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. 2024 Sep 9;14:20936. doi: 10.1038/s41598-024-69024-z

Enhanced visualization of nuclear staining and cell cycle analysis for the human commensal Malassezia

Jayaprakash Sasikumar 1, Suparna Laha 1, Bharati Naik 1, Shankar Prasad Das 1,✉
PMCID: PMC11385215  PMID: 39251619

Abstract

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

Keywords: Malassezia, Nuclear staining, Cell cycle, DAPI, Propidium iodide, Nocodazole, Zymolyase, FACS

Subject terms: Biological techniques, Cell biology

Introduction

Nuclear staining holds a fundamental significance in the realm of cell biology and plays a vital role in diverse applications like cell cycle analysis, DNA damage assays checking for chromatin integrity, genetic research, and the exploration of cellular structures. While it is considered a straightforward procedure in mammalian and other yeast cells, standard staining methods prove ineffective in the fungi Malassezia1. In nature, the commensal Malassezia is a genus of lipophilic fungi that is associated with warm-blooded animals. In humans, it is one of the microbes that is now known to be present in a wide range of organs, and in some of them like mouth, skin, and gut, it shows unique predominance and usually remains undetected due to difficulties in culture conditions and there are several barriers to studying this fungus such as slow growth, specific nutritional requirements2, difficulties in revival3, and lipid-rich cell walls4 making it difficult for genetic manipulations5. Malassezia is widely considered a commensal organism6, however in certain instances due to its immunomodulatory properties7 and because of possible host genetic defects8, it is implicated in a spectrum of diseases9, spanning skin conditions from Seborrheic and Atopic dermatitis, Pityriasis versicolor, and Malassezia folliculitis10,11, to chronic ailments such as Crohn's disease, Ulcerative colitis, and several cancers such as Pancreatic Ductal Adenocarcinoma, Cervical, Colorectal and Oral cancers12–14. Additionally, our recent study provided evidence of its coexistence with other fungi, which might exacerbate its pathogenicity towards severe infections15. Earlier research showed it to be the primary inhabitant of the epidermal region but recent findings revealed its presence in multiple organs of the human body16–23. Intriguingly, several Malassezia species and their DNA have been found in the brain of individuals24–27, particularly in patients with neurodegenerative disorders21,28–31, highlighting its potential pathogenic role in such conditions32. In addition to its pathogenic potential associated with yeast-to-hyphal conversion33, dysbiotic populations of this fungus have been implicated in numerous diseases, emphasizing the critical need for deeper exploration of this commensal. Particularly, investigations into its associations with several cancers highlight the complexity of this commensal microbe.

