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 Aspergillusniger,Aspergillusoryzae,Aspergillusclavatus, Aspergillusnidulans and Aspergillusterreus 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

Host defence peptides identified in human apolipoprotein B as promising antifungal agents

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents

Impact of maturation and growth temperature on cell-size distribution, heat-resistance, compatible solute composition and transcription profiles of Penicillium roqueforti conidia

Penicillium roqueforti is a major cause of fungal food spoilage. Its conidia are the main dispersal structures of this fungus and therefore the main cause of food contamination. These stress resistant asexual spores can be killed by preservation methods such as heat treatment. Here, the effects of cultivation time and temperature on thermal resistance of P. roqueforti conidia were studied. To this end, cultures were grown for 3, 5, 7 and 10 days at 25 °C or for 7 days at 15, 25 and 30 °C. Conidia of 3- and 10-day-old cultures that had been grown at 25 °C had D56-values of 1.99 ± 0.15 min and 5.31 ± 1.04 min, respectively. The effect of cultivation temperature was most pronounced between P. roqueforti conidia cultured for 7 days at 15 °C and 30 °C, where D56-values of 1.12 ± 0.05 min and 4.19 ± 0.11 min were found, respectively. Notably, D56-values were not higher when increasing both cultivation time and temperature by growing for 10 days at 30 °C. A correlation was found between heat resistance of conidia and levels of trehalose and arabitol, while this was not found for glycerol, mannitol and erythritol. RNA-sequencing showed that the expression profiles of conidia of 3- to 10-day-old cultures that had been grown at 25 °C were distinct from conidia that had been formed at 15 °C and 30 °C for 7 days. Only 33 genes were upregulated at both prolonged incubation time and increased growth temperature. Their encoded proteins as well as trehalose and arabitol may form the core of heat resistance of P. roqueforti conidia.</p

Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae

The FlbA-regulated predicted transcription factor Fum21 of <i>Aspergillus niger</i> is involved in fumonisin production

Aspergillus niger secretes proteins throughout the colony except for the zone that forms asexual spores called conidia. Inactivation of flbA that encodes a regulator of G-protein signaling results in colonies that are unable to reproduce asexually and that secrete proteins throughout the mycelium. In addition, the ΔflbA strain shows cell lysis and has thinner cell walls. Expression analysis showed that 38 predicted transcription factor genes are differentially expressed in strain ΔflbA. Here, the most down-regulated predicted transcription factor gene, called fum21, was inactivated. Growth, conidiation, and protein secretion were not affected in strain Δfum21. Whole genome expression analysis revealed that 63 and 11 genes were down- and up-regulated in Δfum21, respectively, when compared to the wild-type strain. Notably, 24 genes predicted to be involved in secondary metabolism were down-regulated in Δfum21, including 10 out of 12 genes of the fumonisin cluster. This was accompanied by absence of fumonisin production in the deletion strain and a 25% reduction in production of pyranonigrin A. Together, these results link FlbA-mediated sporulation-inhibited secretion with mycotoxin production

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end, > 300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO3, Na2HPO4/NaH2PO4, MgSO4 and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (Pmax) that had formed germ tubes was 12.7%, while time needed to reach 0.5 Pmax (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a Pmax of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage

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 (Pmax) 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, Pmax 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 Pmax of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these Pmax 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

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger

Aspergillus niger is used by the industry to produce enzymes and metabolites such as citric acid. In liquid cultures, it can grow as a dispersed mycelium or as micro-colonies with a width in the micrometer to millimeter range. Here, it was assessed whether expression of genes encoding secreted enzymes depends on mycelium morphology. To this end, expression of the reporter gene gfp from the promoters of the glucoamylase gene glaA, the feruloyl esterase gene faeA and the α-glucuronidase gene aguA was causally related to micro-colony size within a liquid shaken culture. Data could be fitted by hyperbolic functions, implying that the genes encoding these secreted proteins are expressed in a shell at the periphery of the micro-colony. The presence of such a shell was confirmed by confocal microscopy. Modelling predicted that the width of these zones was 13 to 156 µm depending on growth medium and micro-colony diameter. Together, data indicate that the highest productive micro-colonies are those colonies that have a radius ≤ the width of the peripheral expression zone

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 Aspergillusniger,Aspergillusoryzae,Aspergillusclavatus, Aspergillusnidulans and Aspergillusterreus 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

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end, > 300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO3, Na2HPO4/NaH2PO4, MgSO4 and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (Pmax) that had formed germ tubes was 12.7%, while time needed to reach 0.5 Pmax (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a Pmax of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage