title: Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.
abstract: Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Liliana
authors.initials: L
authors.lastname: Losada
authors.affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Janyce A
authors.initials: JA
authors.lastname: Sugui
authors.affiliation: Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Eckhaus
authors.affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Yun C
authors.initials: YC
authors.lastname: Chang
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Mounaud
authors.affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Abigail
authors.initials: A
authors.lastname: Figat
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Vinita
authors.initials: V
authors.lastname: Joardar
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Suman B
authors.initials: SB
authors.lastname: Pakala
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Suchitra
authors.initials: S
authors.lastname: Pakala
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Pratap
authors.initials: P
authors.lastname: Venepally
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Natalie
authors.initials: N
authors.lastname: Fedorova
authors.affiliation: J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: William C
authors.initials: WC
authors.lastname: Nierman
authors.affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Kyung J
authors.initials: KJ
authors.lastname: Kwon-Chung
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001228:Aspergillosis / Q000188:drug therapy / Q000382:microbiology / Q000473:pathology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism / Q000472:pathogenicity; D001393:Azoles / Q000494:pharmacology* / Q000627:therapeutic use; D003433:Crosses, Genetic; D026141:Drug Resistance, Multiple, Fungal / Q000187:drug effects; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D049770:Genes, Mating Type, Fungal / Q000187:drug effects; D056426:Genetic Loci / Q000187:drug effects; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D017964:Itraconazole / Q000494:pharmacology / Q000627:therapeutic use; D007814:Larva / Q000187:drug effects; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009036:Moths / Q000187:drug effects; D009154:Mutation; D058886:Sterol 14-Demethylase / Q000235:genetics / Q000378:metabolism*; D016019:Survival Analysis; D014230:Triazoles / Q000494:pharmacology / Q000627:therapeutic use; D014774:Virulence / Q000187:drug effects; D065819:Voriconazole / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D014230:Triazoles; D017964:Itraconazole; C101425:posaconazole; D006899:Mixed Function Oxygenases; D006903:Hydroxymethylglutaryl CoA Reductases; D058886:Sterol 14-Demethylase; C027532:methylsterol monooxygenase; D065819:Voriconazole