Sirtulin–Ypk1 regulation axis governs the TOR signaling pathway and fungal pathogenicity in Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is a life-threatening fungal pathogen that is a causative agent for pulmonary infection and meningoencephalitis in both immunocompetent and immunodeficient individuals. Recent studies have elucidated the important function of the target of rapamycin (TOR) signaling pathway in the modulation of C. neoformans virulence factor production and pathogenicity in animal infection models. Herein, we discovered that Ypk1, a critical component of the TOR signaling pathway, acts as a critical modulator in fungal pathogenicity through post-translational modifications (PTMs). Mass spectrometry analysis revealed that Ypk1 is subject to protein acetylation at lysines 315 and 502, and both sites are located within kinase functional domains. Inhibition of the C. neoformans TOR pathway by rapamycin activates the deacetylation process for Ypk1. The YPK1Q strain, a hyper-acetylation of Ypk1, exhibited increased sensitivity to rapamycin, decreased capsule formation ability, reduced starvation tolerance, and diminished fungal pathogenicity, indicating that deacetylation of Ypk1 is crucial for responding to stress. Deacetylase inhibition assays have shown that sirtuin family proteins are critical to the Ypk1 deacetylation mechanism. After screening deacetylase mutants, we found that Dac1 and Dac7 directly interact with Ypk1 to facilitate the deacetylation modification process via a protein–protein interaction. These findings provide new insights into the molecular basis for regulating the TORC–Ypk1 axis and demonstrate an important function of protein acetylation in modulating fungal pathogenicity. IMPORTANCE Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin–Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.

suggestions that will further improve the quality of the manuscript and strengthen the manuscript.1.The authors show the difference in capsule in the Ypk1 mutants but do not include any other phenotypic characterization of the mutants.It will be important to have all other necessary phenotypes in the paper, mainly the ones that are directly relevant for TOR1 signaling such as nutrient limitation conditions.Furthermore, the images for capsule formation defects should be presented in the manuscript.2. The authors mention additional quantifiable tests were done in liquid cell cultures but no further details are provided.These methods need to be described properly in the methods section.3.In their growth experiments, the YPK1R mutant always exhibits more robust growth than the YPK1WT strains in both their spotting and liquid cultures experiments.What are the possible reasons and implications for this phenotype?These should be discussed in the revised manuscript.4. The authors generated double mutants for dac1, 7, and 9 combinations.However, they do not discuss why only these three mutants were focused on for the generation of double mutants.Additionally, what would be the outcome in the triple mutant for dac1, dac7 and dac9? 5. How do the double mutants for dac1 and dac7 behave in a capsule formation assay? 6.The presentation of their statistical analysis is rather confusing.In their figure legends, the authors mention *-based annotations whereas they write actual p-values in their figures.I would suggest they resort to a single consensus approach and describe their results.
Reviewer #2 (Comments for the Author): Chai Z, et al. investigate the impact of acetylation of two lysin residues from the Ypk1 protein on growth and pathogenicity of C. neoformans.Ypk1 has been previously shown to be involved in pathogenicity of C. neoformans and its role in TOR signaling has been predicted based on other fungal models.The same group has shown previously that Ypk1 is acetylated on two lysins, K315, and K502.Here the Authors demonstrate that the mutant, in which the two K residues of Ypk1 are changed to Q to mimic acetylation, has attenuated virulence in the murine model of cryptococcosis.The same mutant has a mild growth defect in the presence of TOR inhibitor rapamycin, pointing to relevance of this modification to TOR pathway.Furthermore, rapamycin diminishes acetylation of Ypk1.The Authors provide evidence that two sirtuins, Dac1 and Dac7, redundantly act as deacetylases towards Ypk1 and interact with Ypk1.While some of the effects are rather mild, this study provides cohesive evidence that Dac1 and Dac7 act as deacetylases towards Ypk1 and this deacetylation contributes to virulence of C. neoformans.Below are specific comments that should help to further improve this manuscript.

Response to reviewer comments:
General response: We thank the reviewers and the editor for taking the time to review our manuscript.We sincerely appreciate your thoughtful feedback and constructive suggestions, which have undoubtedly improved the quality of our work.Based on your suggestion and request, we have made corrected modifications on the revised manuscript.And more detailed methods and steps have been modified We hope that our work can be improved again.Furthermore, we would like to show the details as follows: Reviewer #1 (Comments for the Author): In this paper, Chai et al explore the impact of acetylation on Ypk1, a key component of the TOR signaling pathway.Authors build on their previous finding that Ypk1 is modified at two places and these acetylation modifications are crucial for its function.Overall, the experiments are well conducted and the conclusions are well supported by their experiments.I have a few suggestions that will further improve the quality of the manuscript and strengthen the manuscript.Response:Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits.
