A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.
Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.
Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Laura
L
Alcazar-Fuoli
Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Timothy
T
Cairns
Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDÆA), Consejo Superior de Investigaciones Científicas, c/Jordi Girona, Barcelona, Spain.
Jordi F
JF
Lopez
Water and Soil Quality Research Group, Institute of Environmental Assessment and Water Research (IDÆA), Consejo Superior de Investigaciones Científicas, c/Jordi Girona, Barcelona, Spain.
Bozo
B
Zonja
Water and Soil Quality Research Group, Institute of Environmental Assessment and Water Research (IDÆA), Consejo Superior de Investigaciones Científicas, c/Jordi Girona, Barcelona, Spain.
Sandra
S
Pérez
Water and Soil Quality Research Group, Institute of Environmental Assessment and Water Research (IDÆA), Consejo Superior de Investigaciones Científicas, c/Jordi Girona, Barcelona, Spain; Catalan Institute of Water Research, ICRA, C/Emili Grahit, 101, edifici H2O Parc Científic i Tecnològic de la Universitat de Girona, Girona, Spain.
Damià
D
Barceló
Biotechnology Research Center, Toyama Prefectural University 5180 Kurokawa, Imizu, Toyama, Japan.
Yasuhiro
Y
Igarashi
Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Paul
P
Bowyer
Manchester Fungal Infection Group, Institute for Inflammation and Repair, Faculty of Medicine and Human Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom.
Elaine
E
Bignell
D000483:Alleles; D000818:Animals; D001232:Aspergillus fumigatus / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D023061:Gene Order; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods*; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D005810:Multigene Family