Recent technological advancements are gradually overcoming the hurdles and illuminating the true nature of these fungi. Hence, there is a need for more studies to explore this wolf in sheep-clothing pathogen11,14. As part of the ongoing investigation, a novel methodology has been developed to unveil Malassezia's genome through nuclear staining. To achieve this, specific agents targeting the nucleus, like DAPI (4',6-diamidino-2-phenylindole) and Hoechst dyes are utilized which are traditionally used for nuclear staining. Both DAPI and Hoechst bind DNA by intercalating between base pairs, causing fluorescence enhancement upon binding34,35. Their distinctive blue fluorescence emission allows for specific visualization of DNA in cells, making them a widely used fluorescent stain for nuclear and chromosomal studies. DAPI has a preference for AT-rich regions of DNA due to the presence of more accessible grooves in these regions whereas Hoechst binds to GC-rich regions36,37. Moreover, Hoechst dyes are frequently used in conjunction with other fluorescent markers to distinguish between cell populations or identify subcellular structures, facilitating complex analyses such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH)38,39. In addition to nuclear staining, an attempt was made to analyze the cell cycle of Malassezia. For this, Propidium iodide (PI) holds significant importance in nuclear staining and cell cycle analysis owing to its distinctive properties. PI is used to stain dead cells as live cells can flush out PI which doesn’t happen in the case of DAPI40. As a fluorescent dye, PI selectively binds to DNA by intercalating between base pairs, emitting red fluorescence upon binding41. This property makes PI an excellent choice for nuclear staining, allowing for visualization and quantification of DNA content within cells. In cell cycle analysis, PI thus serves as a vital tool for discerning different cell cycle phases based on DNA content. By staining cells with PI and subjecting them to flow cytometry, researchers can accurately determine the proportion of cells in the different cell cycle phases, thus gaining insights into cell cycle progression and dynamics42. The cell cycle, a highly regulated process, consists of four main phases: G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis). Checkpoints tightly control progression through these phases, ensuring accurate DNA replication, repair, and cell division43. With the developed PI staining methodology for Malassezia, we tried to arrest the cells at a particular phase of the cell cycle, something that has not been attempted before. Various arresting agents, such as Nocodazole (targeting microtubule polymerization for metaphase arrest), hydroxyurea (blocking DNA replication for S phase arrest), and α-Factor (for arresting budding yeast at G1), are routinely used for cell-cycle-based studies. Nocodazole, a microtubule-depolymerizing agent, interferes with microtubule polymerization dynamics, leading to the disruption of the mitotic spindle apparatus and subsequent arrest of cells in mitosis. This arrest enables synchronization of cells at a specific cell cycle stage (G2/M), facilitating detailed analysis of molecular events and dynamics associated with that phase and also for studying effects in other cell cycle phases when cells are released into the cell cycle post-arrest44. For our methodology, we chose Nocodazole to arrest the cells at the G2/M transition which disrupts microtubule dynamics45. Microtubules are essential components of the cytoskeleton and play pivotal roles in various cellular processes, including chromosome segregation during mitosis46. By combining established methodologies with innovative techniques, our approach represents a significant step towards understanding cell biology and unraveling the complexities associated with the human commensal Malassezia.

Materials and methods

Materials and reagent preparation

25 mg of DAPI (Sigma) was dissolved in 1 ml of molecular-grade water and the stock solution was stored at − 20 °C. 40 μl from the 25 mg/ml DAPI stock solution was dissolved in 960 μl of molecular-grade water to make a working solution (1 mg/ml). This working solution was stored at 4 °C. 25 mg of Hoechst (Sigma) was dissolved in 1 ml of molecular-grade water and the stock solution was stored at − 20 °C. 40 μl from the 25 mg/ml Hoechst stock solution was dissolved in 960 μl of molecular-grade water to make a working solution (1 mg/ml). This working solution was stored at 4 °C. 10 mg of Zymolyase powder (MP Biomedicals) was dissolved in 1 ml of 0.1 M sodium phosphate solution and the stock solution (10 mg/ml) was stored at 4 °C. For 0.1 M Sodium phosphate solution 339.4 mg of sodium phosphate monobasic (HiMedia) and 2.021 g of sodium phosphate dibasic (HiMedia) were added with 80 ml of Milli-Q water. pH was adjusted to 7.4, volume made up to 100 ml, and stored at 4 °C. For 1X PBS solution, NaCl (0.8 g), KCl (20 mg), Na2HPO4 (0.114 g), and KH2PO4 (0.24 mg), were mixed with 80 ml of Milli-Q water. Volume was made up to 100 ml, autoclaved at 121 °C for 20 min, and stored at room temperature. The Triton X-100 solution was prepared by dissolving Triton X-100 in Milli-Q-Water at a 1:9 ratio. 1 mg of RNase powder was added to 1 ml of freshly prepared 1X PBS solution to prepare a stock solution (1 mg/ml) which was kept at − 20 °C. 100 μl from the 1 mg/ml RNase A stock was added to 900 μl of 1X PBS (100 μg/ml working solution) and stored at 4 °C. 1 mg of Propidium Iodide powder was dissolved in 1 ml of molecular-grade water, covered with aluminium foil, and stored at − 20 °C. 50 μl from the 1 mg/ml PI stock was dissolved in 950 μl of molecular-grade water (50 μg/ml working solution), covered with aluminium foil, and stored at 4 °C. The Cell Permeabilizing Solution (CP Solution) 545 μl, used per sample comprised 40 μl of Zymolyase (10 mg/ml), 500 μl of Triton X-100 solution, and 5 μl of β-mercaptoethanol. 10 mg of Nocodazole was mixed in 1 ml of DMSO to make a 10 mg/ml stock and stored at 4 °C until use. 25 mg of chloramphenicol is mixed in 1 ml of 95% ethanol to make a 25 mg/ml stock solution. Yeast cultures were grown in Sabouraud Dextrose (SD) media in an Incubator Shaker (Innova 44R, Eppendorf). Optical density was measured using a Digital photo colorimeter (Sky Technologies). Fluorescent images were captured using the fluorescence microscope (Oxion fluorescence, Euromex). Cell cycle analysis was done using the Flow cytometer (DxFlex, Beckman Coulter) and for data acquisition CytExpert (v2.6) was used. For flow cytometry data analysis Kaluza analysis (v1.6) was used and Adobe Illustrator (2023) was used for designing graphics and artwork.

