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.

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.

Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC.

The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all β-lactam antibiotics. To avoid the use of β-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics.

The Rab GTPase YPT-1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa.

Vesicle traffic involves budding, transport, tethering and fusion of vesicles with acceptor membranes. GTP-bound small Rab GTPases interact with the membrane of vesicles, promoting their association with other factors before their subsequent fusion. Filamentous fungi contain at their hyphal apex the Spitzenkörper (Spk), a multivesicular structure to which vesicles concentrate before being redirected to specific cell sites.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family.

Six hydrophobins are involved in hydrophobin rodlet formation in Aspergillus nidulans and contribute to hydrophobicity of the spore surface.

Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter.

Neurospora crassa NKIN2, a kinesin-3 motor, transports early endosomes and is required for polarized growth.

Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious.

The Aspergillus nidulans kinesin-3 tail is necessary and sufficient to recognize modified microtubules.

Posttranslational microtubule modifications (PTMs) are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The Aspergillus nidulans kinesin-3 UncA uses preferably modified microtubules (MTs) as tracks for vesicle transportation. Here, we show that a positively charged region in the tail of UncA (amino acids 1316 to 1402) is necessary for the recognition of modified MTs.