Three water restriction schedules used in rodent behavioral tasks transiently impair growth and differentially evoke a stress hormone response without causing dehydration

Water restriction is commonly used to motivate rodents to perform behavioral tasks; however, its effects on hydration and stress hormone levels are unknown. Here, we report daily body weight and bi-weekly packed red blood cell volume and corticosterone in adult male rats across 80 days for three commonly used water restriction schedules. We also assessed renal adaptation to water restriction using post-mortem histological evaluation of renal medulla. A control group received ad libitum water. After one week of water restriction, rats on all restriction schedules resumed similar levels of growth relative to the control group. Normal hydration was observed, and water restriction did not drive renal adaptation. An intermittent restriction schedule was associated with an increase in corticosterone relative to the control group. However, intermittent restriction evokes a stress response which could affect behavioral and neurobiological results. Our results also suggest that stable motivation in behavioral tasks may only be achieved after one week of restriction.Peer reviewe

Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in \u3ci\u3eCandida albicans\u3c/i\u3e

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21∆/∆ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filamentinducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21∆/∆ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21∆/∆mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen

A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts

A non-canonical melanin biosynthesis pathway protects Aspergillus terreus conidia from environmental stress

Pro- and eukaryotes produce melanin for protection from environmental stress or as virulence determinant. The human pathogenic fungus Aspergillus fumigatus and related Ascomycetes produce dihydroxynaphthalene (DHN) melanin in conidia, which is essential for inhibiting phagolysosome acidification. In contrast, Aspergillus terreus lacks genes for biosynthesis of DHN-melanin. Therefore, the origin of the pigment in A. terreus conidia was elucidated. Expression analyses from conidiation conditions identified genes coding for an unusual NRPS-like enzyme (MelA) and a tyrosinase. MelA produces aspulvinone E as precursor, which is activated for polymerisation by the tyrosinase TyrP as shown by heterologous in vivo and in vitro reconstitution of pigment formation. Functional studies revealed that the pigment confers resistance against UV-light and hampers phagocytosis by soil amoeba, but does not inhibit acidification of phagolysosomes. Since A. terreus conidia prefer persistence at acidic pH, this uncommon type of melanin, termed Asp-melanin, might specifically contribute to survival in the environment

Correlative extinction and single fluorophore bleaching microscopy for ligand quantification on gold nanoparticles

Nanoparticles (NPs) are promising therapeutic delivery agents, with the number and manner of presentation of cell‐binding ligands on the NP affecting the eventual fate of the therapeutic. Whenever NPs are conjugated with biomolecules, a heterogeneous population of decorated NPs will be produced and these subpopulations of particle‐ligand structures need to be characterized for a reliable interpretation of NP‐based data. An optical microscopy method is reported to quantitatively evaluate the conjugation on a single‐particle basis in samples consisting of gold NPs (GNPs) decorated with holo‐transferrin fluorescently labeled with Alexa647 (Tf). Widefield fluorescence and extinction microscopy are employed on NP‐ligand constructs, alongside a correlative analysis that spatially co‐localizes diffraction‐limited sources of fluorescence with the optical extinction by individual GNPs. A photobleaching step analysis estimates the number of fluorophores contributing to the detected emission rate. The method quantifies the number of fluorescent biomolecules attached per GNP, the numbers of unconjugated GNPs and unbound Tf present within the mixed population, and the size and intraparticle clustering propensity of conjugated GNPs. A high variability is found in the number of Tf ligands per GNP within the GNP population, when analyzed at the single‐particle level, unraveling a non‐trivial statistical distribution not accessible in ensemble‐averaged approaches

Persistence within dendritic cells marks an antifungal evasion and dissemination strategy of Aspergillus terreus