Methods

Fungal growth

Malassezia pachydermatis (CBS 1879) cells (Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands) and Saccharomyces cerevisiae (699) cells47 were streaked on an agar plate containing SD media with the addition of 50 μg/ml chloramphenicol to inhibit bacterial growth. The plate was then incubated at 32 °C for 3 days to facilitate the formation of distinct, visible single colonies. After the incubation period, a single fungal colony was selected and introduced into 5 ml of liquid SD media supplemented with 50 μg/ml chloramphenicol from the 25 mg/ml stock. This liquid culture was incubated overnight at 32 °C with shaking at 120 RPM in an incubator shaker. A subculture was initiated from the starter culture, adjusting the final concentration at OD600 of 0.1 in a liquid SD media supplemented with 50 μg/ml chloramphenicol. This subculture was allowed to grow until it reached an OD600 of 0.4–0.5. The resulting log-phase culture was employed for subsequent experimental procedures.

DAPI staining

To prepare S. cerevisiae cells for DAPI staining, 1 ml of culture with an OD600 of 0.4–0.5 was centrifuged at 5000 RPM for 5 min. The cells were then incubated with 70% ethanol for 30 min at room temperature. Following incubation, the cells were washed twice with 1X PBS, resuspended in 100 μl of 1X PBS, and vortexed. A 0.5 μg/ml DAPI/Hoechst solution was added, and the cells were incubated for 30 min at room temperature. After incubation, the cells were washed once with 1X PBS and observed under a microscope48. For M. pachydermatis, 1 ml of culture with OD600 of 0.4–0.5 was centrifuged at 5000 RPM for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of 1X PBS (pH 7.4) and vortexed. This suspension underwent centrifugation at the same conditions and duration, and the process was repeated twice. Following the PBS washes, the cell pellet was dissolved in 1 ml of 0.1N potassium phosphate solution (pH 7.4) and subjected to centrifugation. After discarding the supernatant, the pellet was resuspended in 1 ml of Triton X-100 solution and centrifuged. Following the removal of the supernatant, to the pellet 545 μl of CP solution (comprising of 500 μl Triton X-100, 40 μl of 10 mg/ml Zymolyase and 5 μl of β-mercaptoethanol (βME)) was added, mixed and incubated at 32 °C, 120 RPM in a shaking incubator for 2 h. After incubation, the cells were centrifuged, and the supernatant was discarded. The obtained pellet was washed once with Triton X-100 solution and once with 1X PBS buffer. Following these washes, post centrifugation the supernatant was removed, and the pellet was dissolved in 100 μl of 1X PBS. To the pellet, 1 μl of 1 mg/ml DAPI/ Hoechst solution was added and mixed. The cells with DAPI were incubated in the dark for 30 min at room temperature. After incubation, the cells were centrifuged, and the supernatant was discarded. The pellet was washed twice with Triton X-100 solution and then resuspended in 100 μl of 1X PBS. For observation, 10 μl of the stained cell suspension was taken and examined under an Oxion fluorescence microscope (Euromex) using a UV channel at 100X magnification.

