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Title: Novel mechanisms of antifungal resistance in Aspergillus fumigatus
Author: Khateb, Aiah
ISNI:       0000 0004 8504 8256
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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Globally there are more than a billion individuals affected by fungal disease with an annual mortality of ~1.5 million. There are few antifungal drugs. The azole class of antifungal is commonly used to treat Aspergillus fumigatus, but resistance is increasing. This project aims to uncover important and novel mechanisms of azole drug resistance. Recently, a genome scale mutagenesis experiment identified a number of genes that were associated with altered azole sensitivity. These genes include; a number of major facilitator superfamily (MFS) and ATP-binding cassette (ABC) transporters, tRNA and tRNA modification enzymes. Few previous studies have determined a role for ABC and MFS azole transporters in azole resistance in A. fumigatus. Eleven knockouts were generated based on the transposon mutagenesis results. Knockouts were tested for minimal inhibitory concentration (MIC), growth rate on solid media, and gene expression under Itraconazole (ITZ). Five knockouts grew significantly faster in the presence of ITZ compared to the parental isolate. Four of the MFS genes were up-regulated under ITZ exposure. One ABC transporter showed higher growth than A1160 under all ITZ at 0.12, 0.5 mg/L with P values 0.01, 0.04.One MFS transporter showed characteristics of being an azole importer. This gene(AziA) was deleted. Increased growth (11.5 fold) was observed for the ΔAziA strain on ITZ plates, peaking at 0.5mg/L (P < 1x106 ). MICs were 4 fold higher for ITZ and 2 fold for VOR and POS than in the parental strain A1160. Deletion of orthologues in N. crassa and S. cerevisiae showed similar increase. Association analysis of A. fumigatus AziA variants in clinical and environmental genomes (n=87) showed three mutations associated with resistance (P < 0.05). AziA expression was induced by ITZ with maximum increase of 5.3 fold at 1 mg/L (P=3.7x10-36). Finally, ITZ concentrations in ΔAziA hyphae were 2.5 times lower compared to A1160 (P < 0.001). Orthologues showing 1-1 reciprocity were identified in all ascomycetes. This study identified the first fungal azole importer in A. fumigatus and in other fungi. Transfer RNA (tRNA) & tRNA modifying enzymes (tRNA ME) regulate translation during cellular stress. This mechanism is explored in realtion to azole resistance. Ten preselected tRNA and tRNA ME genes were deleted. All tRNA knockouts showed similar growth on solid media compared to A1160 except Δmethionyl tRNA formyltransferase (tRNA-Meth) which had 70% lower growth. The highest growth across ITZ concentrations was found in ΔtRNA splicing endonuclease (tRNA-Spl) fallowed by tRNA dihydrouridine synthase (tRNA-DUS) and only tRNA-Spl and tRNA-DUS knockouts were able to grow on 1mg/L ITZ. ΔtRNA-DUS had 4 fold increase in ITZ, 8 fold in POS, and 4 fold in VOR compared to A1160 ΔtRNA-Spl had 2 fold increase in ITZ and 4 fold in VOR but POS remaind unchanged. We hypothesized that some of resistance is linked to an altered tRNA regulation in stress response in the A. fumigatus mutants where tRNA mediates the initiation of the translational process. Culture methods can only recover a small fraction of all lung resident fungi, hence resistant isolates are often missed. Azole resistance is associated with poorer disease outcome. This study aims to detect resistance-associated mutations in cyp51A in the context of uncultured Aspergillus strains present in clinical samples. To achieve this a metagenomics - based approach to directly detect mutations in cyp51A directly from human respiratory samples was developed. DNA was extracted directly from 35 BAL samples. Sixty one SNPS were identified, 29 resulted in amino acid changes. The SNPs M220I/M220T and F46L matched previously reported azole resistance mutations. It also adds comprehensive information about species identification, natural mutations in healthy and chronically colonized patients and resistance mutations. This demonstrates the utility of NGS as a diagnostic tool in detecting hard to culture drug resistant uisolates in complex samples. Chronic pulmonary aspergillosis (CPA) affects more than 3 million individuals with ~15-80% mortality rate. Long term treatment with azoles is known to lead to the development of resistance. We investigated how A.fumigatus genomes evolved over a long term treatment course in a CPA patient (2008-2018). MICs showed variable resistance patterns independent of drug treatment. A genome based phylogenetic tree showed three related clades infecting this patient. The analysis showed high variation in nucleic acid binding, catalyses, transcription factors, protein kinases and MFS transporters. Analysis of copy number variation revealed several large genomic deletions and duplications. Resistance to azoles is a major problem in isolates from patients with CPA. Our current data suggest that CPA in this patient consists of a mixed population of A. fumigatus possibly arising from the same isolate which has evolved in the lung.
Supervisor: Bromley, Michael ; Bowyer, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: wholegenome ; cyp51a ; aspergillosis ; sequencing ; transporters ; mechanism ; itraconazole ; resistance ; fumigatus ; aspergillus ; chronic pulmonary aspergillosis