Aspergillus terreus is an airborne human fungal pathogen causing life-threatening invasive aspergillosis in immunocompromised patients. In contrast to Aspergillus fumigatus, A. terreus infections are associated with high dissemination rates and poor response to antifungal treatment. Here, we compared the interaction of conidia from both fungal species with MUTZ-3-derived dendritic cells (DCs). After phagocytosis, A. fumigatus conidia rapidly escaped from DCs, whereas A. terreus conidia remained persisting with long-term survival. Escape from DCs was independent from DHN-melanin, as A. terreus conidia expressing wA showed no increased intracellular germination. Within DCs A. terreus conidia were protected from antifungals, whereas A. fumigatus conidia were efficiently cleared. Furthermore, while A. fumigatus conidia triggered expression of DC activation markers such as CD80, CD83, CD54, MHCII and CCR7, persistent A. terreus conidia were significantly less immunogenic. Moreover, DCs confronted with A. terreus conidia neither produced pro-inflammatory nor T-cell stimulating cytokines. However, TNF-α addition resulted in activation of DCs and provoked the expression of migration markers without inactivating intracellular A. terreus conidia. Therefore, persistence within DCs and possibly within other immune cells might contribute to the low response of A. terreus infections to antifungal treatment and could be responsible for its high dissemination rates

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Multiphoton microscopy and ultrafast spectroscopy: Imaging meets quantum (MUSIQ) roadmap

In April 2019 the EU Marie Skłodowska-Curie Actions (MSCA) Innovative Training Networks (ITN) MUSIQ officially started. The network brought together a unique team of world-leading academics and industrial partners at the forefront of optical micro-spectroscopy and ultrafast laser technology developments merged with fundamental studies of coherent light-matter interaction phenomena, development of quantitative image analysis tools beyond state-of-the-art, and biomedical/pharmaceutical real-world applications. The unique vision of MUSIQ has been to develop and apply the next-generation optical microscopy technologies exploiting quantum coherent nonlinear phenomena. This Roadmap has been written collectively by the MUSIQ early-stage researchers and their supervisors. It provides a summary of the achievements within MUSIQ to date, with an outlook towards future directions

Etablierung von Infektionsmodellen und Einblicke in die Pathogenese der invasiven Aspergillose durch Aspergillus terreus