Catalase, urease bile esculin, and viability test

Both the treated and the untreated (control) cells were checked for Catalase, Urease, and Bile esculin tests as performed in49 to confirm that the cells are metabolically active. To evaluate the viability of cells following the treatment, the control (untreated) and treated cells were counted and plated onto SD plates. Viable colonies were counted and plotted as percent viable cells based on the average of two independent experiments.

Cell cycle arrest

0.5 ml of the budding yeast cultures (OD600 of 0.4–0.5) were taken and to that 15 μg/ml nocodazole solution was added from 10 mg/ml stock and mixed well. This mixture was kept in a shaking incubator for 6 h at 32 °C for M. pachydermatis and 3 h for S. cerevisiae which are approximately two doubling times and kept at 120 RPM. After nocodazole incubation, the culture was taken and mixed with an equal volume of 70% ethanol and incubated at 4 °C overnight. After ethanol incubation, the mixture was centrifuged at 5000 RPM for 5 min, and the supernatant was discarded. The pellet was then washed twice with 1 ml of 1X PBS. After the washing steps, the cells were resuspended in 500 μl of PBS and supplemented with 100 μl of 100 μg/ml RNase A. The mixture was incubated at 50 °C in a dry bath for 1 h. Following the RNase treatment, 200 μl of 50 μg/ml PI solution was added, and the mixture was incubated at room temperature in the dark for 30 min.

Flow cytometry

The PI-stained cell suspension was diluted 1/10th using 1X PBS. The diluted cell suspension was loaded into the flow cytometer (Cytoflex, Beckman Coulter) sample tube. Data was acquired by running the samples through the flow cytometer at a suitable flow rate ranging between 250 and 500 cells/second, ensuring a minimum of 5000 events were recorded for reliable analysis. Appropriate laser and filter settings were selected for detecting PI fluorescence, as PI typically emits red fluorescence upon excitation by a blue laser. Firstly, cell populations were gated based on Forward Scatter-Area (FSC-A) and Side Scatter-Area (SSC-A) to exclude debris and select single cells in the FSC-A vs. SSC-A plot. Then, a plot of Forward Scatter-Area (FSC-A) vs. PI fluorescence filter-Area (PE-A) was created, and a gate was delineated to include only PI-positive cells. Subsequently, a histogram of PI fluorescence intensity (PE-A) vs. cell count for the PI-positive gated population was generated. The acquired data was further analyzed using flow cytometry software (Kaluza 1.6).

Results and discussion

We have developed a methodology for nucleus staining in Malassezia, something which was attempted earlier but didn’t find much success1. Due to its thick cell wall, Malassezia is refractory to genetic manipulation as direct transformation is still a challenge. New genera are still being discovered with the help of next-generation sequencing technologies50,51. To investigate Malassezia cell biology initially, we employed standard yeast DNA staining protocols with DAPI and Hoechst48,52–54, using the test strain Malassezia pachydermatis (CBS 1879) and Saccharomyces cerevisiae 699 strain as control. Subsequently, the cultured cells were ethanol-fixed, washed, stained with DAPI or Hoechst, and subjected to microscopic examination. The standard staining method yielded clear nuclear visualization in the budding yeast S. cerevisiae (Fig. 1a). However, upon adhering to this procedure, a prominent stained nucleus is not observable in Malassezia, as the dye components tend to adsorb to the cell wall, resembling whole-cell staining rather than highlighting the nucleus (Fig. 1b). Repeated washing of the cells led to the removal of the whole cell staining giving a ghost-like appearance (Supplementary Fig. 1a) confirming the fact that DAPI tends to adsorb to the cell wall instead of entering into the cells, with the conventional protocol. This non-specific binding of DAPI may be attributed to its interaction with membrane lipids (phospholipids), as indicated in previous studies55. Given that phospholipids are major components of the Malassezia cell membrane56 it strengthens the understanding of why DAPI staining in Malassezia can lead to increased non-specific fluorescence. Therefore, we developed a new staining method to visualize the Malassezia nucleus involving multiple standardization steps (Supplementary Fig. 2). This revised methodology involves growing yeast cells in SD media, pelleting, and washing them with PBS and potassium phosphate solution to remove the impurities. To permeabilize the cell walls, a key step in this procedure involves incubation with Zymolyase, which facilitates cell wall disruption by enzymatically cleaving β-1,3-glucan linkages, effectively weakening the fungal cell wall structure57, along with βME, that promotes the action of Zymolyase. Then these partially permeabilized cells were washed with PBS to remove the traces of the enzyme because prolonged exposure to Zymolyase leads to damage to cellular integrity. Finally, the cells were treated with DAPI. This method partially reduced the non-specific staining compared to cells treated with the conventional protocol but prominent nucleus was still not observed (Fig. 2a). When employing the Triton X-100 solution, a non-ionic detergent, which can disrupt cell membranes, enhancing permeability and lipid dissolution58 instead of Zymolyase also led to similar results (Fig. 2b). To enhance the effect of specific staining of the nucleus, we next incubated the cells with both Zymolyase, βME, and Triton X-100 (which we termed Cell Permeabilization or CP solution) to ensure proper permeabilization of the cells (Fig. 2c) which yielded better results than the individual treatments. Further to give enhanced visualization (clarity of the nucleus against low background staining) we washed the cells with Triton X-100 twice to obtain a distinct, prominent nuclei (Fig. 3a,b). In this case, Hoechst is more prone to photobleaching (2–3 s) compared to DAPI (5–6 s). So preferably DAPI can be a better choice for observing Malassezia nucleus using this methodology.

