A Signature-Tagged Mutagenesis (STM)-based murine-infectivity assay for Cryptococcus neoformans
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PROTOCOL A Signature-Tagged Mutagenesis (STM)-based murine-infectivity assay for Cryptococcus neoformans Kwang-Woo Jung1, Kyung-Tae Lee2, and Yong-Sun Bahn2* 1
Radiation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea 2
(Received Jul 6, 2020 / Accepted Aug 12, 2020)
Signature-tagged mutagenesis (STM) is a high-throughput genetic technique that can be used to investigate the function of genes by constructing a large number of mutant strains with unique DNA identification tags, pooling them, and screening them for a particular phenotypic trait. STM was first designed for the identification of genes that contribute to the virulence or infectivity of a pathogen in its host. Recently, this method has also been applied for the identification of mutants with specific phenotypes, such as antifungal drug resistance and proliferation. In the present study, we describe an STM method for the identification of genes contributing to the infectivity of Cryptococcus neoformans using a mutant library, in which each strain was tagged with a unique DNA sequence. Keywords: fungal pathogens, quantitative PCR, virulence, mutant library Overview Microbial pathogens are equipped with both evolutionarily conserved and divergent signaling and metabolic pathways, which allow them to develop a repertoire of virulence determinants to survive and proliferate within their corresponding host. With the recent advances in molecular biological and genetic techniques as well as bioinformatics, many researchers have tried to elucidate the mechanisms of microbial pathogenicity by constructing large-scale gene-deletion mutant libraries to analyze their virulence-related functions. However, given that the conditions of the infectious and pathogenic stages within a host are dynamic and complex, in vitro approaches to identify virulence determinants have limitations that do not allow for a complete understanding of the mech*For correspondence. E-mail: [email protected]; Tel.: +82-2-2123-5558; Fax: +82-2-362-7265 Copyright G2020, The Microbiological Society of Korea
anisms of microbial pathogenicity. To overcome such limitations, the signature-tagged mutagenesis (STM) approach has been widely employed to identify genes involved in infectivity and/or virulence. This largescale approach, which is based on negative selection within a host system, was originally designed to identify virulence genes of Salmonella typhimurium using transposons carrying unique DNA sequence tags (Hensel et al., 1995). In the STM approach, each mutant is tagged with a unique DNA sequence. Mutants with different DNA tags can be pooled together and co-infected into a corresponding host model. Since this approach allows for the screening of a myriad of clones simultaneously within an animal model, it is an attractive method in terms of cost and labor compared to the classical virulenc
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