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An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.

ABSTRACT: Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free "safe haven" (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.

SUBMITTER: Fan Y

PROVIDER: S-EPMC7726056 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:C. neoformans is an encapsulated fungal pathogen with defined asexual and sexual life cycles. Due to the availability of genetic and molecular tools for its manipulation, it has become a model organism for studies of fungal pathogens, even though it lacks a reliable system for maintaining DNA fragments as extrachromosomal plasmids. To compensate for this deficiency, we identified a genomic gene-free intergenic region where heterologous DNA could be inserted by homologous recombination without adverse effects on the phenotype of the recipient strain. Since such a site in the C. neoformans genome at a different location has been named previously as "safe haven", we named this locus second safe haven site (SH2). Insertion of DNA into this site in the genome of the KN99 congenic strain pair caused minimal change in the growth of the engineered strain under a variety of in vitro and in vivo conditions. We exploited this 'safe' locus to create a genetically stable highly fluorescent strain expressing mCherry protein (KN99mCH); this strain closely resembled its wild-type parent (KN99α) in growth under a variety of in vitro stress conditions and in the expression of virulence traits. The efficiency of phagocytosis and the proliferation of KN99mCH inside human monocyte-derived macrophages were comparable to those of KN99α, and the engineered strain showed the expected organ dissemination after inoculation, although there was a slight reduction in virulence. The mCherry fluorescence allowed us to measure specific association of cryptococci with leukocytes in the lungs and mediastinal lymph nodes of infected animals and, for the first-time, to assess their live/dead status in vivo. These results highlight the utility of KN99mCH for elucidation of host-pathogen interactions in vivo. Finally, we generated drug-resistant KN99 strains of both mating types that are marked at the SH2 locus with a specific drug resistant gene cassette; these strains will facilitate the generation of mutant strains by mating.

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An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.

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