Parallel fitness using barcode sequencing in A. fumigatus: Assessment of the role of Aspergillus Kinases in azole resistance and as Targets for Antifungal Drug Discovery

N. ALFURAIJIa, J. Mabey a, M. Bromley a and P. Bowyera

Author address: 

aManchester Fungal Infection Group,Division of Immunity, Infection and Respiratory Medicine,Faculty of Biology, Medicine and Health Sciences, University of Manchester

Abstract: 

Fungi cause a wide range of infections including invasive and life threatening disease, superficial
infections of the skin and mucosal membranes, as well as allergic disorders. The mortality rate due to
fungal diseases remains unacceptably high, and is thought to exceed one million patients annually.
Currently, only four groups of antifungals are available to treat systemic fungal infections: the polyenes,
flucytosine, triazoles and echinocandins. The emergence of resistance to the available antifungal drugs
and toxicity associated with some classes necessitates the exploration of novel pharmacologically
effective antifungal drugs. Protein phosphorylation by protein kinases (PK) impacts all areas of cellular
activity. Their critical roles in a wide range of cellular functions, along with the relative ease in which drug
inhibitor assays can be developed have highlighted this class of enzymes as potential drug targets.
A bioinformatic analysis of the genomes of 9 Aspergillus species has revealed, in contrast to a previous
study, that the number of protein kinases in each species is relatively consistent ranging from a minimum
of 140 in A. nidulans up to 175 in A. niger. The kinases are distributed over 11 sub-groups including
genes which apparently encode tyrosine like kinases and tyrosine kinases. We present comparative
analysis which suggests that there are a sub-group of kinases that are specific to filamentous fungi.
To assess the role of these kinases in growth, virulence and drug tolerance, we have generated a library
of 90 genetically barcoded knockout mutants. We have validated a barcode-sequencing approach to
assess in pooled cultures, the fitness defect in each null mutant when in standard culture conditions and
in the presence of the antifungal drug itraconazole. We show that 2 genes, the mitogen-activated protein
kinase mpkB (AFUB_078810) and a serine/threonine protein kinase ssn3 (AFUB_035220) are critical for
azole tolerance. In addition, we have identified 25 genes that encode kinases that are essential for
viability. Our assessment of virulence of these strains is currently in progress.

2017

abstract No: 

5

Full conference title: 

The Fourteenth International Aspergillus Meeting, Asilomar Conference Center, Pacific Grove, CA, USA
    • Asperfest 14 (2017)