The RAM signaling pathway links morphology, thermotolerance, and CO2 tolerance in the global fungal pathogen Cryptococcus neoformans

  1. Benjamin J Chadwick
  2. Tuyetnhu Pham
  3. Xiaofeng Xie
  4. Laura C Ristow
  5. Damian J Krysan
  6. Xiaorong Lin

  • Curated by eLife

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    This study provides compelling evidence for the involvement of RAM pathway in the survival of C. neoformans in high CO2 concentrations. The work is important to understand how this fungus adapts to the high CO2 concentrations in host tissues. The experimental approach combines genetic and biochemical approaches to explore a complex topic that is of essential for cryptococcal pathogenesis.

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Abstract

The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO 2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO 2 -tolerant clinical strain, we found mutations leading to CO 2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and R egulator of A ce2 and M orphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO 2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans , transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1 Δ in C. neoformans that partially rescued defects in CO 2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1 , which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1 Δ partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO 2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.

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  1. Version published to 10.7554/elife.82563 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The research investigates the genetic basis for resistance to high CO2 levels in the human pathogenic fungus Cryptococcus neoformans. Screening collections of over 5,000 gene deletion strains revealed 96 with impaired growth, including a set of genes all related to the same RAM signaling pathway. Further genetic dissection was able compellingly to place where this pathway lies relative to upstream inputs and through the isolation of suppressor mutants as potential downstream targets of the pathway. Given the high levels of CO2 encountered by fungi in the human host, this work may provide new directions for the control of disseminated fungal disease.

    The research presents both strengths and weaknesses.

    Strengths include:

    (1) One of the largest scale analyses of genes involved in growth under high CO2 concentrations in a fungus, revealing a set of just under 100 mutants with impaired growth.

    (2) Elegant genetic epistasis analysis to show where different components fit within a pathway of transmission of CO2 exposure. For example, over expression of one of the kinases, Cbk1, can overcome the CO2-sensitivity of mutations in the CDC24 or CNA1 genes (but not in the reciprocal overexpression direction).

    (3) Isolation of suppressor mutations in the cbk1 background, now able to grow at high CO2 levels, was able to lead to the identification of two genes. Follow up characterization, which included examining in vitro phenotypes, gene expression analysis, and impact during mouse infection was able to reveal that the two suppressors restore a subset of the phenotypes impacted by mutation of CBK1. Indeed, one conclusion from this careful work is that the reduced virulence of the cbk1 mutant is not due to its sensitivity to high levels of CO2, perhaps an unexpected finding given the original goals of the study towards linking CO2 sensitivity with decreased virulence.

    Weaknesses include:

    (1) What is the rationale for examining gene expression using the NanoString technology of 118 genes rather than a more genome-wide approach such as RNA-sequencing?

    (2) Without additional species examined, some of the conclusions about differences in impact between ascomycetes and basidiomycetes might instead reflect differences between species. For example, RAM mutants in other strains of C. neoformans do not exhibit so strong a temperature sensitive phenotype. Or to extend the comparison further, one might assume given the use of CO2 for Drosophila manipulations that the RAM pathway components in an insect would not be required for surviving high CO2.

    (3) Given the relative ease of generate progeny of this species, it would have been informative to explore if the suppressors of cbk1 also suppressed the loss of genes like CDC24, CNA1, etc, equivalent to the experiment performed of overexpression of CBK1 in those backgrounds.

    We thank the reviewer for the kind summary of our work and the highlights of the major findings. We chose NanoString because we have already generated a probe set of 118 genes that are differentially expressed in response to CO2 based on RNA-seq profiles of multiple natural cryptococcal isolates in a separate study. Nanostring allowed us to focus on CO2 relevant transcripts and do multiple replicates and conditions in a way that is not practical using RNA-Seq.

    Although the RAM pathway has not been extensively characterized in different species of Cryptococcus, we do know that RAM pathway mutants lead to pseudohyphal growth in multiple strain backgrounds including two different species of Cryptococcus (Magditch, Liu, Xue, & Idnurm, 2012; Walton, Heitman, & Idnurm, 2006). We have added corresponding references and discussed this point on lines 167-169.

    We agree with the reviewer that it would be interesting to test the effects of the cbk1Δ suppressor mutations in the backgrounds of other CO2-sensitive gene knockout strains. This is part of our plan for future investigation in characterizing the signaling pathways involved in CO2 tolerance.

