Genetic Manipulation of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, C. neoformans, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system. In particular, genetic manipulation is the first step in the identification and characterization of the function of genes for understanding the mechanisms underlying the pathogenicity of C. neoformans. This unit describes protocols for constructing target gene deletion mutants using double-joint (DJ) PCR, constitutive overexpression strains using the histone H3 gene promoter, and epitope/fluorescence protein-tagged strains in C. neoformans. © 2018 by John Wiley & Sons, Inc.

Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen, Cryptococcus neoformans

“…Deletion mutant strains were constructed in the C. neoformans serotype A H99S strain background using split marker/double-joint PCR strategies [ 19 ]. The genetic information for each gene was obtained from FungiDB ( , accessed on 5 May 2018) The primers used in this study are described in Table S2 .…”

“…It then disseminates to the brain via the bloodstream and infects the central nervous system by crossing the blood–brain barrier [ 17 , 18 ]. Due to the availability of its genetic mutants and molecular tools, C. neoformans has become a model species to study fungal pathogenicity and virulence mechanisms [ 19 ]. In this study, using a reverse genetic approach, we found that thymol not only induced intracellular calcium imbalance in C. neoformans but also inhibited N -glycosylation modification of the Cpy protein.…”

Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans

“…We constructed phosphatase and retromer mutant strains in the C. neoformans serotype A H99S strain background through homologous recombination using gene-disruption cassettes containing the nourseothricin-resistance marker (nourseothricin acetyl transferase; NAT ) using NAT -split marker/double joint PCR (DJ-PCR) strategies 99 (see Supplementary Data 4 for primers). We amplified 5′- and 3′-flanking regions of target genes by PCR with primer pairs L1/ L2 and R1/R2, respectively, from H99S genomic DNA.…”

Cryptococcus neoformans Mating and Genetic Crosses

“…Numerous tools and resources are available for Cryptococcus sexual reproduction and genetic studies. Genetic manipulation in Cryptococcus can be achieved by biolistic transformation, Agrobacterium ‐mediated random insertional mutagenesis, or a novel CRISPR‐mediated electroporation method (Davidson et al., ; Esher, Granek, & Alspaugh, ; Fan & Lin, ; Jung, Lee, So, & Bahn, ; Toffaletti & Perfect, ; Toffaletti, Rude, Johnston, Durack, & Perfect, ). Congenic strain pairs (i.e., strains that are genetically identical with the exception of the MAT locus) have been constructed for C. neoformans , C. deneoformans , and C. deuterogattii (Heitman, Allen, Alspaugh, & Kwon‐Chung, ; Nielsen et al., ; Zhai et al., ).…”