Hyphal Growth in Trichosporon asahii Is Accelerated by the Addition of Magnesium

ABSTRACT Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

The study by Aoki et al. describes the identification of magnesium as a limiting factor for hyphal growth in Sabouraud medium. By exploiting the presence and absence of magnesium in the growth medium, they described defects in the lipid droplets distribution and distinct mitochondrial and vacuolar morphologies in Trichosporon asahii. The authors described enlarged vacuoles in media supplemented with MgSO4. The authors also proposed that yeast cells have a fragmented mitochondrial pattern, while in hypha the morphology of mitochondria was associated with filaments of actin and organized beside of the plasm membrane. In Candida albicans, the deprivation of magnesium inhibits the hypha formation, cell adherence and results in diminished hyphal damage during macrophage infection (PMID: 30659981;PMID: 35834579). In accordance, the absence of the micronutrient magnesium seems to be essential to the formation of hyphal forms associated with the virulence in Trichosporon asahii. The manuscript is technically sound and describes an interesting finding for Trichosporon asahii that can be valuable for future virulence studies. However, there are points that the authors might consider: -The authors could add arrows to highlight the lipid droplets that are not associated to DNA in figure 1. -The figure 4A and the text do not the design of the experiment.
-The authors suggested that the vacuoles are bigger after the addition of MgSO4 compared with YPD. While such observation is supported by the image in figure 5, the microscopy on figure 7 shows vacuoles of similar in sizes for cells grown in YPD and Sabouraud supplemented with MgSO4. Did the authors try somehow to quantify this phenomenon of enlarged vacuoles? -In the passage: "Therefore, actin polymerization may be downstream in a signalling pathway...", please clarify the association made between actin polymerization signalling and the findings related to YPD and magnesium-containing medium.
-Is the fragmented morphology of mitochondria observed in yeast a deficiency or a pattern? Cell ghosts also had the fragmented morphology, how was evaluated the viability of the cells cultivated in Sabouraud for the microscopy? -If morphology of mitochondria observed in yeast is different from hypha and actin is essential for hypha formation, it seems correct that the morphology of mitochondria would be the same as observed in yeast with the treatment of latrunculin A. Would the mitochondria morphology change if, after hypha formation, it was added latrunculin A and incubated for 4-5 hours? -In the passage: "The length of T. asahii cells gradually decreased..", are the cells diminishing or the higher concentrations of magnesium giving better support for hyphal growth? -I would suggest that the authors revise their conclusions about the role of Mg2+, transporters and gene expression, as some of them sound very speculative and are not supported by the findings described in the manuscript.
Reviewer #2 (Comments for the Author): Hyphal growth in Trichosporon asahii is accelerated by the addition of magnesium. Aoki et al.
This paper seeks to examine factors contributing to hyphal formation of Trichosporon asahii as well as intracellular organisation in defined and rich media. The authors show that the addition of magnesium accelerates hyphal growth for a limited time. Distribution and morphology of lipid droplets, vacuoles, cortical actin patches, and mitochondria were examined in cells cultured in different growth media. However, authors compare organelle distribution in yeast cells with polarised hyphal forms, attributing difference in intracellular organisation to media composition, while failing to compare the same morphological forms. Microscopy images are not sufficiently clear to serve as evidence behind the conclusions drawn.
Major comments: 1. Cell length measurements seem to be highly variable, with SD exceeding mean for many conditions. Since the authors aimed to examine hyphal induction, there could be more suitable ways to present the data -for example, in a form of a morphology index. 7. Fig 2D. Actual concentrations should be included on the figure and in the supplemental table. 8. Fig 3C. Hyphal width looks significantly different in Sabouraud medium and Sabouraud with MgSO4. This should be acknowledged in the results. 9. Page 11, paragraph 2. Fig 4 shows that magnesium can accelerate hyphal germination and elongation of the cells preincubated in Sabouraud medium. Claiming that these data show that cells retain the ability to form hyphae after 18h in Sabouraud medium is incorrect, when Fig 3 clearly shows hyphae are able to form in Sab alone. 10. Page 12 line 1. Dynamics of lipid droplets -"morphology" might be better suited for describing these differences. 11. Use of language: vitamin mix or vitamins -not vitamin in a singular form to describe combination of compounds. 12. Fig 7B: a cross section of the hyphal form should also be shown for cells grown in Sabouraud medium alone. 13. P17 line 18-22. These conclusions are confusing -consider revising.
Reviewer #3 (Comments for the Author): The manuscript presents data on the mechanism of yeast-to-hypha transition of Trichosporon asahii and identifies the factors that induce its hyphal growth. The data are original and there are no reports in the literature about the factors that stimulate the dimorphism of T. asahii. The results found are interesting and will contribute significantly to the target scientific community. The manuscript is well written and organized. Here are some considerations. 1. Format the list of references according to the style of the journal. 2. Figure 3: Insert the image of cells at time 0 in Sabouraud + MgSO4 medium Staff Comments:

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The manuscript "Hyphal growth in Trichosporon asahii accelerated by addition of magnesium" is a well-conducted work, data are interesting, and the authors have contributed to increasing the knowledge of the Trichosporon genus over the last years. I have a few commentaries to improve the research, but considering that the submission wasn´t page or line numbered, it will be difficult to address notes at specific parts.
1. Abstract: succinct and informative, but needs to be adjusted to the journal style. 2. Introduction: despite the lack of data on the role of magnesium in the dimorphism of the genus Trichosporon, could the authors consider including its role (and other related compounds) in fungi/yeasts? This is more important than explaining the biochemical processes involved. 3. Material and methods: I understand the complexity of the tests, but the sample size is minimal. Would it have been relevant to use at least a few more isolates to confirm the present findings? I believe it is relevant to mention it as a limitation of the study. 4. Material and methods: Please note that except in the "strains and media" subsection, references were not included. Please check and include the references used. 5. Material and methods: the used culture media, compounds, and methods were difficult to be understood. They are better described in the results (maybe because this section appears earlier according to the editorial style), but it will be worthwhile to the audience and also easier to be understood if the methods were better explained. 6. Measurement of growth rate (MM): why the authors used 72h or 144h once a single T.
asahii isolate was used? Were the measures automatically taken every 10 minutes? 7. Figures´ captions: some figures are shown incubation times different than those mentioned in the methods. Please check and correct. 8. Please, consider perform modifications in the introduction and discussion to make the manuscript easier to be comprehend. We newly examined the cell viabilities grown in YPD, Sabouraud, and Sabouraud+Mg media and found that cells grown in YPD and Sabouraud+Mg media showed 100% viabilities; however, cells grown in Sabouraud medium showed 52% viability. We suppose that the lost viability of 48% was attributed to the fragmented mitochondria in the short cells of <10 µm in Sabouraud medium.
Therefore, we think that the short cells having the fragmented mitochondria is Ø We already showed that actin was important for hyphal formation because cell length apparently shortened, and a swollen-shaped phenotype was produced when latrunculin A was added into each media (Fig. 6). When latrunculin A was added, the mitochondrial distribution also changed to dot-like distributions (Supplemental Ø Previous reports showed that the changes associated with Mg 2+ and magnesium transporters was allosteric; however, hyphal growth induced by adding MgSO4 was not allosteric in Fig. 2D. Therefore, we speculate that the morphological changes in Major comments: 1. Cell length measurements seem to be highly variable, with SD exceeding mean for many conditions. Since the authors aimed to examine hyphal induction, there could be more suitable ways to present the data -for example, in a form of a morphology index. Ø We showed the data of cell length measurements in the form of morphology index in Fig. 2A Ø To show the medial cell plane in Fig. 7, we used Calcofluor White reagent that stained the cell wall of fungi. A proportion of the mitochondria was observed to be distributed adjacent to the cell walls, detected using Calcofluor White, in YPD and Sabouraud+Mg media (Fig. 7A and B). In addition, a proportion of mitochondria was also distributed throughout the cell cytoplasm in hyphal and yeast-like cell in YPD and Sabouraud+Mg media. Therefore, we revised the mitochondrial distribution as throughout the cell cytoplasm and adjacent to the cell walls. The distribution of mitochondria adjacent to the cell walls is still a marker showing the mitochondrial distribution in the cells supplemented with MgSO4. The cell images of hyphal cells and yeast-like cells were described in Fig. 7A and B, respectively.
Concerning co-staining of actin and mitochondria, we deleted all the data because paraformaldehyde-fixed T. asahii cells produced background signals with the green filter.

