Ca Signalling Differentially Regulates Germ-Tube Formation and Cell Fusion in .

is an important plant pathogen and an emerging opportunistic human pathogen. Germination of conidial spores and their fusion via conidial anastomosis tubes (CATs) are significant events during colony establishment in culture and on host plants and, hence, very likely on human epithelia. CAT fusion exhibited by conidial germlings of Fusarium species has been postulated to facilitate mitotic recombination, leading to heterokaryon formation and strains with varied genotypes and potentially increased virulence.

Characterisation of Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy.

The precise characterization of the mechanisms modulating survival within airway epithelial cells has been impaired by the lack of live-cell imaging technologies and user-friendly quantification approaches. Here we described the use of an automated image analysis pipeline to estimate the proportion of spores taken up by airway epithelial cells, those contained within phagolysosomes or acidified phagosomes, along with the fungal factors contributing to these processes. Coupling the use of fluorescent strains and fluorescent epithelial probes targeting lysosomes, acidified compartments and cell membrane, we found that both the efficacy of lysosome recruitment to phagosomes and phagosome acidification determines the capacity of airway epithelial cells to contain growth.

Live-cell imaging of rapid calcium dynamics using fluorescent, genetically-encoded GCaMP probes with Aspergillus fumigatus.

Calcium signalling plays a fundamental role in fungal intracellular signalling. Previous approaches (fluorescent dyes, bioluminescent aequorin, genetically encoded cameleon probes) with imaging rapid subcellular changes in cytosolic free calcium ([Ca]) in fungal cells have produced inconsistent results. Recent data obtained with new fluorescent, genetically encoded GCaMP probes, that are very bright, have resolved this problem.

Phagolysosomal Survival Enables Non-lytic Hyphal Escape and Ramification Through Lung Epithelium During Infection.

is the most important mould pathogen in immunosuppressed patients. Suboptimal clearance of inhaled spores results in the colonisation of the lung airways by invasive hyphae. The first point of contact between and the host is the lung epithelium.

Calcium homeostasis plays important roles in the internalization and activities of the small synthetic antifungal peptide PAF26.

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance.

Cell Wall Composition Heterogeneity between Single Cells in Aspergillus fumigatus Leads to Heterogeneous Behavior during Antifungal Treatment and Phagocytosis.

can cause a variety of lung diseases in immunocompromised patients, including life-threatening invasive aspergillosis. There are only three main classes of antifungal drugs currently used to treat aspergillosis, and antifungal resistance is increasing. Experimental results in fungal biology research are usually obtained as average measurements across whole populations while ignoring what is happening at the single cell level.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of .

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce.

Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus.

Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors.

Lung colonization by Aspergillus fumigatus is controlled by ZNF77.

Aspergillus fumigatus is a critical pathogen of humans. Exposure to A. fumigatus conidia occurs frequently but is normally cleared from the respiratory airways.

Development of an adaptable headspace sampling method for metabolic profiling of the fungal volatome.

Pulmonary aspergillosis can cause serious complications in people with a suppressed immune system. Volatile metabolites emitted by Aspergillus spp. have shown promise for early detection of pathogenicity.

Effect of the Novel Antifungal Drug F901318 (Olorofim) on Growth and Viability of Aspergillus fumigatus.

F901318 (olorofim) is a novel antifungal drug that is highly active against species. Belonging to a new class of antifungals called the orotomides, F901318 targets dihydroorotate dehydrogenase (DHODH) in the pyrimidine biosynthesis pathway. In this study, the antifungal effects of F901318 against were investigated.

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains.

Spatio-temporal MAPK dynamics mediate cell behavior coordination during fungal somatic cell fusion.

Mitogen-activated protein kinases (MAPKs) are conserved regulators of proliferation, differentiation and adaptation in eukaryotic cells. Their activity often involves changes in their subcellular localization, indicating an important role for these spatio-temporal dynamics in signal transmission. A striking model illustrating these dynamics is somatic cell fusion in Germinating spores of this fungus rapidly alternate between signal sending and receiving, thereby establishing a cell-cell dialog, which involves the alternating membrane recruitment of the MAPK MAK-2 in both fusion partners.

Mutual independence of alkaline- and calcium-mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus.

Functional coupling of calcium- and alkaline responsive signalling occurs in multiple fungi to afford efficient cation homeostasis. Host microenvironments exert alkaline stress and potentially toxic concentrations of Ca , such that highly conserved regulators of both calcium- (Crz) and pH- (PacC/Rim101) responsive signalling are crucial for fungal pathogenicity. Drugs targeting calcineurin are potent antifungal agents but also perturb human immunity thereby negating their use as anti-infectives, abrogation of alkaline signalling has, therefore, been postulated as an adjunctive antifungal strategy.

Caspofungin-Mediated Growth Inhibition and Paradoxical Growth in Aspergillus fumigatus Involve Fungicidal Hyphal Tip Lysis Coupled with Regenerative Intrahyphal Growth and Dynamic Changes in β-1,3-Glucan Synthase Localization.

Caspofungin targets cell wall β-1,3-glucan synthesis and is the international consensus guideline-recommended salvage therapy for invasive aspergillosis. Although caspofungin is inhibitory at low concentrations, it exhibits a paradoxical effect (reversal of growth inhibition) at high concentrations by an undetermined mechanism. Treatment with caspofungin at either the growth-inhibitory concentration (0.

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences.

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function.

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus.

Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood.

Searching for the Optimal Fluorophore to Label Antimicrobial Peptides.

With the advent of antimicrobial resistance, there is an urgent need for new strategies to treat infectious diseases. Antimicrobial peptides are considered as promising candidates, and therefore there is a need to understand their mechanism of action in order to exploit their therapeutic potential. To this end, fluorescent analogs are powerful tools to analyze their behavior and subcellular localization in cells and in vivo.

Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines.

Herein we report the preparation of BODIPY mesoionic acid fluorides through a short sequence involving an isocyanide multicomponent reaction as the key synthetic step. These novel BODIPY acid fluorides are water-stable electrophilic reagents that can be used for the fluorescent derivatization of amine-containing biomolecules using mild and activation-free reaction conditions. As a proof of principle, we have labeled the antifungal natamycin and generated a novel fluorogenic probe for imaging a variety of human and plant fungal pathogens, with excellent selectivity over bacterial cells.

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited.

Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue.

Fluorescent antimicrobial peptides are promising structures for in situ, real-time imaging of fungal infection. Here we report a fluorogenic probe to image Aspergillus fumigatus directly in human pulmonary tissue. We have developed a fluorogenic Trp-BODIPY amino acid with a spacer-free C-C linkage between Trp and a BODIPY fluorogen, which shows remarkable fluorescence enhancement in hydrophobic microenvironments.