one of the eight known Rid subfamilies, and its members act by preventing the accumulation of 2-aminoacrylate, a highly reactive enamine generated during the metabolism of some amino acids, by hydrolyzing the 2-iminopyruvate tautomer to pyruvate and ammonia. RidA members are homotrimers exhibiting a remarkable thermal stability. Recently, a novel subclass of RidA was identified in teleosts, which differs for stability and substrate specificity from the canonical RidA. In this study we structurally and functionally characterized RidA from Apis mellifera (AmRidA) as the first example of an invertebrate RidA to assess its belonging to the canonical RidA group, and to further correlate structural and functional features of this novel enzyme class. Circular dichroism revealed a spectrum typical of the RidA proteins and the high thermal stability. AmRidA exhibits the 2-imino acid hydrolase activity typical of RidA family members with a substrate specificity similar to that of the canonical RidA. The crystal structure confirmed the homotrimeric assembly and the presence of the typical structural features of RidA proteins, such as the proposed substrate recognition loop, and the ß-sheets ß1-ß9 and ß1-ß2. In conclusion, our data define AmRidA as a canonical member of the well-conserved RidA family and further clarify the diagnostic structural features of this class of enzymes.

Poacic acid, a ß-1,3-glucan-binding antifungal agent, inhibits cell-wall remodeling and activates transcriptional responses regulated by the cell-wall integrity and high-osmolarity glycerol pathways in yeast.

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.

Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis.

Myocardial aging increases the cardiovascular risk in the elderly. The Receptor for Advanced Glycation End-products (RAGE) is involved in age-related disorders. The soluble isoform (sRAGE) acts as a scavenger blocking the membrane-bound receptor activation. This study aims at investigating RAGE contribution to age-related cardiac remodeling. We analyzed the cardiac function of three different age groups of female Rage-/- and C57BL/6N (WT) mice: 2.5- (Young), 12- (Middle-age, MA) and 21-months (Old) old. While aging, Rage-/- mice displayed an increase in left ventricle (LV) dimensions compared to age-matched WT animals, with the main differences observed in the MA groups. Rage-/- mice showed higher fibrosis and a larger number of α-Smooth Muscle Actin (SMA)+ cells with age, along with increased expression of pro-fibrotic Transforming Growth Factor (TGF)-ß1 pathway components. RAGE isoforms were undetectable in LV of WT mice, nevertheless, circulating sRAGE declined with aging and inversely associated with LV diastolic dimensions. Human cardiac fibroblasts stimulated with sRAGE exhibited a reduction in proliferation, pro-fibrotic proteins and TGF-beta Receptor 1 (TGFbR1) expression and Smad2-3 activation. Finally, sRAGE administration to MA WT animals reduced cardiac fibrosis. Hence, our work shows that RAGE associates with age-dependent myocardial changes and indicates sRAGE as an inhibitor of cardiac fibroblasts differentiation and age-dependent cardiac fibrosis.

Using D- and L-Amino Acid Oxidases to Generate the Imino Acid Substrate to Measure the Activity of the Novel Rid (Enamine/Imine Deaminase) Class of Enzymes.

This chapter describes a method to assay the activity of reactive intermediate deaminases (Rid), a large family of conserved soluble enzymes, which have been proposed to prevent damages from metabolic intermediates such as the highly reactive and unstable compounds enamines/imines. In this method, the flavin adenine dinucleotide-dependent L- or D-amino acid oxidases generate an imino acid starting from a L- or D- amino acid, respectively. This reaction is coupled to the hydrolysis of the imino acid to the corresponding α-keto acid and ammonium ion catalyzed by a Rid enzyme. The spectrophotometric assay consists of measuring the decrease of the initial rate of formation of the semicarbazone, derived from the spontaneous reaction of the imino acid and semicarbazide, caused by the presence of the Rid enzyme. The set-up and testing of this method imply a preliminary characterization of the ability of the amino acid oxidase to release the imino acid required for the subsequent reactions. To this purpose, the activity of the L- or D-amino acid oxidases with different amino acids can be measured as production of hydrogen peroxide or formation of semicarbazone in parallel assays. The advantages and limitations of this assay of Rid activity are discussed.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage.

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products.

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10-7 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. Graphical abstract.

The Glucan-Remodeling Enzyme Phr1p and the Chitin Synthase Chs1p Cooperate to Maintain Proper Nuclear Segregation and Cell Integrity in Candida albicans.