    Reviewer #2 (Public Review):

    In the paper by Chadwick et al., the authors identify the molecular determinants of CO2 tolerance in the human fungal pathogen Cryptococcus neoformans. The authors have screened a collection of deletion mutants to identify the genes that are sensitive at 37oC (host temperature) and elevated CO2 levels. The authors identified that the genes responsible for CO2 sensitivity are involved in the pathways responsible for thermotolerance mechanisms such as Calcineurin, Ras1-Cdc24, cell wall integrity, and the Regulator of Ace2 and Morphogenesis (RAM) pathways. Moreover, they identified that the mutants of the RAM pathway effector kinase Cbk1 were most sensitive to elevated temperature and CO2 levels. This study uncovers the previously unknown role of the RAM pathway in CO2 tolerance. Transcriptome data indicates that the deletion of CBK1 results in an alteration in the expression of CO2-related genes. To identify the potential downstream targets of Cbk1, the authors performed a suppressor screen and obtained the spontaneous suppressor mutants that rescued the sensitivity of cbk1 mutants to elevated temperature and CO2. Through this screen, the authors identified two suppressor groups that showed a modest improvement in growth at 37˚C and in presence of CO2.

    Interestingly, from the suppressor screen, the authors identified a previously known interactor of Cbk1 which is SSD1, and an uncharacterized gene containing a putative Poly(A)-specific ribonuclease (PARN) domain named PSC1 (Partial Suppressor of cbk1Δ) which acts downstream of Cbk1. Deletion of these two genes in cbk1 null mutants rescued the sensitivity to elevated CO2 levels and temperature but did not fully rescue the ability to cause disease in mice.

    This study highlights the underappreciated role of the host CO2 tolerance and its importance in the ability of a fungal pathogen to survive and cause disease in host conditions. The authors claim to gain insight into the genetic components associated with carbon dioxide tolerance. The experimental results including the data presented, and conclusions drawn do justice to this claim. Overall, it is a well-written manuscript. However, some sections need improvement in terms of clarity and experimental design.

    • One major drawback of the study is the virulence assay performed to test the ability of cbk1 mutants to cause the disease in the mouse model. The cbk1 null mutants are thermosensitive in nature. Using these mutants, establishing the virulence attributes in mice would undermine the mutants' ability to infect mice as they won't be able to survive at the host body temperature.

    • The rationale for choosing the genes to test further is not clear in two instances in the study. a) From a list of 96 genes, how do the authors infer the pathways involved? Was any pathway analysis performed that helped them in shortlisting the pathways that they subsequently tested? A GO term analysis of the list of genes identified through the genetic screen would be more helpful to get an overview of the pathways involved in CO2 tolerance. b) The authors do not clearly mention why they chose only four genes to test for the CO2 sensitivity out of 16 downregulated genes identified from the nano string analysis.

    • It would be more useful to the readers if the authors could also include a thorough analysis of the presence of the putative PARN domain-containing protein across various fungal species rather than mentioning that it is only observed in C. neoformans and S. pombe. Also, the authors may want to discuss the known role(s) of SSD1, if any, in pathogenic ascomycetous yeasts so that the proposed functional divergence is supported further.

    We are glad that the reviewer appreciated the approach, the findings, and the significance of this research, and we are grateful for the helpful suggestions to improve the manuscript.

    To remove temperature sensitivity as a variable when testing virulence, we have added a new infection model in the revised manuscript to test the cbk1Δ mutant and its suppressors. This infection model uses the Galleria mellonella larvae as a host. G. mellonella larvae are commonly used to test virulence for temperature sensitive strains as the body temperature of the larvae is similar to that of the environment. We performed cryptococcal infection in this model and the larvae were kept at 30°C rather than at 37°C. The results of these experiments are now described in results section 5 and shown in Figure 6 of the manuscript. The data using the larva-infection model supports our original conclusion about the virulence of these strains observed in mouse models.

    We performed a GO term analysis of the hits from our screening, but did not find any significant or outstanding pathways. From our list of 96 genes, we chose to focus on the RAM pathway because the mutants were among the most sensitive to CO2. We have added an explanation for the genes we decided to test for host CO2 level sensitivity from the 16 downregulated genes on lines 139-141.

    Through Blast searching, we have found that the PARN domain-containing protein has homologs in other basidiomycetes. There might be some homologs in a few zygomycetes and ascomycetes but the confidence scores were so low that we deemed unlikely. We now report this in the manuscript on lines 210-213, “This domain was previously reported to be found in S. pombe (Marasovic, Zocco, & Halic, 2013). Interestingly, through a Blast search of the PARN domain, we did not identify this domain in the genomes of S. cerevisiae, C. albicans or other ascomycetes, but found it in Basidiomycetes and higher eukaryotes”.

