Caroline Barelle

Niche-specific regulation of central metabolic pathways in a fungal pathogen

To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six‐carbon compounds via the glycolytic pathway, and two‐carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis.

GFP as a quantitative reporter of gene regulation in Candida albicans

A system has been developed for the quantitative analysis of gene expression within individual Candida albicans cells in infected tissue. The system is based on the plasmid pGFP, which contains the codon‐optimized yeast enhanced green fluorescent protein (yEGFP; Cormack et al., 1997 ) cloned between a basal CaADH1 promoter and the ScCYC1 terminator on an integrating vector. Promoters were inserted into pGFP and GFP levels measured in individual cells by quantitative fluorescence microscopy. Analysis of pPCK1–GFP and pMET3–GFP fusions revealed that GFP folds rapidly following gene induction, and is turned over rapidly following gene repression. Hence, single cell fluorescence measurements are likely to reflect ongoing gene expression levels with reasonable accuracy. pACT1–GFP expression levels were relatively constant during growth of C. albicans in both yeast and hyphal forms, and during growth in vivo in the mouse model of systemic infection. Therefore, pACT1–GFP provides a useful control for this quantitative GFP‐based system in future analyses of C. albicans molecular responses during fungal infections. Copyright

Asynchronous Cell Cycle and Asymmetric Vacuolar Inheritance in True Hyphae of Candida albicans

ABSTRACT Candida albicans forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G1 phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of C. albicans and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.

Antibody-targeted myofibroblast apoptosis reduces fibrosis during sustained liver injury

BACKGROUND/AIMS Myofibroblast apoptosis promotes the resolution of liver fibrosis. However, retaining macrophages may enhance reversal. The effects of specifically stimulating myofibroblast apoptosis in vivo were assessed. METHODS A single chain antibody (C1-3) to an extracellular domain of a myofibroblast membrane protein was injected as a fluorescent- or gliotoxin conjugate into mice with liver fibrosis. RESULTS C1-3 specifically targeted alpha-smooth muscle actin positive liver myofibroblasts within scar regions of the liver in vivo and did not co-localise with liver monocytes/macrophages. Injection of free gliotoxin stimulated a 2-fold increase in non-parenchymal cell apoptosis and depleted liver myofibroblasts by 30% and monocytes/macrophages by 50% but had no effect on fibrosis severity in the sustained injury model employed. In contrast, C1-3-targeted gliotoxin stimulated a 5-fold increase in non-parenchymal cell apoptosis, depleted liver myofibroblasts by 60%, did not affect the number of monocytes/macrophages and significantly reduced fibrosis severity. Fibrosis reduction was associated with increased metalloproteinase-13 levels. CONCLUSIONS These data demonstrate that specific targeting of liver myofibroblast apoptosis is the most effective anti-fibrogenic therapy, supporting a role for liver monocytes and/or macrophages in the promotion of liver fibrosis reduction.

Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

Background: Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates. Results: A V-NAR specific for human serum albumin was humanized, and its crystal structure in complex with the antigen was solved, revealing an unusual recognition mode. Conclusion: Humanization preserved antigen binding properties and activity of the parental shark antibody. Significance: A structural framework for humanization of shark antibodies was established. The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

Candida albicans VAC8 Is Required for Vacuolar Inheritance and Normal Hyphal Branching

ABSTRACT Hyphal growth is prevalent during most Candida albicans infections. Current cell division models, which are based on cytological analyses of C. albicans, predict that hyphal branching is intimately linked with vacuolar inheritance in this fungus. Here we report the molecular validation of this model, showing that a specific mutation that disrupts vacuolar inheritance also affects hyphal division. The armadillo repeat-containing protein Vac8p plays an important role in vacuolar inheritance in Saccharomyces cerevisiae. The VAC8 gene was identified in the C. albicans genome sequence and was resequenced. Homozygous C. albicans vac8Δ deletion mutants were generated, and their phenotypes were examined. Mutant vac8Δ cells contained fragmented vacuoles, and minimal vacuolar material was inherited by daughter cells in hyphal or budding forms. Normal rates of growth and hyphal extension were observed for the mutant hyphae on solid serum-containing medium. However, branching frequencies were significantly increased in the mutant hyphae. These observations are consistent with a causal relationship between vacuolar inheritance and the cell division cycle in the subapical compartments of C. albicans hyphae. The data support the hypothesis that cytoplasmic volume, rather than cell size, is critical for progression through G1.

Evidence for an active fibrinolytic system in normal human bone marrow

Normal human bone marrow from patients undergoing heart surgery was analysed quantitatively for components of the fibrinolytic system, using functional and immunological assays. Marrow was found to contain considerable fibrinolytic activity, reflecting high levels of t‐PA (tissue‐type plasminogen activator). The t‐PA was in an active form, despite the presence of the inhibitors PAI‐1 and PAI‐2. Plasminogen and α2‐antiplasmin (α2‐AP) were also present in marrow. The balance of proteases and inhibitors differed dramatically from that observed in plasma, with higher levels of t‐PA, PAI‐1 and PAI‐2, and lower levels of u‐PA (urokinase), plasminogen, α2‐AP and t‐PA‐PAI‐1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin–α2‐AP complex at concentrations of the same order of magnitude as total plasminogen and α2‐AP demonstrated that active generation of plasmin was indeed occurring. A role for the active fibrinolytic system in normal human bone marrow may be the removal of unnecessary fibrin deposits formed in the cavities of the marrow, in order to maintain flow through this tissue.

Targeting liver myofibroblasts: a novel approach in anti-fibrogenic therapy

Chronic liver disease results in a liver-scarring response termed fibrosis. Excessive scarring leads to cirrhosis, which is associated with high morbidity and mortality. The only treatment for liver cirrhosis is liver transplantation; therefore, much attention has been directed toward therapies that will slow or reverse fibrosis. Although anti-fibrogenic therapies have been shown to be effective in experimental animal models, licensed therapies have yet to emerge. A potential problem for any anti-fibrogenic therapy in the liver is the existence of the body’s major drug metabolising cell (the hepatocyte) adjacent to the primary fibrosis-causing cell, the myofibroblast. This article reviews the development of a human recombinant single-chain antibody (scAb) that binds to the surface of myofibroblasts. This antibody binds specifically to myofibroblasts in fibrotic mouse livers. When conjugated with a compound that stimulates myofibroblast apoptosis, the antibody directs the specific apoptosis of myofibroblasts with greater specificity and efficacy than the free compound. The antibody also reduces the adverse effect of liver macrophage apoptosis and—in contrast to the free compound—reversed fibrosis in the sustained injury model used. These data suggest that specifically stimulating the apoptosis of liver myofibroblasts using a targeting antibody has potential in the treatment of liver fibrosis.