GH72 family of ß-(1,3)-glucanosyltransferases is unique to fungi and is required for cell wall biogenesis, morphogenesis, virulence, and in some species is essential for life. Candida albicans PHR1 and PHR2 are pH-regulated genes that encode GH72 enzymes highly similar to Gas1p of Saccharomyces cerevisiae. PHR1 is expressed at pH ≥ 5.5 while PHR2 is transcribed at pH ≤ 5.5. Both are essential for C. albicans morphogenesis and virulence. During growth at neutral-alkaline pH, Phr1p-GFP preferentially localizes to sites of active cell wall formation as the incipient bud, the mother-daughter neck, the bud periphery, and concentrates in the septum at cytokinesis. We further investigated this latter localization. In chs3Δ cells, lacking the chitin of the chitin ring and lateral cell wall, Phr1p-GFP still concentrated along the thin line of the primary septum formed by chitin deposited by chitin synthase I (whose catalytic subunit is Chs1p) suggesting that it plays a role during formation of the secondary septa. RO-09-3143, a highly specific inhibitor of Chs1p activity, inhibits septum formation and blocks cell division. However, alternative septa are produced and are crucial for cell survival. Phr1p-GFP is excluded from such aberrant septa. Finally, we determined the effects of RO-09-3143 in cells lacking Phr1p. PHR1 null mutant was more susceptible to the drug than the wild type. The phr1Δ cells were larger, devoid of septa, and underwent endomitosis and cell death. Phr1p and Chs1p cooperate in maintaining cell integrity and in coupling morphogenesis with nuclear division in C. albicans.

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris.

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

Advanced lipoxidation end products (ALEs) as RAGE binders: Mass spectrometric and computational studies to explain the reasons why.

Advanced Lipoxidation End-products (ALEs) are modified proteins that can act as pathogenic factors in several chronic diseases. Several molecular mechanisms have so far been considered to explain the damaging action of ALEs and among these a pathway involving the receptor for advanced glycation end products (RAGE) should be considered. The aim of the present work is to understand if ALEs formed from lipid peroxidation derived reactive carbonyl species (RCS) are able to act as RAGE binders and also to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. ALEs were produced in vitro, by incubating human serum albumin (HSA) with 4-hydroxy-trans- 2-nonenal (HNE), acrolein (ACR) and malondialdehyde (MDA). The identification of ALEs was performed by MS. ALEs were then subjected to the VC1 Pull-Down assay (VC1 is the ligand binding domain of RAGE) and the enrichment factor (the difference between the relative abundance in the enriched sample minus the amount in the untreated one) as an index of affinity, was determined. Computation studies were then carried out to explain the factors governing the affinity of the adducted moieties and the site of interaction on adducted HSA for VC1-binding. The in silico analyses revealed the key role played by those adducts which strongly reduce the basicity of the modified residues and thus occur at their neutral state at physiological conditions (e.g. the MDA adducts, dihydropyridine-Lysine (DHPK) and N-2-pyrimidyl-ornithine (NPO), and acrolein derivatives, N-(3-formyl-3,4-dehydro-piperidinyl) lysine, FDPK). These neutral adducts become unable to stabilize ion-pairs with the surrounding negative residues which thus can contact the RAGE positive residues. In conclusion, ALEs derived from lipid peroxidation-RCS are binders of RAGE and this affinity depends on the effect of the adduct moiety to reduce the basicity of the target amino acid and on the acid moieties surrounding the aminoacidic target.

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism.

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

The Dual Activity Responsible for the Elongation and Branching of ß-(1,3)-Glucan in the Fungal Cell Wall.

ß-(1,3)-Glucan, the major fungal cell wall component, ramifies through ß-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify ß-(1,6)-branching on ß-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced ß-(1,6)-branching on the ß-(1,3)-oligomers following its ß-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear ß-(1,3)-oligomers as well as Bgl2p-catalyzed products [short ß-(1,3)-oligomers linked by a linear ß-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual ß-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual ß-(1,3)-glucan elongating and branching activity. Our report unravels the ß-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core ß-(1,3)-glucan ramification, is poorly understood; the ramified ß-(1,3)-glucan interconnects other cell wall components. Once linear ß-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the ß-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that ß-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual ß-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall ß-(1,3)-glucan branching mechanism. The essentiality of ß-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

The PHR Family: The Role of Extracellular Transglycosylases in Shaping Candida albicans Cells.

Candida albicans is an opportunistic microorganism that can become a pathogen causing mild superficial mycosis or more severe invasive infections that can be life-threatening for debilitated patients. In the etiology of invasive infections, key factors are the adaptability of C. albicans to the different niches of the human body and the transition from a yeast form to hypha. Hyphal morphology confers high adhesiveness to the host cells, as well as the ability to penetrate into organs. The cell wall plays a crucial role in the morphological changes C. albicans undergoes in response to specific environmental cues. Among the different categories of enzymes involved in the formation of the fungal cell wall, the GH72 family of transglycosylases plays an important assembly role. These enzymes cut and religate ß-(1,3)-glucan, the major determinant of cell shape. In C. albicans, the PHR family encodes GH72 enzymes, some of which work in specific environmental conditions. In this review, we will summarize the work from the initial discovery of PHR genes to the study of the pH-dependent expression of PHR1 and PHR2, from the characterization of the gene products to the recent findings concerning the stress response generated by the lack of GH72 activity in C. albicans hyphae.

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE).

The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.

Genomic and functional analyses unveil the response to hyphal wall stress in Candida albicans cells lacking ß(1,3)-glucan remodeling.

BACKGROUND: The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, ß(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH. We exploited the pH-conditional nature of a PHR1 null mutant to analyze the impact of glucan remodeling on the hyphal transcriptional program and the role of chitin synthases in the hyphal wall stress (HWS) response. RESULTS: In hyphal growth inducing conditions, phr1Δ germ tubes are defective in elongation, accumulate chitin, and constitutively activate the signaling pathways mediated by the MAP kinases Mkc1p, Cek1p and Hog1p. The transcriptional profiles revealed an increase of transcript levels for genes involved in cell wall formation (CHS2 and CHS8, CRH11, PGA23, orf19.750, RBR1, RBT4, ECM331, PGA6, PGA13), protein N-glycosylation and sorting in the ER (CWH8 and CHS7), signaling (CPP1, SSK2), ion transport (FLC2, YVC1), stress response and metabolism and a reduced expression of adhesins. A transient up-regulation of DNA replication genes associated with entry into S-phase occurred whereas cell-cycle regulating genes (PCL1, PCL2, CCN1, GIN4, DUN1, CDC28) were persistently up-regulated. To test the physiological relevance of altered CHS gene expression, phr1Δ chsxΔ (x = 2,3,8) mutant phenotypes were analyzed during the Y-H transition. PHR1 deletion was synthetic lethal with CHS3 loss on solid M199 medium-pH 7.5 and with CHS8 deletion on solid M199-pH 8. On Spider medium, PHR1 was synthetic lethal with CHS3 or CHS8 at pH 8. CONCLUSIONS: The absence of Phr1p triggers an adaptive response aimed to reinforce the hyphal cell wall and restore homeostasis. Chs3p is essential in preserving phr1Δ cell integrity during the Y-H transition. Our findings also unveiled an unanticipated essential role of Chs8p during filamentation on solid media. These results highlight the flexibility of fungal cells in maintaining cell wall integrity and contribute to assessments of glucan remodeling as a target for therapy.

Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition.

Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen Candida albicans, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope ß-glucan in its cell wall from host recognition. It has been demonstrated previously that ß-glucan becomes exposed during infection in vivo but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 ß-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.

Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH.

Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of ß(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively ß-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. The pH optimum was 5.8 for Phr1p and 3 for Phr2p. Overall, adaptation to pH of C. albicans appears to involve a fine interplay among the pH-dependent activity of Phr1p and Phr2p, the pH-regulated expression of their genes and protein stability. Unexpectedly, Pga4p was inactive suggesting that it turned into a structural mannoprotein.

Expression and phylogenetic analyses of the Gel/Gas proteins of Tuber melanosporum provide insights into the function and evolution of glucan remodeling enzymes in fungi.

The ß(1,3)-glucanosyltransferases of the GH72 family are redundant enzymes that are essential for the formation and dynamic remodeling of the fungal wall during different stages of the life cycle. Four putative genes encoding glycosylphosphatidylinositol (GPI)-anchored ß(1,3)-glucanosyltransferases, designated TmelGEL1, TmelGEL2, TmelGEL4 and TmelGAS4, have been annotated in the genome of Tuber melanosporum, an ectomycorrhizal fungus that also produces a hypogeous fruiting body (FB) of great commercial value (black truffle). This work focuses on the characterization and expression of this multigene family by taking advantage of a laser microdissection (LMD) technology that has been used to separate two distinct compartments in the FB, the hyphae and the asci containing the ascospores. Of the four genes, TmelGEL1 was the most up-regulated in the FB compared to the free-living mycelium. Inside the FB, the expression of TmelGEL1 was restricted to the hyphal compartment. A phylogenetic analysis of the Gel/Gas protein family of T. melanosporum was also carried out. A total of 237 GH72 proteins from 51 Ascomycotina and 3 Basidiomycota (outgroup) species were analyzed. The resulting tree provides insight into the evolution of the T. melanosporum proteins and identifies new GH72 paralogs/subfamilies. Moreover, it represents a starting point to formulate new hypotheses on the significance of the striking GH72 gene redundancy in fungal biology.