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

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

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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

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

Embryonated Eggs as an Alternative Infection Model To Investigate Aspergillus fumigatus Virulence▿

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the “gold standard” for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity

Distinct roles of Candida albicans-specific genes in host-pathogen interactions

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Distinct roles of Candida albicans-specific genes in host-pathogen interactions

Peer reviewedPreprin

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts