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1.  Histidine Degradation via an Aminotransferase Increases the Nutritional Flexibility of Candida glabrata 
Eukaryotic Cell  2014;13(6):758-765.
The ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humans Candida glabrata. Hemiascomycete fungi, like C. glabrata or Saccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show that C. glabrata instead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. Although ARO8 is also present in S. cerevisiae and is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore, C. glabrata relies only on Aro8 for phenylalanine and tryptophan utilization, since ARO8, but not its homologue ARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, an ARO9 deletion had no effect on growth with aromatic amino acids. In contrast, in S. cerevisiae, ARO9 is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of the ARO9 gene between C. glabrata and S. cerevisiae indicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast to S. cerevisiae, C. glabrata has adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.
doi:10.1128/EC.00072-14
PMCID: PMC4054274  PMID: 24728193
2.  Illuminating Fungal Infections with Bioluminescence 
PLoS Pathogens  2014;10(7):e1004179.
doi:10.1371/journal.ppat.1004179
PMCID: PMC4092138  PMID: 25010008
3.  Inflammatory cytokines in pulmonary hypertension 
Respiratory Research  2014;15(1):47.
Pulmonary hypertension is an “umbrella term” used for a spectrum of entities resulting in an elevation of the pulmonary arterial pressure. Clinical symptoms include dyspnea and fatigue which in the absence of adequate therapeutic intervention may lead to progressive right heart failure and death. The pathogenesis of pulmonary hypertension is characterized by three major processes including vasoconstriction, vascular remodeling and microthrombotic events. In addition accumulating evidence point to a cytokine driven inflammatory process as a major contributor to the development of pulmonary hypertension.
This review summarizes the latest clinical and experimental developments in inflammation associated with pulmonary hypertension with special focus on Interleukin-6, and its role in vascular remodeling in pulmonary hypertension.
doi:10.1186/1465-9921-15-47
PMCID: PMC4002553  PMID: 24739042
Pulmonary hypertension; Inflammation; Immune cells; Experimental models; Cytokines; microRNAs
4.  Regulatory Networks Controlling Nitrogen Sensing and Uptake in Candida albicans 
PLoS ONE  2014;9(3):e92734.
Nitrogen is one of the key nutrients for microbial growth. During infection, pathogenic fungi like C. albicans need to acquire nitrogen from a broad range of different and changing sources inside the host. Detecting the available nitrogen sources and adjusting the expression of genes for their uptake and degradation is therefore crucial for survival and growth as well as for establishing an infection. Here, we analyzed the transcriptional response of C. albicans to nitrogen starvation and feeding with the infection-relevant nitrogen sources arginine and bovine serum albumin (BSA), representing amino acids and proteins, respectively. The response to nitrogen starvation was marked by an immediate repression of protein synthesis and an up-regulation of general amino acid permeases, as well as an up-regulation of autophagal processes in its later stages. Feeding with arginine led to a fast reduction in expression of general permeases for amino acids and to resumption of protein synthesis. The response to BSA feeding was generally slower, and was additionally characterized by an up-regulation of oligopeptide transporter genes. From time-series data, we inferred network interaction models for genes relevant in nitrogen detection and uptake. Each individual network was found to be largely specific for the experimental condition (starvation or feeding with arginine or BSA). In addition, we detected several novel connections between regulator and effector genes, with putative roles in nitrogen uptake. We conclude that C. albicans adopts a particular nitrogen response network, defined by sets of specific gene-gene connections for each environmental condition. All together, they form a grid of possible gene regulatory networks, increasing the transcriptional flexibility of C. albicans.
doi:10.1371/journal.pone.0092734
PMCID: PMC3961412  PMID: 24651113
5.  Assessment of Efficacy of Antifungals against Aspergillus fumigatus: Value of Real-Time Bioluminescence Imaging 
Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 × 105 and 2.5 × 105 conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.
doi:10.1128/AAC.01660-12
PMCID: PMC3697358  PMID: 23587947
6.  Combination of erythropoietin and sildenafil can effectively attenuate hypoxia-induced pulmonary hypertension in mice 
Pulmonary Circulation  2013;3(4):898-907.
Pulmonary hypertension (PH) is an incurable disease that often leads to right ventricular hypertrophy and right heart failure. This study investigated single versus combined therapy with sildenafil and erythropoietin on hypoxia-induced pulmonary hypertension in mice. Mice were randomized into 5 groups and exposed to either hypoxia (10% oxygen) or normoxia for a total of 5 weeks. Hypoxic mice were treated with saline solution, erythropoietin (500 IU/kg 3 times weekly), sildenafil (10 mg/kg daily), or a combination of the two drugs for the last 2 weeks of hypoxic exposure. We measured right ventricular pressures using right heart catheterization, and the ventilatory response to hypoxia was recorded via whole-body plethysmography. Histological analyses were performed to elucidate changes in pulmonary morphology and appearance of right heart hypertrophy. Plasma levels of cardiotrophin-1 and atrial natriuretic peptide were quantified. Treatment with either erythropoietin or sildenafil alone lowered the hypoxia-induced increase of pulmonary pressure and reduced pulmonary edema formation, pulmonary vascular remodeling, and right ventricular hypertrophy. Notably, the combination of the two drugs had the most prominent effect. Changes in cardiotrophin-1 and atrial natriuretic protein levels confirmed these observations. The combination treatment with erythropoietin and sildenafil demonstrated an attenuation of the development of hypoxia-induced PH in mice that was superior to that observed for either drug when given alone.
doi:10.1086/674758
PMCID: PMC4070833  PMID: 25006406
edema; pulmonary remodeling; right ventricular hypertrophy
7.  Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion 
PLoS Pathogens  2012;8(6):e1002777.
The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution.
Author Summary
The capacity of disease-causing microbes to acquire nutrients from their host is one of the most fundamental aspects of infection. Host organisms therefore restrict microbial access to certain key nutrients in a process known as nutritional immunity. Recently, it was found that infected vertebrates sequester zinc from invading microorganisms to control infection. Therefore, the mechanisms of microbial zinc acquisition represent potential virulence attributes. Here we report the molecular mechanism of host-derived zinc acquisition by the major human fungal pathogen, Candida albicans. We show that C. albicans utilises a secreted protein, the pH-regulated antigen 1 (Pra1), to bind zinc from its environment. Pra1 then reassociates with the fungal cell via a syntenically encoded (genetically-linked) membrane transporter (Zrt1) to acquire this essential metal. Deletion of PRA1 prevented utilisation of host zinc and damage of host cells in the absence of exogenous zinc. Finally, we demonstrate that this zinc-scavenging locus arose in an ancient fungal lineage and remains conserved in many contemporary species. Syntenically arranged zinc acquisition systems have evolved independently in the fungal and bacterial kingdoms, suggesting that such an arrangement is evolutionary beneficial for microorganisms.
doi:10.1371/journal.ppat.1002777
PMCID: PMC3386192  PMID: 22761575
8.  Pulmonary hypertension in patients with Martorell hypertensive leg ulcer: a case control study 
Respiratory Research  2012;13(1):45.
Background
Martorell hypertensive ischemic leg ulcer (Martorell ulcer) is characterized by distinct alterations in the arteriolar wall of subcutaneous vessels, leading to progressive narrowing of the vascular lumen and increase of vascular resistance. These changes are similar to the alterations observed in pulmonary arterioles in patients with chronic pulmonary hypertension (PH). This study was aimed to assess an association between the two disorders.
Methods
In this case–control study, 14 patients with Martorell ulcer were clinically assessed for the presence of pulmonary hypertension using transthoracic Doppler echocardiography. Data from patients were compared to 28 matched hypertensive controls.
Results
Systolic pulmonary arterial pressure (sPAP) in patients with Martorell ulcer was significantly higher than in the control group (33.8 ± 16.9 vs 25.3 ± 6.5 mmHg, p = 0.023); the prevalence of pulmonary hypertension was 31% (5/14) in patients and 7% (2/28) in controls (p = 0.031). No differences were seen in left heart size and function between patients and controls.
