SATAY-based chemogenomic screening uncovers antifungal resistance mechanisms and key determinants of ATI-2307 and chitosan sensitivity

Reviewer #1 (Public review):

Summary:

In this study, the authors employed Saturated Transposon Analysis in Yeast (SATAY) in the model yeast Saccharomyces cerevisiae to uncover mutations conferring resistance to 20 different antifungal compounds. These screens revealed novel resistance mechanisms and the modes of action for the antifungal compounds Chitosan and HTI-2307. The authors discovered that Chitosan electrostatically interacts with cell wall mannosylphosphate and identified Hol1 as the transporter of HTI-2307.

Strengths:

The study highlights the power of SATAY in uncovering drug-resistance mechanisms, modes of action, and cellular processes influencing fungal responses to drugs. Identifying novel resistance mechanisms and modes of action for various compounds in this model yeast provides valuable insights for further investigating these compounds in fungal pathogens and developing antifungal strategies. This study thus represents a significant resource for exploring cellular responses to chemical stresses.

The manuscript is well-written and highly clear.

Weaknesses:

As the study was conducted using highly modified non-pathogenic laboratory yeast strains, verification of the findings in fungal pathogens would greatly enhance its relevance and applicability.

Reviewer #2 (Public review):

The study begins by exposing wild-type yeast libraries to some well-understood antifungals (amphotericin B, caspofungin, myriocin) to illustrate the complexity and power of the analytical method. These toxins are positively selected for loss-of-function transposon (CDS) insertions in many of the genes identified previously in earlier studies. The outlier genes were visually evident in scatter plots (Figure 1A, 1B, 1C) but the magnitude and statistical significance of the effects were not presented in tables. There were some unexplained and unexpected findings as well. For example, caspofungin targets the product of the GSC2 gene, and yet transposon insertions in this gene were positively selected rather than negatively selected (seemingly discordant from other studies).

Interestingly, transposon insertions immediately upstream of toxin targets (Figure 1D) and toxin efflux transporters or their regulators (Figure 1E) were visibly selected by exposure to the toxins, suggesting gain-of-expression. Most of these findings are convincing, even without statistical tests. However, some were not (for example, Soraphen A on YOR1). A relevant question emerges here: Do both ends of the transposon confer the same degree of cryptic enhancer/promoter activity? If one end contains strong activity on downstream gene expression while the other does not, the effects of one may be obscured by the other. The directionality of transposon insertions (not provided) would then be important to consider when interpreting the raw data.

A masterful rationalization of transposon insertion selection in the YAP1 and FLR1 genes was presented wherein loss of C-terminal auto-inhibitory domain of the Yap1 transcription factor resulted in FLR1 overexpression and resistance to Cerulenin. Transposon insertions in the CDS of YAP1 and FLR1 were negatively selected in Chlorothalonil while the gain-of-function and -expression insertions (enriched in Cerulenin) were not. The rationalization of these findings - that Chlorothalonil activates Yap1 while Cerulenin does not - was much less convincing and should be tested directly with a simple experiment such as Q-PCR.

Moving to specially engineered yeast strains (Figure 2) where multiple efflux transporters were eliminated (for Prochloraz testing) or new drug targets were inserted (for Fludioxonil and Iprodione), numerous interesting observations were obtained. For instance, transposon insertions in totally different sets of genes were enriched by prochloraz depending on the strain background. Conversely, almost the exact same genes were selected in Fludioxonil and Iprodione, including genes in the well-known HOG pathway. Because several candidate receptors of these compounds were not significant in the Tn-seq dataset, the authors add new evidence to the field suggesting that the introduced gene (BdDRK1) represents the direct, or near-direct, target of these compounds.

Chitosan effectiveness was studied by Tn-seq in yet another specialized strain of yeast that is uniquely susceptible to the toxin. Once again, the authors masterfully rationalize the complex effects, leading to a simple model where chitosan interacts with mannosyl-phosphate in the cell wall and membrane, which is deposited by Mnn4 and Mnn6 and masked by Mnn1 enzymes in the Golgi complex (themselves regulated or dependent on a number of additional gene products such as YND1. This research compellingly adds to our understanding of an industrial antifungal.