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in severely immunocompromised patients. Although A. fumigatus is the most common cause of IBPA, infections with A. terreus are emerging and mortality rates are comparable to A. Fumigates infections. Although A. terreus is distributed ubiquitously, the incidence of A. Terreus mediated IBPA is comparably low. To elucidate the pathogenesis of A. terreus aspergillosis in comparison to A. Fumigates mediated IBPA, an alternative infection model and two distinct pulmonary murine infection models were established and characterised in this study. To induce lethal infections by A. terreus an at least 100 times higher infectious dose was required than for A. fumigatus, but even with the highest infectious dose only 50% mortality was observed in corticosteroidtreated mice. However, surviving mice transiently displayed clinical symptoms and bioluminescence imaging revealed transient fungal growth, suggesting that the immune system of surviving animals was able to control the infection. In moribund animals, the disease pattern largely resembled A. fumigatus IBPA. However, persistent, ungerminated but viable conidia were frequently found in alveolar macrophages and pulmonary epithelial cells of surviving animals. In contrast to A. fumigatus infections, all mice infected with A. Terreus developed a fatty liver degeneration, possibly due to the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage represent unique features of A. terreus mediated IBPA. Furthermore, these models provide the necessary tools to investigate the pathogenesis of A. terreus mediated aspergillosis in detail. The persistence and incomplete germination of conidia in macrophages and epithelial cells observed in vivo led to the hypothesis that the initial steps of disease establishment might be fundamentally different between A. terreus and A. fumigatus. Since alveolar macrophages represent the first immune cells facing inhaled conidia in the lung, the interaction of A. terreus and A. fumigatus conidia with these phagocytes was investigated. Interestingly, A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia. This was likely due to the higher exposure of β-1,3-glucan and galactomannan on the surface of A. Terreus resting and pre-swollen conidia. In agreement with the increased PAMP exposure observed on conidia, blocking of dectin-1 and the mannose receptor, the ligand-specific PRRs, significantly reduced phagocytosis of A. terreus to basal levels, but had only a moderate effect on phagocytosis of A. fumigatus. While A. fumigatus prevents phagolysosomal acidification which allows germination within this organelle, A. terreus conidia persisted in fully matured and acidified phagolysosomes after phagocytosis. The acidic pH of the phagolysosome prevented germination of A. terreus conidia, thus resulting in significantly reduced macrophage cytotoxicity. Blocking phagolysosome acidification by the specific v- ATPase inhibitor bafilomycin A increased A. terreus germination within phagolysosomes and consequently cytotoxicity. Thus, it appears that the two fungal species have evolved different interaction strategies with macrophages: While A. fumigatus interferes with phagosome maturation and escapes from phagocytes by germination, A. terreus remains viable but trapped within acidified phagolysosomes. However, possibly because germlings are more sensitive than conidia, A. fumigatus is inactivated to a higher extent by macrophages. To determine factors involved in phagolysosome interaction, this work focused on pigments which are frequently used for morphological species discrimination in Aspergillus diagnostics. These pigments are part of the cell wall, provide defence against physiological stresses and can moreover interact with PRRs of immune cells. In A. fumigatus, the polyketide synthase PksP produces the pigment DHN-melanin, which prevents phagolysosome acidification, but is absent in A. terreus. However, recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, in A. terreus inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, this study demonstrates for the first time that A. terreus and A. Fumigates interact fundamentally different with the immune system during disease establishment. Although aspergillosis caused by A. terreus resembles A. fumigatus IBPA, significant differences in the interaction with macrophages suggest a modified pathogenicity strategy. While A. fumigatus hides from phagocytosis and prevents phagolysosome acidification which allows escape from macrophages by germination, A._terreus is rapidly phagocytosed, does not interfere with phagolysosomal maturation but persists within macrophages. Consequently, the pathogenicity strategy of A. fumigatus cannot be taken as a general model for all aspergilli but requires species-specific investigation.Die invasive, bronchopulmonale Aspergillose (IBPA) stellt eine lebensbedrohliche Komplikation in immunsupprimierten Patienten dar. Hauptverursacher dieses Krankheitsbildes ist A. fumigatus, jedoch nimmt die Häufigkeit von A. terreus Erkrankungen mit ähnlichen Letalitätsraten zu. Obwohl