Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 24;120(4):e2209831120.
doi: 10.1073/pnas.2209831120. Epub 2023 Jan 20.

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans

Asiya Gusa  1 , Vikas Yadav  1 , Cullen Roth  2 , Jonathan D Williams  1 , Eva Mei Shouse  1 , Paul Magwene  2 , Joseph Heitman  1 , Sue Jinks-Robertson  1
Affiliations

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans

Asiya Gusa et al. Proc Natl Acad Sci U S A. .

Abstract

We recently reported transposon mutagenesis as a significant driver of spontaneous mutations in the human fungal pathogen Cryptococcus deneoformans during murine infection. Mutations caused by transposable element (TE) insertion into reporter genes were dramatically elevated at high temperatures (37° vs. 30°) in vitro, suggesting that heat stress stimulates TE mobility in the Cryptococcus genome. To explore the genome-wide impact of TE mobilization, we generated transposon accumulation lines by in vitro passage of C. deneoformans strain XL280α for multiple generations at both 30° and at the host-relevant temperature of 37°. Utilizing whole-genome sequencing, we identified native TE copies and mapped multiple de novo TE insertions in these lines. Movements of the T1 DNA transposon occurred at both temperatures with a strong bias for insertion between gene-coding regions. By contrast, the Tcn12 retrotransposon integrated primarily within genes and movement occurred exclusively at 37°. In addition, we observed a dramatic amplification in copy number of the Cnl1 (Cryptococcus neoformans LINE-1) retrotransposon in subtelomeric regions under heat-stress conditions. Comparing TE mutations to other sequence variations detected in passaged lines, the increase in genomic changes at elevated temperatures was primarily due to mobilization of the retroelements Tcn12 and Cnl1. Finally, we found multiple TE movements (T1, Tcn12, and Cnl1) in the genomes of single C. deneoformans isolates recovered from infected mice, providing evidence that mobile elements are likely to facilitate microevolution and rapid adaptation during infection.

Keywords: Cryptococcus; fungal pathogen; heat stress; temperature; transposons.

PubMed Disclaimer

Conflict of interest statement

The authors have additional information to disclose, Author J.H. and reviewer E.H.S. are co-authors on a 2020 review together (DOI: 10.1128/mBio.00449-20). They have not collaborated directly.

Figures

Fig. 1.
Fig. 1.
Location and copy number of full-length and truncated T1 and Tcn12 sequences in the C. deneoformans XL280α genome.
Fig. 2.
Fig. 2.
Mapping of TE changes in XL280α TA lines passaged ~800 generations. (A) Southern analysis of BspEI-digested genomic DNA probed for T1 (SI Appendix, Fig. S2A) in TA lines passaged at 30° and (B) passaged at 37°. Arrowheads indicate de novo T1 insertions, and asterisks indicate the loss of T1 copies relative to the XL280α progenitor genome. (C) T1 mapping using Illumina read-pair analysis. Read depths for T1 in the passaged lines were normalized against the XL280α progenitor genome (Top) to indicate insertion of new T1 copies (peaks) or the loss of T1 copies (troughs) in representative TA lines 37-01, 37-02, and 37-03. The variation in height of each peak or trough corresponds to the number of supporting reads from the read-pair analysis.
Fig. 3.
Fig. 3.
T1 and Tcn12 movement in TA lines passaged at 30° and 37° compared to the XL280α progenitor strain. (A and B): Composite distribution of de novo T1 insertions (n = 46) and Tcn12 insertions (n = 19) mapped on chromosomes 1 to 14 by whole-genome sequencing in eight lines passaged at 30° and 12 lines passaged at 37°. (C) Total TE changes detected in TA lines for 20 genomes passaged at 30° (Left) and 22 genomes passaged at 37° (Right), evaluated using Southern analysis or whole-genome sequencing; sequenced genomes are indicated by asterisks.
Fig. 4.
Fig. 4.
Cnl1 copy number changes in passaged lines. (A) Full-length and truncated copies of Cnl1 in the progenitor XL280α genome compared to TA line 37-02. Cnl1 copies present on the subtelomeric regions of each chromosomal arm are represented by block arrows whose size approximates the length relative to the 3.4 kb full-length element. Full-length and truncated copies are indicated in black and gray, respectively. (B) Fold change in Cnl1 copy number for all TA lines passaged at 30° (n = 20) and 37° (n = 22) relative to the XL280α strain as determined by qPCR of the 5′-end of Cnl1 from genomic DNA. The middle dotted line of each violin plot represents the median fold change value while the top and bottom dotted lines represent the upper and lower quartile values, respectively. The fold change in copy number was calculated using the comparative ΔΔCT method with GPD1 as the internal control gene. The Mann–Whitney U test was applied to compare the difference for each set of mean ΔCT values from three biological replicates (*** indicates P < 0.0001). (C) The estimated Cnl1 copy number for each TA line is plotted, determined by multiplying the fold change value by nine copies, which equals the number of full-length Cnl1 copies in the XL280α genome.
Fig. 5.
Fig. 5.
Southern analysis of TE fragments in 5FOA-resistant XL280α mutants recovered from the lungs, kidneys, and brain of two mice 10 d post-infection. Each mutant had an independent Tcn12 mutation in the URA5 gene. Genomic DNA was digested with PvuII and probed for (A) Tcn12, (B) T1, or (C) Cnl1. The XL280α inoculum strain is shown for comparison. Arrows indicate new TE fragments and asterisks indicate TE fragment loss.
See this image and copyright information in PMC

References

    1. Hagen F., et al. , Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal. Genet. Biol. 78, 16–48 (2015). - PubMed
    1. Lin X., Heitman J., The biology of the Cryptococcus neoformans species complex. Annu. Rev. Microbiol. 60, 69–105 (2006). - PubMed
    1. Casadevall A., Kwon-Chung K. J., Perfect J. R., Kozel T. R., Heitman J., Cryptococcus: From Human Pathogen to Model Yeast (ASM Press, 2011).
    1. Rajasingham R., et al. , The global burden of HIV-associated cryptococcal infection in adults in 2020: A modelling analysis. Lancet Infect. Dis. 22, 1748–1755 (2022). - PMC - PubMed
    1. Kwon-Chung K. J., et al. , Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harb. Perspect. Med. 4, a019760 (2014). - PMC - PubMed

Publication types