Background Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. Results We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. Conclusions We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

List of differential expressed genes (DEGs) related to the significantly enriched categories from the FunCat and GO functional analysis. A p-value of 0.05 was used for the enrichment method. For FunCat the first and second level is shown and when appropriate even higher levels. (XLS 28782Â kb)

The highly dynamic development of sequencing technologies, earlier powered from whole genome sequencing, opens new approaches to analysis. The wide differences of the currently available techniques provide a great opportunity for transcriptional analysis. In this application note we would like to introduce a technique developed in-house that combines short and long read technologies for expression profiling which will vastly improve your sequencing and analysis results.

Die Kombination massiv-paralleler long-read-Sequenzierungstechnologie mit short-read-Technologie eröffnet neue Wege in der Identifikation und quantitativen Analyse von mRNA-Transkripten und hilft so beim Verständnis von Genexpression.

Moulds are characterized by their saprophytic lifestyle that is based on osmotrophy. Among them, Aspergillus fumigatus has emerged as the leading cause of fungal infections in the presence of an underlying immunodeficiency. To assess the role of its nutritional versatility for virulence, transcriptional profiling studies in the presence of varying sources of nitrogen were carried out and revealed an extensive reprogramming of the fungal transcriptome when shifting to a proteinaceous growth substrate. Transcripts encoding metabolic activities were predominantly upregulated, as were proteinases and transport activities. To probe whether fundamental aspects of its osmotrophic lifestyle, that is, extracellular proteolysis and uptake of oligopeptides, are required for A. fumigatus pathogenicity, serial gene replacements were carried out, which eventually yielded an octuple deletion mutant ablated for the opt gene family. This strain displayed no growth defect on various substrates, but supplementary reduction of extracellular proteolytic activity by additional deletion of the prtT gene revealed a synthetic phenotype on porcine lung tissue agar. Virulence studies in a murine model of pulmonary aspergillosis did not disclose any attenuation in virulence of these deletants. Our data emphasize a high degree of redundancy encoded by the A. fumigatus genome that secures nutrient supply for growth and, therefore, virulence.

ABSTRACT Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing β serine recombinase acting on six recognition sequences (β-rec/ six ) in a fungal host, the human pathogen Aspergillus fumigatus , and its use in establishing a self-excising resistance marker cassette for serial gene replacement.