Mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus: therapeutic and evolutionary implications

Mitochondria within eukaryotic cells continuously fuse and divide. This phenomenon is called mitochondrial dynamics and crucial for mitochondrial function and integrity. We performed a comprehensive analysis of mitochondrial dynamics in the pathogenic mold Aspergillus fumigatus. Phenotypic characterization of respective mutants revealed the general essentiality of mitochondrial fusion for mitochondrial genome maintenance and the mold's viability. Surprisingly, it turned out that the mitochondrial rhomboid protease Pcp1 and its processing product, s‐Mgm,1 which are crucial for fusion in yeast, are dispensable for fusion, mtDNA maintenance and viability in A. fumigatus. In contrast, mitochondrial fission mutants show drastically reduced growth and sporulation rates and increased heat susceptibility. However, reliable inheritance of mitochondria to newly formed conidia is ensured. Strikingly, mitochondrial fission mutants show a significant and growth condition‐dependent increase in azole resistance. Parallel disruption of fusion in a fission mutant partially rescues growth and sporulation defects and further increases the azole resistance phenotype. Taken together, our results indicate an emerging dispensability of the mitochondrial rhomboid protease function in mitochondrial fusion, the suitability of mitochondrial fusion machinery as antifungal target and the involvement of mitochondrial dynamics in azole susceptibility.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus

Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.     

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".     

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co‐occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co‐incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.     

Aspergillus fumigatus: virulence genes in a street-smart mold

Infections with the filamentous fungus Aspergillus fumigatus are among the most devastating of the systemic mycoses. Unlike most primary pathogens, which possess virulence traits that developed in association with a host organism, evidence suggests that the virulence of A. fumigatus entails a collection of 'street-smart' attributes that have evolved to resist the adverse selection pressures encountered in decaying vegetation. These features enhance the overall competitiveness of the organism in its environmental niche but are also thought to promote growth and survival in a human host. Although many of the genes that are responsible for these characteristics do not fit into the classical definition of a virulence factor, they are nonetheless important to the pathogenesis of aspergillosis and may therefore provide novel opportunities for antifungal development.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Gliotoxin from Aspergillus fumigatus affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils

Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10min with 30-100ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia.     

Secretome profiling reveals temperature-dependent growth of Aspergillus fumigatus

Aspergillus fumigatus is a ubiquitous opportunistic fungus. In this study, systematic analyses were carried out to study the temperature adaptability of A. fumigatus. A total of 241 glycoside hydrolases and 69 proteases in the secretome revealed the strong capability of A. fumigatus to degrade plant biomass and protein substrates. In total, 129 pathogenesis-related proteins detected in the secretome were strongly correlated with glycoside hydrolases and proteases. The variety and abundance of proteins remained at temperatures of 34°C–45°C. The percentage of endo-1,4-xylanase increased when the temperature was lowered to 20°C, while the percentage of cellobiohydrolase increased as temperature was increased, suggesting that the strain obtains carbon mainly by degrading xylan and cellulose, and the main types of proteases in the secretome were aminopeptidases and carboxypeptidases. Only half of the proteins were retained and their abundance declined to 9.7% at 55°C. The activities of the remaining β-glycosidases and proteases were merely 35% and 24%, respectively, when the secretome was treated at 60°C for 2 h. Therefore, temperatures 60°C restrict the growth of A. fumigatus.     

Metabolism of progesterone by Aspergillus fumigatus

Metabolism of progesterone by a typical strain of Aspergillus fumigatus was studied. The four metabolites isolated were characterized as 5α-pregnane-3ß-ol-20-one, 15ß-hydroxy-1,4-pregnadiene-3,20-dione, 7ß,15ß-dihydroxy-4-pregnene-3,20-dione and 11α,15ß-dihydroxy-4-pregnene-3,20-dione by the application of various spectrometric techniques.     

