Sterol metabolism during germination of conidia of Aspergillus fumigatus.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.     

Detection of early phase specific surface appendages during germination of Aspergillus fumigatus conidia

During the past 15 years the saprophytic fungus Aspergillus fumigatus has become the most prevalent airborne fungal pathogen, causing severe and often fatal infections especially in immuno-compromised patients. Germination of inhaled conidia is an early and crucial event in the infection process of A. fumigatus. In this study we have analyzed morphological changes that take place during this differentiation process using scanning electron microscopy. Our data show that (i) the hydrophobic surface layer of resting conidia seems to be shed before the cells start to swell and (ii) that filamentous surface appendages are expressed at a very early phase of the germtube formation. These surface structures were only found on the first few microm of the germtube, but were absent from the surface of mycelial hyphae and resting or swollen conidia. The highly regulated expression of these novel surface organelles suggests that they may play an important role during early germination and represent a potential target for future anti-A. fumigatus therapies.     

Inter- and intra-species heterogeneity in germination of Aspergillus conidia

Aspergilli are among the most abundant fungi worldwide. They degrade organic material and can be pathogens of plants and animals. Aspergilli spread by forming high numbers of conidia. Germination of these stress resistant asexual spores is characterized by a swelling and a germ tube stage. Here, we show that conidia of Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus, Aspergillus nidulans and Aspergillus terreus show different swelling and germ tube formation dynamics in pure water or in water supplemented with (in)organic nutrients. Apart from inter-species heterogeneity, intra-species heterogeneity was observed within spore populations of the aspergilli except for A. terreus. Sub-populations of conidia differing in size and/or contrast showed different swelling and germ tube formation dynamics. Together, data imply that aspergilli differ in their competitive potential depending on the substrate. Moreover, results suggest that intra-species heterogeneity provides a bet hedging mechanism to optimize survival of aspergilli.

     

High-resolution cell surface dynamics of germinating Aspergillus fumigatus conidia

We used real-time atomic force microscopy with a temperature-controlled stage (37°C) to probe the structural and physicochemical dynamics of single Aspergillus fumigatus conidia during germination. Nanoscale topographic images of dormant spores revealed the presence of a layer of rodlets made of hydrophobins, in agreement with earlier electron microscopy observations. Within the 3-h germination period, progressive disruption of the rodlet layer was observed, revealing hydrophilic inner cell wall structures. Using adhesion force mapping with hydrophobic tips, these ultrastructural changes were shown to correlate with major differences in cell surface hydrophobicity. That is, the rodlet surface was uniformly hydrophobic due to the presence of hydrophobins, whereas the cell wall material appearing upon germination was purely hydrophilic. This study illustrates the potential of real-time atomic force microscopy imaging and force spectroscopy for tracking cell-surface dynamics.     

Germination of Aspergillus fumigatus conidia in the lungs of normal and cortisone-treated mice.

Inhaled conidia of Aspergillus fumigatus germinated in the lungs of mice at a low rate but both germinated and ungerminated spores were cleared. Spores germinated at a high rate in the lungs of cortisone-treated mice.     

Polyamine and ornithine metabolism during the germination of conidia of Aspergillus nidulans.

1. The activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase and ornithine-2-oxoglutarate aminotransferase were studied during the first 24 h of conidial germination in Aspergillus nidulans. 2. Increases (over 100-fold) in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase occurred during the emergence of the germ-tube and before the doubling of DNA and this was followed by a sharp fall in the activities of both enzymes by 16h. 3. The increase in ornithine decarboxylase could be largely suppressed if 0.6 mM-putrescine was added to the growth medium. 4. Low concentrations of cycloheximide, which delayed germination by 2h, caused a corresponding delay in the changes in ornithine decarboxylase activity. 5. Ornithine-2-oxoglutarate aminotransferase activity increased steadily during the first 24h of germination. 6. Ornithine or arginine in the growth medium induced higher activity of ornithine-2-oxoglutarate aminotransferase, but did not affect ornithine decarboxylase activity. 7. The significance of these enzyme changes during germination is discussed.     

Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for severe, life-threatening infections in immunocompromised patients. Airborne conidia are the infectious agent and can reach the lower parts of the respiratory system. In the lung, phagocytes represent the first line of defence. Resident macrophages are able to track down, engulf and kill the invading spores. Phagocytosis of the conidia is therefore a prerequisite for their efficient elimination. Using human and murine macrophages we analysed the phagocytic uptake of A. fumigatus conidia. We found that conidial phagocytosis is an actin-depending process that additionally requires myosin motor, phosphoinositide-3-phosphate kinase and tyrosine kinase activity. Both broad range tyrosine kinase inhibitors and inhibitors that specifically block src kinases had a strong impact on the conidial uptake. Immunofluorescence data demonstrate the recruitment of tyrosine-phosphorylated proteins to the vicinity of engulfed conidia. Uptake of the conidia was accompanied by a strong and local reorganisation of the actin cytoskeleton, whereas no prominent reorganisation was apparent for the microtubules. Both confocal immunofluorescence and electron microscopic data revealed the presence of large ruffle-like structures engaged in the uptake of conidia. This suggests that the internalisation of A. fumigatus spores can be mediated by a process resembling macropinocytosis, which is furthermore supported by the detection of intracellular conidia within spacious vacuoles. Taken together, our data provide new insights into the internalisation of A. fumigatus spores by macrophages, a key process in the early immune defence against an Aspergillus infection.     

Methods for reducing particle concentrations of Aspergillus fumigatus conidia and mouldy hay dust

The effectiveness of commercially available domestic air purifiers to reduce airborne Aspergillus fumigatus conidia and mouldy hay dust was investigated. It was found that the rate of particle clearance is a function of the volume of air passing through the purifiers but that the low throughflow of air makes their use of little value in clearing particles from a normal sized room. Vacuum cleaners were more effective than air purifiers because of their higher air throughput, so too were high volume fan systems in conjunction with simple filtration units. Ionizers had no effect but steam condensation was very efficient at clearing airborne particles.     

Methylcitrate synthase from Aspergillus fumigatus. Propionyl-CoA affects polyketide synthesis, growth and morphology of conidia

Methylcitrate synthase is a key enzyme of the methylcitrate cycle and required for fungal propionate degradation. Propionate not only serves as a carbon source, but also acts as a food preservative (E280-283) and possesses a negative effect on polyketide synthesis. To investigate propionate metabolism from the opportunistic human pathogenic fungus Aspergillus fumigatus, methylcitrate synthase was purified to homogeneity and characterized. The purified enzyme displayed both, citrate and methylcitrate synthase activity and showed similar characteristics to the corresponding enzyme from Aspergillus nidulans. The coding region of the A. fumigatus enzyme was identified and a deletion strain was constructed for phenotypic analysis. The deletion resulted in an inability to grow on propionate as the sole carbon source. A strong reduction of growth rate and spore colour formation on media containing both, glucose and propionate was observed, which was coincident with an accumulation of propionyl-CoA. Similarly, the use of valine, isoleucine and methionine as nitrogen sources, which yield propionyl-CoA upon degradation, inhibited growth and polyketide production. These effects are due to a direct inhibition of the pyruvate dehydrogenase complex and blockage of polyketide synthesis by propionyl-CoA. The surface of conidia was studied by electron scanning microscopy and revealed a correlation between spore colour and ornamentation of the conidial surface. In addition, a methylcitrate synthase deletion led to an attenuation of virulence, when tested in an insect infection model and attenuation was even more pronounced, when whitish conidia from glucose/propionate medium were applied. Therefore, an impact of methylcitrate synthase in the infection process is discussed.     

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (P_max) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, P_max of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO₃, KH₂PO4, or MgSO4 increased P_max of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these P_max values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO₄. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.     

Changes in lipid composition and carbohydrate composition of Aspergillus niger conidia during germination

Data on the lipid composition and carbohydrate composition of Aspergillus niger conidia make it possible to characterize the individual germination stages and differentiate between the conidia capable of germination and those that lost the germination capacity. The following criteria are proposed: the ratio of phosphatidylcholine and phosphatidylethanolamine, the ratio of mannitol and arabitol, and the levels of sterols and free fatty acids. The role of these compounds in the biochemical background of cell transition from dormancy to active metabolism and their use as indices of the quality of inocula in biotechnological processes are discussed.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Antitumor effect of heat-killed Aspergillus fumigatus mycelium in a mouse model

Summary A suspension of heat-killed Aspergillus fumigatus mycelium inhibited the growth of a chemically-induced mouse bladder tumor (MBT). Tumor growth was inhibited when the mycelium was injected into mice in a mixture with the tumor cells, when injected into growing tumors, and when introduced IP at the time tumor cells were injected into the hind leg muscle. In the concentrations that affected tumor growth no toxicity of the fungus preparation was observed. The fungal suspension was more effective against MBT than a Corynebacterium parvum strain known to be a potent biologic response modifier. A significant increase in the number of mouse peritoneal exudate cells (PEC) was noted following inoculation with the mycelium. The induced PEC were cytotoxic to the tumor cells in vivo, suggesting that at least part of the tumor inhibition by the mycelium is host-mediated.     

Effect of nitric oxide donors on survival of conidia,germination and growth ofAspergillus fumigatus in vitro

The effect of nitric oxide (NO) donors on survival of conidia, germination and growth of the opportunistic pathogenic fungusAspergillus fumigatus was investigated. Most efficient was sodium nitrite in an acidic milieu, (pH 4.5). At a concentration of 5 mmol/L it killed all resting conidia in buffer within 16 h. S-Nitroso derivatives of thiols (cysteine, N-acetylcysteine and N-acetylpenicillamine) at the same concentration killed about 30–50% of spores within 24 h. The NO scavenger, oxyhemoglobin, abolished these effects. S-Nitrosoglutathione had no fungicidal effect and promoted germination. Sodium nitrite and S-nitroso-N-acetylcysteine inhibited germination of conidia in various media from concentration of 0.5 mmol/L and stopped it at concentrations of 1.4–2.9 mmol/L. In media with glucose and casein hydrolyzate or sodium nitrate as nitrogen source, growth inhibition by sodium nitrite (0.5–2 mmol/L) was only weak and mostly transient. In general, the used strainA. fumigatus seems to be less sensitive to nitric oxide donors than dimorphic pathogenic fungi. Thus, nitric oxide is probably not a major effector molecule in killing phagocytized elements of this fungus by host's immunocytes.