Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in <i>Cryptococcus neoformans</i>
Bibliographic record
Abstract
ABSTRACT The opportunistic human fungal pathogen Cryptococcus neoformans has an enormous impact on human health as the causative agent of cryptococcal meningitis, and there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in C. neoformans is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter pCTR4 is the most widely used system to regulate essential gene expression; however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including Saccharomyces cerevisiae , Candida albicans , and Candida auris , the tetracycline-repressible promoter system is a powerful tool to regulate gene expression; however, it has yet to be adapted for C. neoformans . In this study, we successfully implemented the tetracycline-repressible system in C. neoformans to regulate the expression of the essential genes HSP90 and FKS1 . Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted HSP90 at both transcript and protein levels and inhibited C. neoformans growth and viability. Similarly, the depletion of FKS1 with doxycycline enhanced sensitivity of the strain to the echinocandin caspofungin, an antifungal that targets the glucan synthase but is generally ineffective against C. neoformans . Thus, this work unveils a novel approach to generate conditional-expression mutants in C. neoformans, providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen. IMPORTANCE Invasive fungal infections cause millions of deaths annually, while the number of antifungals available to combat these pathogens is limited to only three classes: polyenes, azoles, and echinocandins. The largest source of novel antifungal drug targets are essential gene products, which are required for cellular viability. However, tools to identify and characterize essential genes in C. neoformans are extremely limited. Here, we adapted the tetracycline-repressible promoter system, that has been widely used in other organisms, to study essential gene function in C. neoformans . By placing this regulatable promoter upstream of the essential genes HSP90 and FKS1 , we confirmed that the growth of the strains in the presence of the tetracycline analog doxycycline results in the depletion of essential gene expression. This approach provides a significant advance for the systematic study of essential genes in C. neoformans .
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".