Abstract
Cryptococcus gattii is a primary fungal pathogen, which causes pulmonary and brain infections in healthy as well as immunocompromised individuals. Genetic manipulations in this pathogen are widely employed to study its biology and pathogenesis, and require integration of foreign DNA into the genome. Thus, identification of gene free regions where integrated foreign DNA can be expressed without influencing, or being influenced by, nearby genes would be extremely valuable. To achieve this goal, we examined publicly available genomes and transcriptomes of C. gattii, and identified two intergenic regions in the reference strain R265 as potential “safe haven” regions, named as CgSH1 and CgSH2. We found that insertion of a fluorescent reporter gene and a selection marker at these two intergenic regions did not affect the expression of their neighboring genes and were also expressed efficiently, as expected. Furthermore, DNA integration at CgSH1 or CgSH2 had no apparent effect on the growth of C. gattii, its response to various stresses, or phagocytosis by macrophages. Thus, the identified safe haven regions in C. gattii provide an effective tool for researchers to reduce variation and increase reproducibility in genetic experiments.
Highlights
Cryptococcosis caused by the pathogenic Cryptococcus species complexes is responsible for 181,100 deaths annually [1]
C. gattii is responsible for the Vancouver Island outbreak in 1999, and it has spread to the north–western US [8,9]
Integration in a designated gene free region in the genome is preferred. Several such gene free regions named “safe haven” sites have recently been identified in two reference strains of the C. neoformans species complex (H99 and XL280), and Af293/CEA10 strains of A. fumigatus [18,19,20,21]
Summary
Cryptococcosis caused by the pathogenic Cryptococcus species complexes is responsible for 181,100 deaths annually [1]. In the past two decades, major advances in molecular technology have facilitated our understanding of the pathogenic Cryptococcus species complexes [11,12,13,14] Classic approaches such as gene complementation, gene overexpression, and construction of fluorescent tagged protein strains are routinely used to analyze gene functions and all require introduction of foreign DNA into the genome [15,16]. Several such gene free regions named “safe haven” sites have recently been identified in two reference strains of the C. neoformans species complex (H99 and XL280), and Af293/CEA10 strains of A. fumigatus [18,19,20,21] The identification of these safe haven sites has greatly facilitated genetic research in these fungal species [22,23,24,25]. Together with the previous work for the C. neoformans species complex, this study completes the important task of identifying safe haven regions for the entire pathogenic cryptococcal species complexes
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