Conclusion
This study provides first evidence that subcutaneous arteriolosclerosis, the hallmark of Martorell ulcer, is associated with PH. These findings suggest that patients with Martorell leg ulcer might be at significant risk to develop elevated pulmonary arterial pressure. Patients with leg ulcers who present with dyspnea should be evaluated by echocardiography for the presence of pulmonary hypertension.
doi:10.1186/1465-9921-13-45
PMCID: PMC3471016  PMID: 22686459
Pulmonary hypertension; Echocardiography; Martorell ulcer
9.  7th meeting of the global arthritis research network 
Last October, the 7th meeting of the Global Arthritis Research Network was held in Zurich, Switzerland. European and American experts who have made major recent contributions to molecular biology got together to provide insights into novel technologies and approaches useful for biomedical research, especially for research on arthritis and related conditions.
doi:10.1186/ar3340
PMCID: PMC3239332  PMID: 21892971
10.  Persistence versus Escape: Aspergillus terreus and Aspergillus fumigatus Employ Different Strategies during Interactions with Macrophages 
PLoS ONE  2012;7(2):e31223.
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.
doi:10.1371/journal.pone.0031223
PMCID: PMC3272006  PMID: 22319619
11.  Application of Bioluminescence Imaging for In Vivo Monitoring of Fungal Infections 
Fungi can cause severe invasive infections especially in the immunocompromised host. Patient populations at risk are increasing due to ongoing developments in cancer treatment and transplantation medicine. Only limited diagnostic tools and few antifungals are available, rendering a significant number of invasive fungal infections life threatening. To reduce mortality rates, a better understanding of the infection processes is urgently required. Bioluminescence imaging (BLI) is a powerful tool for such purposes, since it allows visualisation of temporal and spatial progression of infections in real time. BLI has been successfully used to monitor infections caused by various microorganisms, in particular bacteria. However, first studies have also been performed on the fungi Candida albicans and Aspergillus fumigatus. Although BLI was, in principle, suitable to study the infection process, some limitations remained. Here, different luciferase systems are introduced, and current approaches are summarised. Finally, suggestions for further improvements of BLI to monitor fungal infections are provided.
doi:10.1155/2012/956794
PMCID: PMC3205719  PMID: 22121368
12.  Embryonated Eggs as an Alternative Infection Model To Investigate Aspergillus fumigatus Virulence▿  
Infection and Immunity  2010;78(7):2995-3006.
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.
doi:10.1128/IAI.00268-10
PMCID: PMC2897382  PMID: 20421382
13.  Aspergillus fumigatus Catalytic Glucokinase and Hexokinase: Expression Analysis and Importance for Germination, Growth, and Conidiation▿† 
Eukaryotic Cell  2010;9(7):1120-1135.
Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.
doi:10.1128/EC.00362-09
PMCID: PMC2901669  PMID: 20453072
14.  Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies▿† 
Eukaryotic Cell  2010;9(6):878-893.
Aspergillus fumigatus is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal α-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence. A. fumigatus HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and α-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An hcsA deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified A. fumigatus proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.
doi:10.1128/EC.00020-10
PMCID: PMC2901645  PMID: 20363898
15.  In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis 
BMC Microbiology  2010;10:105.
Background
Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain.
Results
In this study, we demonstrate that bioluminescence imaging can track the progression of IA at different anatomic locations in a murine model of disease that recapitulates the natural route of infection. To define the temporal and functional requirements of distinct innate immune cellular subsets in host defense against respiratory A. fumigatus infection, we examined the development and progression of IA using bioluminescence imaging and histopathologic analysis in mice with four different types of pharmacologic or numeric defects in innate immune function that target resident and recruited phagocyte subsets. While bioluminescence imaging can track the progression and location of invasive disease in vivo, signals can be attenuated by severe inflammation and associated tissue hypoxia. However, especially under non-inflammatory conditions, such as cyclophosphamide treatment, an increasing bioluminescence signal reflects the increasing biomass of alive fungal cells.
Conclusions
Imaging studies allowed an in vivo correlation between the onset, peak, and kinetics of hyphal tissue invasion from the lung under conditions of functional or numeric inactivation of phagocytes and sheds light on the germination speed of conidia under the different immunosuppression regimens. Conditions of high inflammation -either mediated by neutrophil influx under corticosteroid treatment or by monocytes recruited during antibody-mediated depletion of neutrophils- were associated with rapid conidial germination and caused an early rise in bioluminescence post-infection. In contrast, 80% alveolar macrophage depletion failed to trigger a bioluminescent signal, consistent with the notion that neutrophil recruitment is essential for early host defense, while alveolar macrophage depletion can be functionally compensated.
doi:10.1186/1471-2180-10-105
PMCID: PMC2859869  PMID: 20377900
16.  Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis▿  
Applied and Environmental Microbiology  2008;74(22):7023-7035.
Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.
doi:10.1128/AEM.01288-08
PMCID: PMC2583481  PMID: 18820063
17.  Back to the roots 
Microbial biotechnology  2009;2(2):132-133.
doi:10.1111/j.1751-7915.2009.00090_4.x
PMCID: PMC3815819  PMID: 21261892
18.  Aspergillus fumigatus Does Not Require Fatty Acid Metabolism via Isocitrate Lyase for Development of Invasive Aspergillosis▿  
Infection and Immunity  2006;75(3):1237-1244.
Aspergillus fumigatus is the most prevalent airborne filamentous fungus causing invasive aspergillosis in immunocompromised individuals. Only a limited number of determinants directly associated with virulence are known, and the metabolic requirements of the fungus to grow inside a host have not yet been investigated. Previous studies on pathogenic microorganisms, i.e., the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, have revealed an essential role for isocitrate lyase in pathogenicity. In this study, we generated an isocitrate lyase deletion strain to test whether this strain shows attenuation in virulence. Results have revealed that isocitrate lyase from A. fumigatus is not required for the development of invasive aspergillosis. In a murine model of invasive aspergillosis, the wild-type strain, an isocitrate lyase deletion strain, and a complemented mutant strain were similarly effective in killing mice. Moreover, thin sections demonstrated invasive growth of all strains. Additionally, thin sections of lung tissue from patients with invasive aspergillosis stained with anti-isocitrate lyase antibodies remained negative. From these results, we cannot exclude the use of lipids or fatty acids as a carbon source for A. fumigatus during invasive growth. Nevertheless, test results do imply that the glyoxylate cycle from A. fumigatus is not required for the anaplerotic synthesis of oxaloacetate under infectious conditions. Therefore, an antifungal drug inhibiting fungal isocitrate lyases, postulated to act against Candida infections, is assumed to be ineffective against A. fumigatus.
doi:10.1128/IAI.01416-06
PMCID: PMC1828595  PMID: 17178786
19.  Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans 
PLoS Pathogens  2007;3(2):e13.
The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.
Author Summary
Aspergillus fumigatus and Candida albicans are the most common of all human pathogenic fungal germs. Normally, inhaled Aspergillus spores are destroyed by alveolar macrophages and polymorphonuclear neutrophils (PMNs), both of which are lung phagocytes, i.e., cells that kill inhaled microbes by ingestion. In contrast, C. albicans is a normal constituent of the human gut flora that is controlled by tissue-resident PMNs. If immune control is lost, both fungi grow into the surrounding tissue and cause life-threatening infections. To investigate how phagocytes function in the disparate environments of lung air sacs (lacking a definite matrix-composition [two-dimensional (2-D)]) or mucosal tissues (providing a three-dimensional [3-D] space), the authors mimicked 2-D and 3-D environments and analyzed the process of ingestion, called phagocytosis, by PMNs and other phagocytes. Phagocytosis was a dynamic cellular process where distinct cells showed vastly different behavior. The environmental setup (2-D versus 3-D) had a profound impact on the cell's ability to phagocytose. Aspergillus conidia were much better ingested in 2-D systems, while Candida yeasts were only ingested in 3-D systems, even if the other pathogen was present. This was true for different 2-D and 3-D systems and for both cells of mice and humans. Besides providing a comprehensive analysis of the cellular movements underlying phagocytosis, the results also suggest an evolution of phagocytes to optimally recognize fungal pathogens in the environment of natural infection.
doi:10.1371/journal.ppat.0030013
PMCID: PMC1790725  PMID: 17274685
20.  Generation and Phenotypic Characterization of Aspergillus nidulans Methylisocitrate Lyase Deletion Mutants: Methylisocitrate Inhibits Growth and Conidiation 
Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the α-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the Ki of (2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 μM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.
doi:10.1128/AEM.71.9.5465-5475.2005
PMCID: PMC1214605  PMID: 16151139
21.  The fungal α-aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate 
Molecular Microbiology  2012;86(6):1508-1530.
Fungi produce α-aminoadipate, a precursor for penicillin and lysine via the α-aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.
doi:10.1111/mmi.12076
PMCID: PMC3556520  PMID: 23106124

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