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. 2022 Feb 11;8(2):178.
doi: 10.3390/jof8020178.

Identification and Characterization of an Intergenic "Safe Haven" Region in Human Fungal Pathogen Cryptococcus gattii

Yeqi Li  1 , Tuyetnhu Pham  2 , Xiaofeng Xie  1 , Xiaorong Lin  1   2
Affiliations

Identification and Characterization of an Intergenic "Safe Haven" Region in Human Fungal Pathogen Cryptococcus gattii

Yeqi Li et al. J Fungi (Basel). .

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.

Keywords: Cryptococcus gattii; complementation; ectopic integration; genome editing; safe haven.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1
Identification of putative safe haven sites in Cryptococcus gattii R265. (A) Flow chart indicating the selection criteria for potential safe haven candidates. (B) Diagram of the three candidate regions including their neighboring genes and the size of intergenic regions. The arrows indicate the transcription direction of the genes. (C) A genome browser shot of the transcript profiles for the three potential safe haven candidates based on the published RNA-seq data of R265 cultured in YNB medium, with or without the zinc chelator TPEN [26]. The red arrows indicate the designed guide RNA target site used in this study.
Figure 2
Figure 2
DNA insertion in the candidate 1 site or the candidate 2 site has no significant impact on the expression of their neighboring genes and chromosomal rearrangement. (A–C) The relative transcript levels of the genes neighboring the three intergenic safe haven regions in the indicated strains were measured by RT-PCR. The transcript level of house-keeping gene ACT1 was used as the internal control in every sample. The transcript level of each gene was compared to that in the wild-type R265, which was set to 1 for normalization. The experiments were performed in three independent biological replicates. Statistical significance was determined using a 2-tailed t test: ns, not significant, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (D) Image of a PFGE gel of the genomic DNA preparations from the indicated strains.
Figure 3
Figure 3
The mNeonGreen integrated into either the candidate 1 site or the candidate 2 site is expressed at a similar level irrespective of the insertion direction. (A) Images of the selected transformants and the wild-type strain R265 were examined microscopically under DIC and the GFP filter. Scale Bar, 5 μm. (B) Quantification of the fluorescence intensity in the indicated isolates by measuring the fluorescence as detailed in the method using Zeiss ZEN 3.0 software. Statistical significance was determined using a one-way ANOVA statistical analysis. * p < 0.05.
Figure 4
Figure 4
Insertion of the foreign DNA construct in the candidate 1 site or the candidate 2 site has no significant impact on growth of C. gattii or its response to various stresses. Cells of the indicated strains were tested for thermotolerance using a spotting assay with serial dilutions on YPD medium. Cells were incubated at 22 °C, 30 °C, and 37 °C for 2 days. The same serial dilutions of these strains were also tested for melaninization on L-Dopa medium and capsule formation on RPMI medium with 10% CO2 (see Figure S3 for Indian ink staining for capsule). Cells were cultured on YPD with Congo Red (0.05%), NaCl (1.5 mM), or KCl (1.5 mM) to test their tolerance of cell wall and osmotic stresses.
Figure 5
Figure 5
DNA insertion in the candidate 1 or the candidate 2 site has no apparent impact on sexual reproduction and phagocytosis of C. gattii. (A) The indicated strains were diluted to equal concentrations and cultured on V8 agar medium with the mating partner JEC20a at the same cell density at 22 °C in the dark for 14 days. Images of the edges of the mating colonies (scale bar: 100 μm) and the fruiting bodies (scale bar: 10 μm) were taken. (B) The indicated strains were opsonized with mouse serum for 30 min before incubation with murine J774A.1 macrophage cells for two hours. Non-adherent C. gattii cells were washed with DPBS. Phagocytosed and adherent C. gattii cells were then released from lysed macrophages and plated on YNB medium for CFU counting. The percentage of phagocytosis was calculated by the number of recovered CFUs versus the starting number of cryptococcus cells. The phagocytosis of R265 was set to 1 for normalization. The experiments were performed in three independent biological replicates. Statistical significance was determined using a 2-tailed t test. ns, not significant.
Figure 6
Figure 6
Comparison of intergenic regions in six other C. gattii strains that represent all four molecular types. The diagrams show the organization of the CgSH1 (A) and CgSH2 (B) equivalent intergenic regions and neighboring genes in the six indicated C. gattii strains that represent four molecular types.
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