A. terreus ubiquitär verbreitet ist, ist die Erkrankungsinzidenz jedoch verhältnismäßig gering. Um die Pathogenese der A. terreus Aspergillose vergleichend zu A. fumigatus aufzuklären, entwickelte und charakterisierte die vorliegende Studie ein alternatives und zwei murine Infektionsmodelle. Um letale Infektionen in diesen Modellen hervorzurufen, benötigte es einer mindestens 100fach höheren Infektionsdosis im Vergleich zu A. fumigatus. Dennoch konnte unter Kortisonbehandlung lediglich eine Mortalität von 50% der infizierten Versuchstiere beobachtet werden. Lumineszenzmessungen zeigten jedoch ein transientes Wachstum des Pilzes in überlebenden Tieren, welches mit klinischer Symptomatik einherging. Dies lässt vermuten, dass eine Kontrolle der Infektion durch das Immunsystem in diesen Tieren möglich war. Der Krankheitsverlauf moribunder Tiere glich weitgehend dem einer IBPA durch A. fumigatus. Allerdings waren in überlebenden Tieren häufig lebende, ungekeimte Konidien in Alveolarmakrophagen und Epithelzellen der Lunge auffindbar. Im Unterschied zu einer A. fumigatus IBPA wurde bei allen mit A. terreus infizierten Tieren, unabhängig vom Infektionsmodell, eine kleinvakuolige, peripherlobulare, fettige Degeneration der Leber beobachtet. Diese ist wahrscheinlich auf den Einfluss pilzlicher Sekundarmetabolite zurückzuführen, die während der Infektion gebildet werden. Somit konnten in dieser Studie, die Persistenz von Konidien und eine fettige Leberdegeneration als Besonderheiten der, durch A. terreus verursachten, invasiven Aspergillose identifiziert werden. Die hier vorliegenden Modelle liefern zudem die Grundlage für die weitere, detailierte Untersuchung der Pathogenese der A. terreus-induzierten IBPA. Die, während der Etablierung der in vivo Infektionsmodelle, beobachtete Persistenz von Sporen in Makrophagen und Epithelzellen führte zu der Hypothese, dass sich die anfänglichen Prozesse des Infektionsverlaufes zwischen beiden Pilzspezies grundlegend unterscheiden. Da Alveolarmakrophagen in der Lunge als professionelle Phagozyten initial mit Pathogenen interagieren, wurde die Interaktion von Makrophagen mit Sporen von A. terreus und A. fumigatus untersucht. Sporen von A. terreus wurden wesentlich schneller und vollständiger phagozytiert als Sporen von A. fumigatus. Dies kann auf eine erhöhte β- 1,3-Glukan und Galaktomannan Präsentation auf der Oberflache der A. terreus Sporen zurückgeführt werden. Damit übereinstimmend wurde gezeigt, dass die Blockierung der, zur Erkennung dieser Zellwandbestandteile notwendigen, Rezeptoren Dectin-1 und des Mannose-Rezeptors zu einer deutlichen Verringerung der Phagozytose von A. terreus Sporen durch Makrophagen führte. Die Phagozytoserate von A. fumigatus wurde durch diese Blockierung nur gering beeinflusst. Sporen von A. fumigatus verhindern zudem nach der Phagozytose die Ansäuerung von Phagolysosomen und können durch Auskeimung aus Makrophagen entkommen, was mit einer Schädigung der Makrophagen einhergeht. Im Gegensatz dazu persistierten phagozytierte Konidien von A. terreus in angesäuerten Phagolysosomen ohne auszukeimen und ohne die Makrophagen zu schädigen. Die Inhibierung der Ansäuerung des Phagolysosoms durch den spezifischen v-ATPase Inhibitor Bafilomycin A führte zu einer deutlich erhöhten Auskeimungsrate und Zytotoxizität von A. terreus. Die beiden Aspergillus-Arten scheinen also zwei unterschiedliche Strategien im Umgang mit Makrophagen entwickelt zu haben: Während A. fumigatus die Ansäuerung von Phagolysosomen verhindert und durch Auskeimen entkommt, persistiert A. terreus als Spore in angesäuerten Phagolysosomen. Dabei wird jedoch ein höherer Anteil von A. fumigatus durch Makrophagen inaktiviert. Dies liegt vermutlich an der verstärkten Empfindlichkeit von Keimlingen und Hyphen gegenüber antimikrobiellen Stoffen im Vergleich zu Sporen. Pigmente als Zellwandbestandteile, haben eine potentielle Rolle in der Interaktion mit dem Phagolysosom und werden in der mikrobiologischen Diagnostik häufig als Speziesdiagnostikum verwandt. A. fumigatus produziert das Pigment DHN-Melanin mit Hilfe der Polyketidsynthase PksP, welches die Phagolysosomansäuerung verhindert jedoch in A. terreus fehlt. Durch rekombinante Expression der Naphthopyronsynthase wA von A. nidulans, einem Homolog der A. fumigatus PksP, in A. terreus konnte die Ansäuerung des Phagolysosom verhindert werden. Dies führte zu einer erhöhten Auskeimungsrate in Makrophagen, einer verstärkten Zytotoxizität und erhöhte die Virulenz im Kortison-Modell. Damit zeigt diese Studie erstmalig, dass A. terreus und A. fumigatus grundlegende Unterschiede in der Interaktion mit Phagozyten aufweisen. Trotz gleicher klinischer Symptomatik, entwickelten A. terreus und A. fumigatus unterschiedliche Pathogenesestrategien, um der Eliminierung durch das Immunsystem zu entgehen. Während A. fumigatus Phagozytose sowie Ansäuerung des Phagolysosom verhindert, wird A. terreus schnell phagozytiert und persistiert in Makrophagen. Folglich kann die IBPA durch A. fumigatus nicht als Modell für alle Aspergillosen dienen und die Pathogenese sollte abhängig von der Aspergillus-Art untersucht werden