Differences in organelle morphology and distribution could largely be explained by the lack of polarised growth and the cell cycle arrest in cells cultured in Sabouraud medium.
For example in Fig 5, cells in the same growth stages need to be examined for vacuolar morphology.
Ø We appreciate the encouragement. T. asahii cells are classified into three morphological types: yeast, hypha, and arthroconidia, and the size of each type is very variable. In the study, it is difficult for us to describe the relationships about polarized growth and cell cycle arrest that Reviewer 2 pointed out. We would like to understand the association between organelle morphology and polarized growth or cell cycle in a future study. Here, we analyzed the vacuolar size and frequency of mitochondrial distribution by ranging each cellular size in Fig. 5C  To examine the fragmented morphology of mitochondria in detail along cell length, we classified the distribution of mitochondria into the following three phenotypes: (1) throughout the cell cytoplasm and adjacent to the cell walls, (2) fragmented within the cytoplasm, and (3) a reduced distribution (Fig. 7C). We found that approximately 45.4% of cells grown in Sabouraud medium were characterized by mitochondria with a fragmented morphology, which was observed in both hyphal and yeast cells (Fig. 7C). Therefore, appearance of the fragmented mitochondria We did not estimate whether lipid droplets were functional or not; however, the accumulation of large lipid droplet in Sabouraud medium (Fig. 5A) indicated that lipid may not disperse throughout the cells cultivated in Sabouraud medium.
Therefore, it is suggested that nutrients may not be supplied normally to the mitochondria in the cells cultivated in Sabouraud medium in line 21-24, page 21.
Conclusion about mitochondrial activity was deleted from the discussion.
6. Fig 2B. Statistical analysis should compare salt drop-out media with media + salt to determine which ions were required for growth, and not with MgSO4∆.
Ø Each statistical analysis of the drop-out media with media + salt was added in the Supplemental Table. Statistical data are described only in the Supplemental Table. 7. Fig 2D. Actual concentrations should be included on the figure and in the supplemental 10. Page 12 line 1. Dynamics of lipid droplets -"morphology" might be better suited for describing these differences.
Ø The term "dynamics of lipid droplets" was corrected to "morphology of lipid droplets" (line 6, page 13).
11. Use of language: vitamin mix or vitamins -not vitamin in a singular form to describe combination of compounds.
Ø The singular form of vitamin was corrected to "vitamins" in the text (line 6, page 8).

Fig 7B: a cross section of the hyphal form should also be shown for cells grown in
Sabouraud medium alone. Ø We observed that the intake of Mg 2+ induced hyphal growth, and that cells were swollen-shaped upon the addition of an actin inhibitor. From these observations, we first thought that actin polymerization pathway was downstream of Mg 2+ signaling.
In contrast, another possibility was also considered. Mg 2+ uptake may increase the cell volume, but not the length of T. asahii cells, because the cell volume of cells grown in Sabouraud+Mg medium was larger than that of those in Sabouraud medium, under the inhibition of actin ( Fig. 6B; Supplemental Fig. 4). In this case, a signaling pathway affected by Mg 2+ may be independent of the actin polymerization.
The possibility was rewritten in the discussion (line 6, page 20). We would like to clarify the mechanism by which an increase of Mg 2+ affects hyphal growth in a future study.

To Reviewer 3
Reviewer #3 (Comments for the Author): The manuscript presents data on the mechanism of yeast-to-hypha transition of Trichosporon asahii and identifies the factors that induce its hyphal growth. The data are original and there are no reports in the literature about the factors that stimulate the dimorphism of T. asahii. The results found are interesting and will contribute significantly to the target scientific community. The manuscript is well written and organized.
Here are some considerations.

Format the list of references according to the style of the journal.
Ø The list of references was formatted according to the style of Microbiology Spectrum.

Figure 3: Insert the image of cells at time 0 in Sabouraud + MgSO4 medium
Ø In Fig. 3D, an image at time 0 was added in the line of Sabouraud + Mg medium. The manuscript is improved from previous version. There are still some issues to resolve: Reviewer #1 raises issues of cell viability in new measurements, which should be relatively easy to perform.
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