Metabolism of p-Cresol by the Fungus Aspergillus fumigatus

The fungus Aspergillus fumigatus ATCC 28282 was shown to grow on p-cresol as its sole source of carbon and energy. A pathway for metabolism of this compound was proposed. This has protocatechuate as the ring-fission substrate with cleavage and metabolism by an ortho-fission pathway. The protocatechuate was formed by two alternative routes, either by initial attack on the methyl group, which is oxidized to carboxyl, followed by ring-hydroxylation, or by ring-hydroxylation as the first step with subsequent oxidation of 4-methylcatechol to the acid. The pathway was elucidated from several pieces of evidence. A number of compounds, including 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, protocatechualdehyde, and 4-methylcatechol, appeared transiently in the medium during growth on p-cresol. These compounds were oxidized without lag by p-cresol-grown cells but not by succinate-grown cells. Enzyme activities for most of the proposed steps were demonstrated in cell extracts after growth on p-cresol, and the products of these activities were identified. None of the activities were found in succinate-grown cells.     

Mastitis by Aspergillus fumigatus in sheep

Several outbreaks of sheep mastitis by Aspergillus fumigatus in Castilla y Leon (Spain), were studied. Only sheep that were treated intramammarily with antibacterial antibiotics during the dry period suffered this mastitis. Mastitis was acute with a morbidity up to 14 % and mortality near 100 %. The udder was markedly enlarged in size, fibrotic, haemorrhagic and with multiple compact nodules, some with purulent material inside; after 30-50 days postpartum, cheesy abscess of several centimetres in diameter were present. Some sheep had granulomatous nodules in the lung. Microscopy and culture shown the presence of A. fumigatus in milk, udder and lung. The route of infection was by intramammary via as a consequence of unhygienic intramammary treatment in the dry period.     

Degradation of melanin by Aspergillus fumigatus.

A strain of Aspergillus fumigatus from composted coffee and garden wastes utilized natural deproteinized insect, banana, hair, octopus, and synthetic tyrosine and dopa melanins as sole sources of carbon. With a sucrose supplement, degradation was essentially complete after 50 days in Czapek medium pH 6.5 at 30 degrees C. The catabolic rate differed for each substrate pigment, as did the molecular weight distribution of products accumulating in the medium. After incubation with L-[U-14C]melanin, over 50% was recovered in a dark fungal pigment, the remainder appearing as cell protein, chitin, lipid, CO2, and polar metabolites. When grown on melanin, the normally pale mycelia darkened with the production of a fungal allomelanin, with infrared spectrum and alkali fusion products differing from those of the substrate pigment. Isotope distribution in amino acids for A. fumigatus grown on labeled melanin supplemented with sucrose suggested separate pools for synthesis of cell proteins and melanoproteins. Deposition of allomelanin increased resistance of conidia, sterigma, and conidiophores to lytic carbohydrases as judged by scanning electron microscopy.     

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.     

Differential localization patterns of septins during growth of the human fungal pathogen Aspergillus fumigatus reveal novel functions

Septins, a conserved family of GTPases, are heteropolymeric filament-forming proteins that associate with the cell membrane and cytoskeleton and serve essential functions in cell division and morphogenesis. Their roles in fungal cell wall chitin deposition, septation, cytokinesis, and sporulation have been well established and they have recently been implicated in tissue invasion and virulence in Candida albicans. Septins have never been investigated in the human pathogenic fungus, Aspergillus fumigatus, which is a leading cause of death in immunocompromised patients. Here we localize all the five septins (AspA–E) from A. fumigatus for the first time, and show that each of the five septins exhibit varied patterns of distribution. Interestingly AspE, which is unique to filamentous fungi, and AspD, belonging to the CDC10 class of septins, localized prominently to tubular structures which were dependent on actin and microtubule networks. Localization of AspD and AspE has never been reported in filamentous fungi. Taken together these results suggest that septins in A. fumigatus might have unique functions in morphogenesis and pathogenicity.