1.The authors show the difference in capsule in the Ypk1 mutants but do not include any other phenotypic characterization of the mutants.It will be important to have all other necessary phenotypes in the paper, mainly the ones that are directly relevant for TOR1 signaling such as nutrient limitation conditions.Furthermore, the images for capsule formation defects should be presented in the manuscript.Response: We very much appreciate the comment from this expert.This is a very good suggestion, indeed it should be tested in the research.TOR1 signaling dynamically responds to nutrient limitation conditions to promote cell survival and maintain cellular homeostasis.During amino acid starvation, TOR operates independently as a positive regulator of autophagy through the conserved TORC2 and its downstream target kinase Ypk1(1).In the revised manuscript, we analyzed the starvation tolerance of these mutants under nitrogen starvation (SD-N) and carbon starvation (SD-C) (2).In the 7-day nitrogen starvation treatment, the ypk1Δ mutant exhibited pronounced growth defects, as expected.Meanwhile, YPK1Q exhibited growth defects almost identical to ypk1Δ.The same phenomenon was also observed during the 14-day carbon starvation treatment (Figure 3F).This phenomenon suggests that the acetylation modification level of Ypk1 plays a crucial regulatory role in responding to starvation tolerance conditions, and it impacts the process of autophagy.And it plays a more significant role in tolerance to nitrogen-only starvation than in to carbon-only starvation.Please see line 185-195 of the "Marked-Up Manuscript" document.Additionally, the images for capsule formation are presented below and are supplemented in Supplemental Figure 1A.Thanks for the reviewer for pointing this out.
2. The authors mention additional quantifiable tests were done in liquid cell cultures but no further details are provided.These methods need to be described properly in the methods section.Response: We appreciate the comment and apologize for not clarifying the methods.The detailed steps have been supplemented in the Materials and Methods section.In summary, We utilizing 96-well microtitre plates to assess the growth of cells in liquid culture mediu.Overnight YPD cultures were washed three times with phosphate-buffered saline (PBS) and diluted to an optical density of 0.02 at 600 nm in fresh YPD, supplemented with 2 ng/ml rapamycin.Following this, 100 µl of the resulting cell suspension was carefully dispensed into individual wells of a 96-well plate.The well plate was subjected to incubation at a temperature of 30°C for either 12, 24 or 48 hr.Subsequently, optical density measurements at a wavelength of 600 nm were obtained using a Synergy HTX microplate reader manufactured by BioTek.The growth of the relevant strain was standardized by normalizing it to the well without rapamycin treatment.Six or twelve biological replicates were conducted for each strain.The data were graphed utilizing GraphPad Prism software.Two tails unpaired t-tests were used.Please see line 391-403 of the "Marked-Up Manuscript" document.
3. In their growth experiments, the YPK1R mutant always exhibits more robust growth than the YPK1WT strains in both their spotting and liquid cultures experiments.What are the possible reasons and implications for this phenotype?These should be discussed in the revised manuscript.
Response: The acetylation level of proteins is a dynamic process during cell growth, regulated by both acetyltransferases and deacetylases.In our data shows that the acetylation modification level of the Ypk1 protein rapidly decreases under stimulation with DNEM+10% FBS or rapamycin (Figure 2B, Figure 3E).Furthermore, the YPK1WT strain exhibits a very low levels of acetylation modification, indicating that Ypk1 protein exists in a non-acetylated state during the growth of Cryptococcus neoformans.Ypk1 will remain in a non-acetylated state when we artificially mutate acetylation sites K ac315 and K ac502 to arginine.It may be more conducive to its function within YPK1R strain.Studies have extensively shown that the non-acetylation state of proteins contributes to the activation of protein functions.For example, Cryptococcus neoformans ISW1, an chromatin remodeling factor, when mutated in its ISW1R form, activates the protein ubiquitination pathway, contributing to the development of antifungal drug resistance(3).The K642R mutation in the mammalian mitochondrial fission protein dynamin-related protein 1 (Drp1) blocked palmitate-induced Drp1 phosphorylation, oligomerization, and activity in adult cardiomyocytes.This ultimately reduced cardiomyocyte death and heart dysfunction(4).In Saccharomyces cerevisiae, Ypk1 protein post-translational modification (PTM), phosphorylation, acts as a critical modulator for its protein activity and function (5).Perhaps the phosphorylation activity of Ypk1 is higher in its non-acetylated state.To further elucidate the interplay between these two modifications, a detailed analysis of Ypk1 phosphorylation and the associated target proteins must be performed using mass spectrometry.And these hypotheses require more in-depth research to be confirmed in the future.Please see Discussion section, line 277-283 of the "Marked-Up Manuscript" document.
4. The authors generated double mutants for dac1, 7, and 9 combinations.However, they do not discuss why only these three mutants were focused on for the generation of double mutants.