    Ssd1 has been studied in the pathogenic yeast Candida albicans and is also regulated by Cbk1 in this organism. We have added a discussion about possible functions of Ssd1 in C. neoformans based on references to studies in C. albicans in the discussion section on lines 401-408. “In C. albicans, Ssd1 plays an important role in polarized growth and hyphal initiation by negatively regulating the transcription factor Nrg1 (H. J. Lee, Kim, Kang, Yang, & Kim, 2015). The observation that cbk1Δpsc1Δ and cbk1Δssd1Δ suppressor mutants partially rescue cell separation defects or depolarized growth suggests that C. neoformans may primarily utilize Ssd1/Psc1 rather than a potential Ace2 homolog to regulate cell separation or polarization. Differential regulation of target mRNA transcripts by Ssd1 and Psc1 may explain the functional divergence of the RAM pathway we observed here between basidiomycete Cryptococcus and the ascomycete yeasts.”

    Reviewer #3 (Public Review):

    In this work the authors identify genes and pathways important for CO2 and thermotolerance in Cryptococcus neoformans. They additionally rule out the contribution of the bicarbonate or cAMPdependent activation of adenylyl cyclase to this pathway, which is important for CO2 sensing in other fungi, further solidifying the need to characterize CO2 sensing in basidiomycetes. The authors establish the importance of focusing on CO2 tolerance by testing the impact of CO2 on fluconazole susceptibility with varied pH, suggesting the ability of CO2 to sensitize cryptococcal cells to fluconazole. Furthermore, the authors compared the CO2 tolerance of clinical reference strains to environmental isolates. The characterization of the RAM pathway Cbk1 kinase illustrated the integration of multiple stress signaling pathways. By using a series of CBK1OE insertions in strains with deletions in other pathways, the ability of Cbk1 over-expression to rescue several strains from CO2 sensitivity was apparent. Additionally, NanoString expression analysis comparing cbk1∆ to H99 validated the author's screen of CO2-sensitive mutants as 16/57 downregulated genes were found in their screen, further confirming the interconnected nature of these pathways. The importance of the RAM pathway in maintaining CO2 and thermotolerance was also incredibly clear.

    Perhaps most interestingly, the authors identify suppressor colonies with distinctive phenotypes that allowed for the characterization of downstream effectors of the RAM pathway. These suppressor colonies were found to have mutations in SSD1 and PSC1 which somewhat restore growth at 37oC with CO2 exposure. Further confirming the importance of the RAM pathway, the cbk1∆ strain had markedly attenuated virulence during infection. Interestingly, the generated suppressor strains had varying impacts on fungal infection in vivo. While the sup1 suppressor was completely cleared from the lungs during both intranasal and IV infection, the sup2 strain, containing mutations in SSD1, maintained a high fungal load in the lungs and was able to disseminate into host tissues during IV infection but not intranasal infection.

    The authors make a strong case for the exploration of thermotolerance and CO2 tolerance as contributors to virulence. Through screening and characterization of RAM pathway kinase CBK1's ability to rescue other mutants from CO2 sensitivity, the overlapping contributions of several signaling pathways and the importance of this kinase were revealed. This work is important and will be valuable to the field. However, the cbk1∆ strain does show reduced melanization, urease secretion, and higher sensitivity to cell wall stressor Congo Red in SI Appendix, Figure S4. While the authors make a strong argument that these well-established virulence factors are not perfect predictors of virulence in vivo, the cbk1∆ strain is not an example of such a case as it does have defects in these important factors in addition to thermotolerance and CO2 tolerance. Not acknowledging the changes in these virulence factors in the cbk1∆ and their potential contribution to phenotypes observed is a weakness of the manuscript. Interestingly, the sup1 and sup2 strains also rescue these virulence factors compared to cbk1∆. Additionally, the assertion that "the observation that only sup2 can survive, amplify, and persist in animals stresses the importance of CO2 tolerance in cryptococcal pathogens" due to the sup2's slightly higher CO2 tolerance compared to sup1, could be better supported by the data. These suppressors did not restore transcript abundances of the differentially expressed genes to WT levels, suggesting post-transcriptional regulation. However, there may be differences in the ability of sup2 to resist stress better than sup1 especially given the known Ssd1 repression of transcript translation in S. cerevisiae. Finally, pH appears to impact the sup1 and sup2 strain's sensitivity to CO2 in SI Appendix Figure 4. This could be better explained and interrogated in the manuscript. Finally, this work includes a variety of genes in several signaling pathways. The paper would be greatly clarified by a graphical abstract indicating how CBK1 may be integrating these pathways or by indicating which genes belong to which pathways in the Figure 1 legend to make this figure easier to follow.

    We thank the reviewer for the thorough summary of the study. We appreciate the reviewer’s enthusiasm about this study as well as constructive critiques on the manuscript. Indeed, the suppressor mutations in the cbk1Δ mutant rescue more phenotypes of cbk1Δ in vitro than just thermotolerance and CO2 tolerance (Supplemental Figure 5), which could benefit the survival of these suppressor strains in vivo compared to the original the cbk1Δ mutant. However, between the sup1 and the sup2 mutants, the only clear difference in growth we observed was in host levels of CO2 and temperature. There was no obvious difference in their resistance to Congo red (cell wall stress), melanization, susceptibility to FK506 (calcineurin pathway inhibitor), sensitivity to H2O2 (ROS), or urease (Supplemental Figure 5). Nonetheless, we agree with the reviewer that there could be other reasons which may influence the outcome in vivo, given that the host environment is more complex than we know. We have changed our wording in the manuscript to make it clear that contribution of better tolerance of CO2 to better survival of the sup2 mutant is only our hypothesis and there could be other unrecognized contributing factors. “The only in vitro difference observed between sup1 and sup2 was better growth of sup2 at host CO2 levels which may explain the difference in their ability to propagate and persist in the mouse lungs. However, due to the complexity of the host environment, there could be other unrecognized factors contributing to their growth difference in vivo.” (Lines 276279).

    About growth at different pH levels, C. neoformans tends to grow better at lower pH, closer to pH 5. This fungus can grow at pH 3, the lowest pH that our lab had tested (it may be able to sustain viability even at pH 2 based on others’ conference presentations). The high temperature/CO2 combined with neutral or high pH likely causes worse growth of both H99 and the mutants tested.

    We tried making a model to integrate all the pathways and factors identified in this work as the reviewer suggested. However, in this process, we found it difficult to propose a model. Although the current findings clearly demonstrate the importance of Cbk1 in thermotolerance and CO2 tolerance (overexpression of CBK1 can partially restore thermotolerance and/or CO2 tolerance in the mutants defective in the cell wall integrity pathway, the calcineurin pathway or the Cdc24-Ras1 pathway, and that the reciprocal overexpression of these genes in the cbk1∆ mutant does not rescue any of the cbk1∆ mutant’s defects), we do not know the exact mechanisms underlying this phenomenon. Do these pathways directly interact with Cbk1, affect its phosphorylation status, or alter its subcellular localization? Or do these pathways act through some other massagers to indirectly activate Cbk1 or maybe Cbk1’s downstream targets? These are the questions that warrant further investigations in the future. To be prudent, we think it is better not to propose a model at this point given the uncertainty of the mechanism. The mutants belonging to each of the pathways are clearly specified in the texts in this revised manuscript to help orient the readers. For example “As the RAM pathway effector kinase mutant cbk1Δ showed the most severe defect in thermotolerance and CO2 tolerance compared to the mutants of the other pathways, we first overexpressed the gene CBK1 in the following mutants, cdc24∆ (Ras1-Cdc24), mpk1∆ (CWI), cna1∆ (Calcineurin), and the cbk1Δ mutant itself, and observed their growth at host temperature and host CO2 (Figure 2B)...”

  3. eLife

    eLife assessment

    This study provides compelling evidence for the involvement of RAM pathway in the survival of C. neoformans in high CO2 concentrations. The work is important to understand how this fungus adapts to the high CO2 concentrations in host tissues. The experimental approach combines genetic and biochemical approaches to explore a complex topic that is of essential for cryptococcal pathogenesis.

  4. eLife

    Reviewer #1 (Public Review):

    The research investigates the genetic basis for resistance to high CO2 levels in the human pathogenic fungus Cryptococcus neoformans. Screening collections of over 5,000 gene deletion strains revealed 96 with impaired growth, including a set of genes all related to the same RAM signaling pathway. Further genetic dissection was able compellingly to place where this pathway lies relative to upstream inputs and through the isolation of suppressor mutants as potential downstream targets of the pathway. Given the high levels of CO2 encountered by fungi in the human host, this work may provide new directions for the control of disseminated fungal disease.

    The research presents both strengths and weaknesses.

    Strengths include:

    (1) One of the largest scale analyses of genes involved in growth under high CO2 concentrations in a fungus, revealing a set of just under 100 mutants with impaired growth.
    (2) Elegant genetic epistasis analysis to show where different components fit within a pathway of transmission of CO2 exposure. For example, over expression of one of the kinases, Cbk1, can overcome the CO2-sensitivity of mutations in the CDC24 or CNA1 genes (but not in the reciprocal overexpression direction).
    (3) Isolation of suppressor mutations in the cbk1 background, now able to grow at high CO2 levels, was able to lead to the identification of two genes. Follow up characterization, which included examining in vitro phenotypes, gene expression analysis, and impact during mouse infection was able to reveal that the two suppressors restore a subset of the phenotypes impacted by mutation of CBK1. Indeed, one conclusion from this careful work is that the reduced virulence of the cbk1 mutant is not due to its sensitivity to high levels of CO2, perhaps an unexpected finding given the original goals of the study towards linking CO2 sensitivity with decreased virulence.

    Weaknesses include:

    (1) What is the rationale for examining gene expression using the NanoString technology of 118 genes rather than a more genome-wide approach such as RNA-sequencing?
    (2) Without additional species examined, some of the conclusions about differences in impact between ascomycetes and basidiomycetes might instead reflect differences between species. For example, RAM mutants in other strains of C. neoformans do not exhibit so strong a temperature sensitive phenotype. Or to extend the comparison further, one might assume given the use of CO2 for Drosophila manipulations that the RAM pathway components in an insect would not be required for surviving high CO2.
    (3) Given the relative ease of generate progeny of this species, it would have been informative to explore if the suppressors of cbk1 also suppressed the loss of genes like CDC24, CNA1, etc, equivalent to the experiment performed of overexpression of CBK1 in those backgrounds.

  5. eLife

    Reviewer #2 (Public Review):

    In the paper by Chadwick et al., the authors identify the molecular determinants of CO2 tolerance in the human fungal pathogen Cryptococcus neoformans. The authors have screened a collection of deletion mutants to identify the genes that are sensitive at 37oC (host temperature) and elevated CO2 levels. The authors identified that the genes responsible for CO2 sensitivity are involved in the pathways responsible for thermotolerance mechanisms such as Calcineurin, Ras1-Cdc24, cell wall integrity, and the Regulator of Ace2 and Morphogenesis (RAM) pathways. Moreover, they identified that the mutants of the RAM pathway effector kinase Cbk1 were most sensitive to elevated temperature and CO2 levels. This study uncovers the previously unknown role of the RAM pathway in CO2 tolerance. Transcriptome data indicates that the deletion of CBK1 results in an alteration in the expression of CO2-related genes. To identify the potential downstream targets of Cbk1, the authors performed a suppressor screen and obtained the spontaneous suppressor mutants that rescued the sensitivity of cbk1 mutants to elevated temperature and CO2. Through this screen, the authors identified two suppressor groups that showed a modest improvement in growth at 37{degree sign}C and in presence of CO2.
    Interestingly, from the suppressor screen, the authors identified a previously known interactor of Cbk1 which is SSD1, and an uncharacterized gene containing a putative Poly(A)-specific ribonuclease (PARN) domain named PSC1 (Partial Suppressor of cbk1Δ) which acts downstream of Cbk1. Deletion of these two genes in cbk1 null mutants rescued the sensitivity to elevated CO2 levels and temperature but did not fully rescue the ability to cause disease in mice.

    This study highlights the underappreciated role of the host CO2 tolerance and its importance in the ability of a fungal pathogen to survive and cause disease in host conditions. The authors claim to gain insight into the genetic components associated with carbon dioxide tolerance. The experimental results including the data presented, and conclusions drawn do justice to this claim. Overall, it is a well-written manuscript. However, some sections need improvement in terms of clarity and experimental design.

    • One major drawback of the study is the virulence assay performed to test the ability of cbk1 mutants to cause the disease in the mouse model. The cbk1 null mutants are thermosensitive in nature. Using these mutants, establishing the virulence attributes in mice would undermine the mutants' ability to infect mice as they won't be able to survive at the host body temperature.

    • The rationale for choosing the genes to test further is not clear in two instances in the study. a) From a list of 96 genes, how do the authors infer the pathways involved? Was any pathway analysis performed that helped them in shortlisting the pathways that they subsequently tested? A GO term analysis of the list of genes identified through the genetic screen would be more helpful to get an overview of the pathways involved in CO2 tolerance. b) The authors do not clearly mention why they chose only four genes to test for the CO2 sensitivity out of 16 downregulated genes identified from the nano string analysis.

    • It would be more useful to the readers if the authors could also include a thorough analysis of the presence of the putative PARN domain-containing protein across various fungal species rather than mentioning that it is only observed in C. neoformans and S. pombe. Also, the authors may want to discuss the known role(s) of SSD1, if any, in pathogenic ascomycetous yeasts so that the proposed functional divergence is supported further.

  6. eLife

    Reviewer #3 (Public Review):

    In this work the authors identify genes and pathways important for CO2 and thermotolerance in Cryptococcus neoformans. They additionally rule out the contribution of the bicarbonate or cAMP-dependent activation of adenylyl cyclase to this pathway, which is important for CO2 sensing in other fungi, further solidifying the need to characterize CO2 sensing in basidiomycetes. The authors establish the importance of focusing on CO2 tolerance by testing the impact of CO2 on fluconazole susceptibility with varied pH, suggesting the ability of CO2 to sensitize cryptococcal cells to fluconazole. Furthermore, the authors compared the CO2 tolerance of clinical reference strains to environmental isolates. The characterization of the RAM pathway Cbk1 kinase illustrated the integration of multiple stress signaling pathways. By using a series of CBK1OE insertions in strains with deletions in other pathways, the ability of Cbk1 over-expression to rescue several strains from CO2 sensitivity was apparent. Additionally, NanoString expression analysis comparing cbk1∆ to H99 validated the author's screen of CO2-sensitive mutants as 16/57 downregulated genes were found in their screen, further confirming the interconnected nature of these pathways. The importance of the RAM pathway in maintaining CO2 and thermotolerance was also incredibly clear.

    Perhaps most interestingly, the authors identify suppressor colonies with distinctive phenotypes that allowed for the characterization of downstream effectors of the RAM pathway. These suppressor colonies were found to have mutations in SSD1 and PSC1 which somewhat restore growth at 37oC with CO2 exposure. Further confirming the importance of the RAM pathway, the cbk1∆ strain had markedly attenuated virulence during infection. Interestingly, the generated suppressor strains had varying impacts on fungal infection in vivo. While the sup1 suppressor was completely cleared from the lungs during both intranasal and IV infection, the sup2 strain, containing mutations in SSD1, maintained a high fungal load in the lungs and was able to disseminate into host tissues during IV infection but not intranasal infection.

    The authors make a strong case for the exploration of thermotolerance and CO2 tolerance as contributors to virulence. Through screening and characterization of RAM pathway kinase CBK1's ability to rescue other mutants from CO2 sensitivity, the overlapping contributions of several signaling pathways and the importance of this kinase were revealed. This work is important and will be valuable to the field. However, the cbk1∆ strain does show reduced melanization, urease secretion, and higher sensitivity to cell wall stressor Congo Red in SI Appendix, Figure S4. While the authors make a strong argument that these well-established virulence factors are not perfect predictors of virulence in vivo, the cbk1∆ strain is not an example of such a case as it does have defects in these important factors in addition to thermotolerance and CO2 tolerance. Not acknowledging the changes in these virulence factors in the cbk1∆ and their potential contribution to phenotypes observed is a weakness of the manuscript. Interestingly, the sup1 and sup2 strains also rescue these virulence factors compared to cbk1∆. Additionally, the assertion that "the observation that only sup2 can survive, amplify, and persist in animals stresses the importance of CO2 tolerance in cryptococcal pathogens" due to the sup2's slightly higher CO2 tolerance compared to sup1, could be better supported by the data. These suppressors did not restore transcript abundances of the differentially expressed genes to WT levels, suggesting post-transcriptional regulation. However, there may be differences in the ability of sup2 to resist stress better than sup1 especially given the known Ssd1 repression of transcript translation in S. cerevisiae. Finally, pH appears to impact the sup1 and sup2 strain's sensitivity to CO2 in SI Appendix Figure 4. This could be better explained and interrogated in the manuscript. Finally, this work includes a variety of genes in several signaling pathways. The paper would be greatly clarified by a graphical abstract indicating how CBK1 may be integrating these pathways or by indicating which genes belong to which pathways in the Figure 1 legend to make this figure easier to follow.

  7. Version published to 10.1101/2022.08.14.503895v1 on bioRxiv