Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi.

While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe morphological changes or molecular events in fungi, especially when long observation periods or high light fluxes are warranted, encounter problems owed to altered regulatory pathways or even cell death caused by various photosensing mechanisms. Consequently, the ever expanding repertoire of visible fluorescent protein toolboxes and high-resolution microscopy methods designed to investigate fungi in vitro and in vivo need to comply with an additional requirement: to decrease the unwanted side effects of illumination. In addition to optimizing exposure, an obvious solution is red-shifted illumination, which, however, does not come without compromises. This review summarizes the interactions of fungi with light and the various molecular biology and technology approaches developed for exploring their functions on the molecular, cellular, and in vivo microscopic levels, and outlines the progress towards reducing phototoxicity through applying far-red and near-infrared light. KEY POINTS: • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity.

Increased Cd2+ biosorption capability of Aspergillus nidulans elicited by crpA deletion.

The P-type ATPase CrpA is an important Cu2+ /Cd2+ pump in the Aspergilli, significantly contributing to the heavy metal stress tolerance of these ascomycetous fungi. As expected, the deletion of crpA resulted in Cu2+ /Cd2+ -sensitive phenotypes in Aspergillus nidulans on stress agar plates inoculated with conidia. Nevertheless, paradoxical growth stimulations were observed with the ΔcrpA strain in both standard Cu2+ stress agar plate experiments and cellophane colony harvest (CCH) cultures, when exposed to Cd2+ . These observations reflect efficient compensatory mechanisms for the loss of CrpA operating under these experimental conditions. It is remarkable that the ΔcrpA strain showed a 2.7 times higher Cd biosorption capacity in CCH cultures, which may facilitate the development of new, fungal biomass-based bioremediation technologies to extract harmful Cd2+ ions from the environment. The nullification of crpA also significantly changed the spatial distribution of Cu and Cd in CCH cultures, as demonstrated by the combined particle-induced X-ray emission and scanning transmission ion microscopy technique. Most important, the centers of gravity for Cu and Cd accumulations of the ΔcrpA colonies shifted toward the older regions as compared with wild-type surface cultures.

Total transcriptome response for tyrosol exposure in Aspergillus nidulans.

Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction.

Effect of cell wall integrity stress and RlmA transcription factor on asexual development and autolysis in Aspergillus nidulans.

The cell wall integrity (CWI) signaling pathway is responsible for cell wall remodeling and reinforcement upon cell wall stress, which is proposed to be universal in fungal cultures. In Aspergillus nidulans, both the deletion of rlmA encoding the RlmA transcription factor in CWI signaling and low concentrations of the cell wall polymer intercalating agent Congo Red caused significant physiological changes. The gene deletion mutant ΔrlmA strain showed decreased CWI and oxidative stress resistances, which indicated the connection between the CWI pathway and the oxidative stress response system. The Congo Red stress resulted in alterations in the cell wall polymer composition in submerged cultures due to the induction of the biosynthesis of the alkali soluble fraction as well as the hydrolysis of cell wall biopolymers. Both RlmA and RlmA-independent factors induced by Congo Red stress regulated the expression of glucanase (ANID_00245, engA) and chitinase (chiB, chiA) genes, which promoted the autolysis of the cultures and also modulated the pellet sizes. CWI stress and rlmA deletion affected the expression of brlA encoding the early conidiophore development regulator transcription factor BrlA and, as a consequence, the formation of conidiophores was significantly changed in submerged cultures. Interestingly, the number of conidiospores increased in surface cultures of the ΔrlmA strain. The in silico analysis of genes putatively regulated by RlmA and the CWI transcription factors AnSwi4/AnSwi6 in the SBF complex revealed only a few jointly regulated genes, including ugmA and srrA coding for UgmA UDP-galactopyranose mutase and SrrA stress response regulator, respectively.

Rare earth element sequestration by Aspergillus oryzae biomass.

The fungus Aspergillus oryzae could be shown to be a viable alternative for biosorption of valuable metals from solution. Fungal biomass can be obtained easily in high quantities as a waste of biofermentation processes, and used in a complex, multi-phase solution mimicking naturally occurring, mining-affected water samples. With test solution formulated after natural conditions, formation of secondary Al and Fe phases co-precipitating Ce was recorded in addition to specific biosorption of rare earth elements. Remarkably, the latter were removed from the solution despite the presence of high concentrations of interfering Fe and Al. The biomass was viable even after prolonged incubation in the metal solution, and minimal inhibitory concentrations for single metals were higher than those in the test solution. While precipitation/biosorption of Ce (maximal biosorption efficiency was 58.0 ± 22.3% after 6 h of incubation) coincided with the gross removal of Fe from the metal solution, Y (81.5 ± 11.3% efficiency, 24 h incubation) and Nd (87.4 ± 9.1% efficiency, 24 h incubation) were sequestered later, similarly to Ni and Zn. The biphasic binding pattern specific to single metals could be connected to dynamically changing pH and NH4+ concentrations, which were attributed to the physiological changes taking place in starving A. oryzae biomass. The metals were found extracellularly in minerals associated with the cell wall, and intracellularly precipitated in the vacuoles. The latter process was explained with intracellular metal detoxification resulting in metal resistance.

Effects of hemin, CO2, and pH on the branching of Candida albicans filamentous forms.

Morphological transitions of wild-type and oxidative stress-tolerant Candida albicans strains were followed in the RPMI-FBS culture medium at pH values and CO2 levels characteristic for the anatomical niches inhabited by this opportunistic human pathogen fungus, including the oral cavity as well as the intestinal and vaginal lumens. Selected cultures were also supplemented with hemin modeling bleedings. Germination as well as elongation and branching of hyphae were monitored in the cultures using time-lapse video microscopy. Unexpectedly, branching time, which is defined as the time taken until the first branch of hypha emerges for the first time after germination, correlated well with alterations in the environmental conditions meanwhile no such correlations were found for germination time (time lasted until the appearance of the germination tube). Based on these observations, hypotheses were set up to estimate the significance of branching time in the pathogenesis of both superficial and systemic candidiases.