Finally, the effects of a preclinical antifungal ATI-2307 were studied for the first time. Remarkably, ATI-2307 efficacy greatly depended on HOL1 coding sequences and an upstream enhancer (Figure 4). After engineering hol1∆ strains, uptake of the compound and sensitivity to the compound were lost and then restored by heterologous expression of CaHOL1 from a pathogenic yeast. HOL1 also conferred susceptibility to polyamines with related structures (Pentamidine, Iminoctadine). Remarkably, separation-of-function mutations were obtained in HOL1 that abolished the uptake of the toxins while preserving the uptake of nutrient polyamines in low nitrogen conditions, which strongly suggests that HOL1 encodes a direct transporter of the toxins. The implications are important for ATI-2307 efficacy in patients, where resistance mutations could arise spontaneously and produce poor clinical outcomes.

Additional comments:

The experiments presented here are often convincing and serve to illustrate the power of Tn-seq approaches in elucidating drug resistance mechanisms in eukaryotic microbes. The gain-of-expression effects (upstream of CDS), gain-of-function effects (elimination of auto-inhibitory domains), and loss-of-function effects were all carefully exposed and discussed, leading to numerous new insights on the action of diverse toxins.

On the other hand, several deficiencies and weaknesses (in addition to the minor ones described above) limit the utility of the data that has been generated.

(1) There was no summary table of Tn-seq data for different genes in the different conditions, so readers could not easily access data for genes and pathways not mentioned in the text. This is especially important because transposon insertions that were negatively selected (of great interest to the community) were barely mentioned. Additionally, the statistical significance of outlier genes was not reported. The same is true for insertions within the DNA segments upstream of CDSs. Users of these data are therefore restricted to visually inspecting insertion sites on a genome browser.

(2) Only one dose of each toxin was studied, which therefore produces a limited perspective on the genetic mechanisms of resistance in each case.

(3) No Tn-seq experiments were performed in diploid yeast strains. The gain-of-expression and gain-of-function insertions under positive selection in haploid strains in the different conditions are expected to be dominant in diploid strains as well, while loss-of-function insertions in CDS are expected to be recessive. Do these expectations hold? Could such experiments potentially confirm the models for Cerulenin and Chlorothalonil effects on YAP1 and FLR1? Pathogenic Candida species are usually diploid where gain-of-function/expression mutants most frequently lead to poor clinical outcomes. Resistance to ATI-2307 through loss of HOL1 may not be as significant for diploid C. albicans with two functional copies of all genes. On a related note, is it possible that transposon insertions in the 3' untranslated region produce anti-sense transcripts that lowers the expression of the upstream gene from both alleles in diploids, thereby producing a strong selective advantage in ATI-2307? This study already touches on exciting new applications of the Tn-seq method but could easily go a bit further.

Reviewer #3 (Public review):

Summary:

This manuscript describes an extensive application of the Yeast (SATAY) transposon mutagenesis and sequencing method to explore loss- and gain-of-function mutations conferring resistance to 20 different antifungal compounds. Impressively, the authors demonstrate that SATAY can be used to identify mutations that lead to antifungal resistance, including promoter mutations that include the direct targets of antifungal compounds and drug efflux pumps. Because SATAY is not tied to a specific genetic background, the sensitivity of an S. cerevisiae strain, AD1-8, that specifically displays Chitosan susceptibility was examined in detail, and the results suggest that Chitosan acts through interactions with the fungal cell wall. Through a series of experiments that expand upon SATAY analysis, the novel antifungal ATI-2307, the authors clearly show that the transporter Hol1 concentrates this compound within yeast.

General Comments:

This is a very impressive application of SATAY, highlighting many different strategies for exploring the mechanism of action of various antifungal compounds. It's clear from the findings presented that SATAY is a powerful and potentially highly productive approach for chemical-genetic analysis.