Community Resources for Aspergillus fumigatus

Summary

Aspergillus fumigatus is the predominant cause of both allergic and invasive aspergillosis possessing particular metabolic capabilities and genetic determinants that differentiate it from most other fungal species. A. fumigatus is also a primary pathogen of the sinuses, lungs, damaged skin, and subcutaneous tissues, and disseminates to other organs including the brain. The key drugs for treating aspergillosis are azoles, especially voriconazole, but unfortunately resistance to these drugs is emerging.

Genomic resources for studying the biology and pathology of A. fumigatus are limited in two critical ways. The first limitation is that we have available the genome sequences of only two strains, Af293 and A1163. Microsatellite typing and similar gene content document that they are closely related, greatly limiting our view of the genetic/genomic variation within the species. To address this limitation we are proposing to sequence and annotate two additional reference strains carefully selected to provide a much broader representation of the species variation. The use of new sequencing technologies will likely allow the sequencing of genomic features such as improved centromeric sequence and structure that were left unsequenced in Af293 and A1163 due to the limitations of Escherichia coli clone libraries used in sequencing these strains.

The low quality of the genome annotation of A. fumigatus is the second major limitation to studies on A. fumigatus. While the annotation of A. fumigatus is probably superior to that of any sequenced filamentous fungus, state-of-the-art gene calling and annotation software lacks the power to produce high quality annotation. Numerous gene models in the A. fumigatus sequenced genomes have undetected errors in translation start and stop sites, and splice boundaries. Identification of 5? and 3? untranslated regions of protein-coding genes and alternative splice sites was not even addressed in the process of the annotation of these genomes. Neither was the identification and genomic locations of non-coding genes including small RNAs (sRNAs) that may regulate A. fumigatus biology. This circumstance forces investigators attempting to delete or modify an A. fumigatus gene to experimentally determine/verify the gene structure of a gene of interest in an inefficient one-gene-at-a-time manner. To address this limitation, the RNA-Seq and sRNA-Seq technologies will be employed to experimentally verify the gene models including the 5? and 3? UTRs for nearly all of the genes in the genome and non-coding genes (sRNAs). A. fumigatus investigators from 7 research groups have agreed to provide RNAs from a range of in vitro and in vivo cultivation conditions to allow the experimental determination of the gene structures of what we expect will be essentially all of the coding and non-coding genes in the genome.

In the clinical treatment of invasive aspergillosis, drug resistance in A. fumigatus has emerged as a major challenge in obtaining good clinical outcomes. Numerous mutations resulting in resistance have been described, but its molecular and evolutionary mechanisms remain to be characterized. We will sequence 42 resistant strains and 6 matched susceptible strains from the same patient to identify additional mechanisms of resistance as this resistance evolves in patients. To achieve a high resolution view of the genome sequence alterations in these strains we will use high sequence coverage of these genomes from a short read length sequencing platform.

This project has been developed in consultation with a large number of US and international A. fumigatus PIs including Michelle Momany, the current elected chair of the Aspergillus Genomics Research Policy Committee (AGRPC). This project thus represents the consensus opinion of the Aspergillus community for addressing the two limitations of Aspergillus fumigatus genomic resources cited above and to explore the mechanism of drug resistance development in invasive aspergillosis clinical practice.

White Paper Access

The initial white paper submitted can be downloaded here. Since white papers are not always approved exactly as submitted, this document may not exactly describe the final form of the project. Please contact gsc@jcvi.org if you have any questions.

All Publications that use data generated and/or are supported by the Sequencing Center at JCVI should acknowledge the sponsor as: This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.


Investigators and Collaborators

Liliana Losada, PhD

Assistant Professor, JCVI

David W. Denning, PhD

Professor of Medicine and Medical Mycology, University Hospital of South Manchester