Additionally, what would be the outcome in the triple mutant for dac1, dac7 and dac9?Response: We appreciate the comment and apologize for not clarifying the rationale of dac1Δ, dac7Δ, and dac9Δ.Li et al. have confirmed that there are two main deacetylase families present in Cryptococcus neoformans: the HDAC (Histone Deacetylase) family and sirtuin family (6).The HDAC family includes Dac2-6, 8, and 11, while the sirtuin family includes Dac1, 7, and 9.In this study, we found that the sirtuin deacetylase family regulates the acetylation of Ypk1 (data from Figure 4 and Figure 5).Moreover, the acetylation modification level of Ypk1 could only be increased when both Dac1 and Dac7 were deleted.However, whether it was a single knockout of Dac9 or a double knockout in combination with Dac1 or Dac7, the acetylation modification level of Ypk1 did not change.It is ruled out that Dac9 is a deacetylase of Ypk1.Therefore, in the subsequent experiments, we focus on the interaction between Dac1, Dac7, and Ypk1, rather than on the triple mutant for Dac1, Dac7 and Dac9.In the revised manuscript, we have provided a more comprehensive explanation for this section.Please see line 199-201 of the "Marked-Up Manuscript" document.

How do the double mutants for dac1Δ and dac7Δ behave in a capsule formation assay?
Response: According to your suggestion, we tested the capsule structure formation ability of dac1Δ and dac7Δ and dac1Δdac7Δ strains.The results showed a significant reduction in the thickness of the capsule structure in all three mutant strains, especially in the dac1Δdac7Δ double knockout strain.It is consistent with the phenotype of the YPK1Q strain's capsule structure shown in Figure 2A.However, the dac1Δdac7Δ double knockout exhibited a more severe defect, indicating the importance of downstream genes regulated by Dac1 and Dac7, which warrants further investigation in the future (Figure .5C).The images for capsule formation are presented below and are supplemented in Supplemental Figure 1B.Meanwhile, we supplemented the experiments on the nutrient starvation tolerance and rapamycin resistance of single knockout strain dac1Δ and dac7Δ, as well as the dac1Δdac7Δ double knockout strain.From the results, it can be seen that under YPD conditions, the growth of dac1Δ and dac7Δ and dac1Δdac7Δ strains was consistent with the wild-type H99 strain.However, after 7-days nitrogen starvation treatment and 14-days carbon starvation treatment, the dac7Δ single knockout strain did not show any growth defects, while the dac1Δ exhibited slight growth defects, and the dac1Δdac7Δ double knockout strain showed significant growth defects (Figure .5D).In the rapamycin resistance experiment, only the dac1Δdac7Δ double knockout strain showed slight growth defects (Supplemental Figure 1C).This phenomenon confirms the functional redundancy of Dac1 and Dac7, indicating that both collectively regulate the TOR pathway and nutrient intake.The deacetylase also positively regulates the process of autophagy, and its absence affects the growth of strains under nutritional stress conditions.Please see line 217-228 of the "Marked-Up Manuscript" document.
6.The presentation of their statistical analysis is rather confusing.In their figure legends, the authors mention *-based annotations whereas they write actual p-values in their figures.I would suggest they resort to a single consensus approach and describe their results.Response:We thank for the comment, and apologize for the confusion in the original manuscript.In the revised manuscript, we use actual p-values consistently for representation.Detailed statistical methods and descriptions will be provided in the Materials and Methods section.Please see line 438-441 of the "Marked-Up Manuscript" document.
Reviewer #2 (Comments for the Author): Chai Z, et al. investigate the impact of acetylation of two lysin residues from the Ypk1 protein on growth and pathogenicity of C. neoformans.Ypk1 has been previously shown to be involved in pathogenicity of C. neoformans and its role in TOR signaling has been predicted based on other fungal models.The same group has shown previously that Ypk1 is acetylated on two lysins, K315, and K502.Here the Authors demonstrate that the mutant, in which the two K residues of Ypk1 are changed to Q to mimic acetylation, has attenuated virulence in the murine model of cryptococcosis.The same mutant has a mild growth defect in the presence of TOR inhibitor rapamycin, pointing to relevance of this modification to TOR pathway.Furthermore, rapamycin diminishes acetylation of Ypk1.The Authors provide evidence that two sirtuins, Dac1 and Dac7, redundantly act as deacetylases towards Ypk1 and interact with Ypk1.While some of the effects are rather mild, this study provides cohesive evidence that Dac1 and Dac7 act as deacetylases towards Ypk1 and this deacetylation contributes to virulence of C. neoformans.Below are specific comments that should help to further improve this manuscript.

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Thank you for submitting your paper to Spectrum.Sincerely, Alexandre Alanio Editor Microbiology Spectrum -24R1 (Sirtulin-Ypk1 regulation axis governs the TOR signaling pathway and fungal pathogenicity in Cryptococcus neoformans.)Dear Dr. Zhijie Zhang: