Ras1-Independent High Iron-Mediated Hyphal Formation in Candida albicans

Round 1

Reviewer 1 Report

The manuscript is clearly written and the experiments support the conclusions. I feel that the main new finding, is that high iron levels repress BCY1 transcripts under the conditions chosen. The interesting question in my view is, how BCY1 levels are repressed by high iron levels. The authors should discuss this in more detail, additional experiments to this would be great. The effect of BCY1 on filamentation in mutants defective of the upper ras pathway has been shown previously, as the authors propperly cite.

The authors show that C. albicans ras1 deletion strains are not able to form hyphae in YNB-GlcNAc media with 2 uM iron, but in the same media containing 100 uM. They could show that levels of BCY1 mRNA are lower under high iron conditions, if compared to low iron conditions. Consequently, they have constructed a strain overexpressing BCY1 which reverts the effect of high iron on filamentation, indicating that BCY1-levels are critical for this process. I would suggest including the supplemental Fig.2 into the main manuscript to visualize the increased transcript levels (e.g. as Fig. 5c).  The effect of BCY1 on filamentation has been shown previously in an Adenylate Cyclase deletion strain in standard SC-GlcNAc media (Konopka Lab). Pseudorevertant strains lacking a functional BCY1 copy regained the ability to form filaments. In the manuscript in Fig 5 under low iron conditions this is not visible. It would be interesting to see how the ras1 deletion + BCY1 overexpression strain behaves under standard conditions (with regard to iron concentration) and in case there is a difference to the work by Konopka, how this could be explained. For an easier comparison to standard media, the authors should give an indication about the iron levels in the standard media used.

The authors furthermore showed an experiment with C. elegans on low and high iron containing plates. Do the authors have any indication on the iron levels within C. elegans under both conditions? It would be excellent to have an indication if there is a difference. I would expect that C. elegans regulates its iron level stringently.

 

Author Response

Reviewer 1

The manuscript is clearly written and the experiments support the conclusions. I feel that the main new finding, is that high iron levels repress BCY1 transcripts under the conditions chosen. The interesting question in my view is, how BCY1 levels are repressed by high iron levels. The authors should discuss this in more detail, additional experiments to this would be great. The effect of BCY1 on filamentation in mutants defective of the upper ras pathway has been shown previously, as the authors properly cite.

We thank the reviewer for the positive comments and insightful suggestion. We have now expanded the Discussion section (Although the present study demonstrates…………. morphogenetic switching) to further address potential mechanisms linking iron signaling and BCY1 regulation and acknowledge this as an important direction for future investigation.

The authors show that C. albicans ras1 deletion strains are not able to form hyphae in YNB-GlcNAc media with 2 uM iron, but in the same media containing 100 uM. They could show that levels of BCY1 mRNA are lower under high iron conditions, if compared to low iron conditions. Consequently, they have constructed a strain overexpressing BCY1 which reverts the effect of high iron on filamentation, indicating that BCY1-levels are critical for this process. I would suggest including the supplemental Fig.2 into the main manuscript to visualize the increased transcript levels (e.g. as Fig. 5c). 

We agree that the BCY1 transcript data are important to support the role of BCY1 repression in high iron-induced filamentation. As suggested, we have now moved Supplemental Fig. 2 into the main manuscript and included it as Fig. 6c to better visualize the increased BCY1 transcript levels.

The effect of BCY1 on filamentation has been shown previously in an Adenylate Cyclase deletion strain in standard SC-GlcNAc media (Konopka Lab). Pseudorevertant strains lacking a functional BCY1copy regained the ability to form filaments. In the manuscript in Fig 5 under low iron conditions this is not visible. It would be interesting to see how the ras1 deletion + BCY1 overexpression strain behaves under standard conditions (with regard to iron concentration) and in case there is a difference to the work by Konopka, how this could be explained. For an easier comparison to standard media, the authors should give an indication about the iron levels in the standard media used.    

The study by Min et al. from the Konopka laboratory (1) demonstrated that loss of one BCY1 copy increases basal PKA activity and serves as a prerequisite for hyphal induction, but additional regulatory mechanisms (protein kinases) are required for robust filamentation. Consistent with this model, we observed that the ras1Δ/Δ strain exhibited limited filamentation under low-iron conditions because Ras1 is the primary upstream activator of the cAMP-PKA signaling pathway. In contrast, robust filamentation occurred only under high-iron conditions, potentially due to iron-mediated effects on chromatin remodeling and MAPK signaling (2). These findings also raise the possibility that iron-dependent BCY1 downregulation require additional iron-responsive signals to fully drive hyphal development. We agree that examining the ras1Δ/Δ + BCY1 overexpression strain under standard iron conditions would provide additional insight into the role of BCY1 in regulating hyphal morphogenesis. While this experiment was beyond the scope of the current study, we have acknowledged this limitation and included it as an important direction for future investigation in the Discussion section as “While BCY1 overexpression…...independently of iron availability.”

The authors furthermore showed an experiment with C. elegans on low and high iron containing plates. Do the authors have any indication on the iron levels within C. elegans under both conditions? It would be excellent to have an indication if there is a difference. I would expect that C. elegans regulates its iron level stringently.

Although we did not directly measure iron levels within C. elegans in the current study, previous studies have demonstrated that iron levels in C. elegans can be altered depending on environmental iron availability. A previous study showed that worms grown under high-iron and low-iron conditions exhibit corresponding changes in iron accumulation and iron-responsive gene regulation, indicating that environmental iron conditions can influence internal iron homeostasis in C. elegans despite tight regulatory control (3). We have now included this point as “Environmental iron availability……. external iron conditions” in the Result section 3.2 to clarify the physiological relevance of our experimental conditions.

 

1. Min K, Jannace TF, Si H, Veeramah KR, Haley JD, Konopka JB. 2021. Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans. PLoS Pathog 17:e1009861.
2. Puri S, Lai WK, Rizzo JM, Buck MJ, Edgerton M. 2014. Iron-responsive chromatin remodelling and MAPK signalling enhance adhesion in Candida albicans. Mol Microbiol 93:291-305.
3. Valentini S, Cabreiro F, Ackerman D, Alam MM, Kunze MB, Kay CW, Gems D. 2012. Manipulation of in vivo iron levels can alter resistance to oxidative stress without affecting ageing in the nematode C. elegans. Mech Ageing Dev 133:282-90.

Reviewer 2 Report

This is  very interesting work that teases out the role of Ras1 in hyphal production in C. albicans which is an important virulence factor for this organism. Iron is known to be an important growth factor, but here its role in signaling through the Ras pathway shows its importance for morphogenesis and virulence. This is a novel feature and important for the understanding of how C. albicans regulates its ability to colonize and invade human tissues.

One concern is that the description of the linking components of the Ras pathway are not well described until Fig. 6 in the discussion. It would be better to move this Figure or some version of it in the introduction to better understand the rationale for examine various mutants. 

It is really unclear why BCY1 was selected for further study from Fig. 4- is it because it is the only iron responsive gene to be regulated in an opposite manner? If this is so then CFL5 should also be examined unless there is an error in the Table. A heat map would be much easier to follow in Fig. 4. Is PKA also similarly regulated by iron? 

Although Fig. 5 is convincing in demonstrating the role of Bcy1 in iron responses, the transition to this regulator is abrupt. More description of Bcy1 is needed.

Fig. 3 shows that iron can induce filamentation independent from cAMP levels in Ras mutants, but it would be interesting to know if adding cAMP can also induce hyphae in Ras mutants without additional iron.  

Author Response

Reviewer 2

This is very interesting work that teases out the role of Ras1 in hyphal production in C. albicans which is an important virulence factor for this organism. Iron is known to be an important growth factor, but here its role in signaling through the Ras pathway shows its importance for morphogenesis and virulence. This is a novel feature and important for the understanding of how C. albicans regulates its ability to colonize and invade human tissues.

One concern is that the description of the linking components of the Ras pathway is not well described until Fig. 6 in the discussion. It would be better to move this Figure or some version of it in the introduction to better understand the rationale for examine various mutants. 

We thank the reviewer for this valuable suggestion. As suggested, we have now added the schematic model (Figure1) summarizing the Ras pathway components (independent of iron) to the Introduction to provide better context for the experimental design.

It is really unclear why BCY1 was selected for further study from Fig. 4- is it because it is the only iron responsive gene to be regulated in an opposite manner? If this is so, then CFL5 should also be examined unless there is an error in the Table. A heat map would be much easier to follow in Fig. 4. Is PKA also similarly regulated by iron? 

BCY1 was selected for further investigation because it encodes the regulatory subunit of the cAMP-PKA pathway, a central regulator of hyphal morphogenesis in C. albicans. Among the cAMP-PKA pathway genes, BCY1 showed inverse expression in the Δ/Δras1 strain compared to the parental strain under high-iron conditions. We also thank the reviewer for pointing out the issue with CFL5 and apologize for this confusion. We have corrected the error in the table accordingly. Also, CFL5 is already well-known to be an iron-regulated gene whereas iron-regulation of BCY1 stood out as a novel finding.  In addition, Fig. 4 has been revised to include a heat map representation, which improves visualization and comparison of iron-responsive transcriptional changes between strains and conditions.

Although Fig. 5 is convincing in demonstrating the role of Bcy1 in iron responses, the transition to this regulator is abrupt. More description of Bcy1 is needed.

To improve clarity and provide better context for the reader, we have now expanded the description of Bcy1 in the Result section 3.5 (Bcy1 serves……...…downstream PKA-dependent pathways), including its role as function in regulating filamentation downstream of Ras signaling.

Fig. 3 shows that iron can induce filamentation independent from cAMP levels in Ras mutants, but it would be interesting to know if adding cAMP can also induce hyphae in Ras mutants without additional iron.  

Previous studies have reported that exogenous cAMP can partially restore filamentation in ras1Δ/Δ mutant under certain hypha-inducing conditions (4). However, in our study, intracellular cAMP levels remained low under high iron conditions despite robust filamentation, suggesting that high iron-induced hyphal formation occurs through a cAMP-independent mechanism. Since the primary focus of this work was to define the role of iron signaling in bypassing the Ras pathway, upstream of Cyr1, we did not further examine the effects of exogenous cAMP supplementation in this study.

 

 

Reference

4.         Davis-Hanna A, Piispanen AE, Stateva LI, Hogan DA. 2008. Farnesol and dodecanol effects on the Candida albicans Ras1-cAMP signalling pathway and the regulation of morphogenesis. Mol Microbiol 67:47-62.

Round 2

Reviewer 1 Report

Thank you very much for the answers and changes provided. The BCY1 transcript-levels have been included in Fig. 5c, as suggested. However, description of Fig. 5c in the text and in the figure legend do not match. Fig. 5c depicts and describes in the legend the difference between BCY1-levels in ras1 and ras1+BCY1 strains. However, the (media) conditions used are not described in the legend. The text describes that we look at the difference between BCY1-levels in ras1 strains under low and high iron conditions. It would be ideal to include both in Fig. 5c. This would enable a direct comparison between the two settings under the same conditions (media, temperature, etc.).

I realized that the overexpression strain constructed in this work is not listed in the strain table. Please add.

Regarding the overexpression expression plasmid, pNIM1, originally by Park and Morschhäuser according to your reference (33), I understand that overexpression using pNIM1 is induced by tetracylin, so it is a Tet-on system. Please give the exact conditions how you induced BCY1 in the overexpression strain. I couldn´t find the tetracycline amount used. Tetracycline is only mentioned once, as part of the selection media for transformants (no concentration given). With the conditions given in your manuscript there would be no BCY1 expression from the Tet-on system designed by Park and Morschhäuser.

Another major point remains to be clarified, which escaped my attention previously: Fig 1S shows that the ras1-deletion strain and CAI4 (ura3/URA3), show almost identical growth rates in low iron conditions and high iron conditions (YNB, as given in MM). You explicitly state this in the text as well. This is in conflict with several publications describing a significant reduction in growth rates of ras1 deletion strains in comparison to the wild type strain in different media (e.g. Leberer et al 2001, Wijnants et al 2023, just to mention two publications). Most ras1 mutant strains are derived from CAI4, thus from the same background used here. My main concern with this point is, that strains deleted for genes of the cAMP-pathway like ras1, cyr1 or cdc25 are prone to genetic alterations resulting in faster growing mutant strains – as nicely described in Min et al 2021. Therefore, you will need to explain why your ras1-deletion strain behaves like WT in the media you used? Do you see differences in growth rates in any media? If this is not the case, you need to clearly state that you cannot exclude background mutations as shown in other publications (see above). Alternatively, I would suggest generating a new ras1 deletion strain or achieve a deletion strain with the originally described phenotypes and confirm your results.

For growth rate measurements you describe that you use 96 well plates measuring absorbance at 600 nm for 24h. Did you shake the plates? As you compare strains growing as hyphae or yeast in the media employed, I wonder how you accurately measure growth kinetics/doubling of cells. With hyphae we never get accurate numbers in absorption measurements. The media used for Fig. S1 is not given.   

 

 

Standard YNB usually is supplemented to contain about 1,2 uM iron, thus the low iron condition used in this manuscript should be comparable to supplemented standard YNB (please confirm if this holds true for your lab as well). Therefore, I am not sure if the term low iron is appropriate here, because it might suggest significant limitations. I would suggest to change this.

With regard to C. elegans the result looks convincing, however, you need to assume that the iron concentration in the gut of the worm is high enough to induce hyphae formation in ras1 deletion strains. You cannot be sure. Please indicate this in the text.

In the abstract you describe the C. elegans infection mode as novel in vivo model. The model is not novel, as C. elegans is used frequently as infection model for various applications, from drug screening to mutant evaluation. Please remove the word novel in the abstract.

Author Response

• Thank you very much for the answers and changes provided. The BCY1 transcript-levels have been included in Fig. 5c, as suggested. However, description of Fig. 5c in the text and in the figure legend do not match. Fig. 5c depicts and describes in the legend the difference between BCY1-levels in ras1 and ras1+BCY1 strains. However, the (media) conditions used are not described in the legend. The text describes that we look at the difference between BCY1-levels in ras1 strains under low and high iron conditions. It would be ideal to include both in Fig. 5c. This would enable a direct comparison between the two settings under the same conditions (media, temperature, etc.).
• We thank the reviewer for these valuable suggestions. To address this concern, we have revised figure legend to accurately describe the experimental conditions used. Now legend of Fig 5C is “Real-time qPCR analysis showed that BCY1 gene was downregulated in Δ/Δras1 under low iron condition compared to high iron condition. Cells were obtained from high iron and low iron hyphal induction medium (YNB-GlcNAc), incubated at 37 °C for 6 h with shaking at 200 rpm.”

 

• I realized that the overexpression strain constructed in this work is not listed in the strain table. Please add.
• We have now added the strain, along with its relevant genotype information, to Table 2.

 

• Regarding the overexpression expression plasmid, pNIM1, originally by Park and Morschhäuser according to your reference (33), I understand that overexpression using pNIM1 is induced by tetracylin, so it is a Tet-on system. Please give the exact conditions how you induced BCY1 in the overexpression strain. I couldn´t find the tetracycline amount used. Tetracycline is only mentioned once, as part of the selection media for transformants (no concentration given). With the conditions given in your manuscript there would be no BCY1 expression from the Tet-on system designed by Park and Morschhäuser.
• We have now added the construction and induction conditions of the BCY1 overexpression strain in the revised Methods section 2.9 as “Transformants were selected on YPD agar plates supplemented with 100 μg/mL tetracycline and 100 μg/mL nourseothricin and incubated at 30°C for 2 days. Positive transformants were screened by colony PCR using BCY1-specific primers, and BCY1 overexpression was confirmed by RT-PCR. For induction of BCY1 expression, doxycycline was added to the culture medium at a final concentration of 20 μg/mL.”

 

• Another major point remains to be clarified, which escaped my attention previously: Fig 1S shows that the ras1-deletion strain and CAI4 (ura3/URA3), show almost identical growth rates in low iron conditions and high iron conditions (YNB, as given in MM). You explicitly state this in the text as well. This is in conflict with several publications describing a significant reduction in growth rates of ras1 deletion strains in comparison to the wild type strain in different media (e.g. Leberer et al 2001, Wijnants et al 2023, just to mention two publications). Most ras1 mutant strains are derived from CAI4, thus from the same background used here. My main concern with this point is, that strains deleted for genes of the cAMP-pathway like ras1, cyr1 or cdc25 are prone to genetic alterations resulting in faster growing mutant strains – as nicely described in Min et al 2021. Therefore, you will need to explain why your ras1-deletion strain behaves like WT in the media you used? Do you see differences in growth rates in any media? If this is not the case, you need to clearly state that you cannot exclude background mutations as shown in other publications (see above). Alternatively, I would suggest generating a new ras1 deletion strain or achieve a deletion strain with the originally described phenotypes and confirm your results.
• In our study, we observed that the Δ/Δ ras1 strain exhibited reduced colony growth on YNB agar plates compared with the parental CAI4 strain, irrespective of iron levels. However, repeated growth analyses in liquid YNB medium under both high- and low-iron conditions showed no statistically significant differences in growth kinetics between the two strains. The studies cited above (Leberer et al., 2001; Wijnants et al., 2023) generally reported growth defects of Δ/Δ ras1strains relative to the SC5314 wild-type background, whereas our experiments compared the mutant with its parental strain, CAI4 that is distinct from SC5314. Furthermore, Wijnants et al. (2023) demonstrated that the growth phenotype of the Δ/Δ ras1 strain is media-dependent, as they did not observe growth defects in liquid RPMI medium or in SC medium containing 5 mM glucose, but defect was visible in solid media (Figure 1D and F and Figure 2 Wijnants et al. (2023)).

• For growth rate measurements you describe that you use 96 well plates measuring absorbance at 600 nm for 24h. Did you shake the plates? As you compare strains growing as hyphae or yeast in the media employed, I wonder how you accurately measure growth kinetics/doubling of cells. With hyphae we never get accurate numbers in absorption measurements. The media used for Fig. S1 is not given.
• Yes, plates were shaken at the interval of 30 minutes for 5 Seconds. Shaking time has now been added in the Material and Method section 2.4. The growth assays were performed in YNB-glucose medium (in place of GlcNAc) with low and high iron at 30°C. Under these conditions (in the absence of inducers like 37 °C and GlcNAc as carbon source), C. albicans predominantly grows in the yeast form and does not undergo significant hyphal development. Media and growth information has been  added in Figure S1 legend as “C. albicans cells and Δ/Δras1 cells were grown in YNB-glucose medium containing low iron (2 µM) and high iron concentrations (100 µM), and growth was monitored at OD₆₀₀ over a period of 24 hours at 30°C. plates were shaken at the interval of 30 minutes for 5 Seconds.”

 

Detailed comments

• Standard YNB usually is supplemented to contain about 1,2 uM iron, thus the low iron condition used in this manuscript should be comparable to supplemented standard YNB (please confirm if this holds true for your lab as well). Therefore, I am not sure if the term low iron is appropriate here, because it might suggest significant limitations. I would suggest to change this.
• In our experiments, initial iron restriction is achieved by supplementation of the medium with the iron chelator bathophenanthroline disulfonate (BPS) to chelate background medium iron and this baseline medium is then supplemented with either low or high iron. We have extensively published with these low and high iron conditions that consistently show the expected changes in the gene expression of iron-related genes, confirming these as appropriate low and high iron conditions (1-3).

 

• With regard to elegans the result looks convincing, however, you need to assume that the iron concentration in the gut of the worm is high enough to induce hyphae formation in ras1 deletion strains. You cannot be sure. Please indicate this in the text.
• We agree that the worm gut iron conditions allowed hyphae formation, albeit only under high iron, bolstering our hypothesis that iron supplementation changes worm iron levels such that worms grown in low iron were unable to induce hyphae in Δ/Δ ras1 strain.

 

• In the abstract you describe the C. elegans infection mode asnovel in vivo model. The model is not novel, as C. elegans is used frequently as infection model for various applications, from drug screening to mutant evaluation. Please remove the word novel in the abstract.
• As suggested the word “novel” has been removed from the abstract.

 

References

1. Sharma R, Gibb AA, Barnts K, Elrod JW, Puri S. 2023. Alternative oxidase promotes high iron tolerance in Candida albicans. Microbiol Spectr 11:e0215723.
2. Tripathi A, Nahar A, Sharma R, Kanaskie T, Al-Hebshi N, Puri S. 2022. High iron-mediated increased oral fungal burden, oral-to-gut transmission, and changes to pathogenicity of Candida albicans in oropharyngeal candidiasis. J Oral Microbiol 14:2044110.
3. Tripathi A, Liverani E, Tsygankov AY, Puri S. 2020. Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response. J Biol Chem 295:10032-10044.