Figure 1.

Figure 1

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Comparison of nuclear staining profiles of yeasts using traditional methodology. Nucleus as observed in (a) S. cerevisiae and (b) M. pachydermatis using the stains DAPI and Hoechst.

Figure 2.

Figure 2

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Critical elements contributing to the development of the novel methodology. DAPI staining using (a) Zymolyase, (b) Triton X-100 solution, and (c) both Zymolyase and Triton X-100 (CP solution). Treatment of the cells was carried out for 0, 1, and 2 h. The yellow highlighted box indicates the obtained distinct nucleus.

Figure 3.

Figure 3

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Nuclear staining profiles of Malassezia using the novel methodology. Nuclear staining protocol for M. pachydermatis using (a) DAPI and (b) Hoechst staining.

DAPI facilitates the visual observation of the nucleus, while an alternative DNA staining method utilizing Propidium Iodide (PI) not only aids in nuclear DNA visualization but also serves as a valuable tool for cell cycle analysis. PI-stained cell cycle analysis involves measuring the DNA content to distinguish the three main phases: G1, S, and G2/M. In G1, cells have a haploid DNA content, while the S phase is characterized by DNA synthesis and an increase in DNA content. G2/M marks the preparation for cell division with a diploid DNA content59. Through flow cytometry analysis, the distinct DNA content in each phase can be quantified by the intensity of PI fluorescence, allowing us to assess the distribution of cells across the cell cycle. In this regard, we have taken the growing culture of M. pachydermatis in SD media and an equal amount of ethanol was added to serve as a fixative for preserving the cellular structures. After this treatment, cells underwent RNase treatment to selectively degrade RNA, preventing interference with DNA fluorescence signals and ensuring accurate measurement of DNA. Subsequently, the RNase-treated cells were supplemented with PI staining to stain the DNA. This simplified method, utilizing minimal consumables, comparable to other cell cycle analysis protocols eliminates the need for additional reagents such as sodium citrate buffer and proteinase K60,61 and has demonstrated reproducibility. We also analyzed the effect of this treatment on the cell viability. While the untreated cells showed no loss in viability, the treated cells using the new protocol showed nearly 50% viability. In addition, the treated cells using this new methodology were also found to be metabolically active as assessed from the Urease, Bile esculin, and Catalase tests (Supplementary Fig. 3). This will certainly add up newer avenues to do live cell imaging studies and other downstream applications using Malasessia as a host.

Having established PI staining methodology, we proceeded to undertake a novel and unprecedented task: cell cycle arrest in Malassezia, not reported before. These investigations involve synchronizing cells in different stages of the cell cycle, enabling a more precise analysis of molecular processes and events occurring at those specific stages. We used Nocodazole which is a microtubule-depolymerizing agent, that disrupts the dynamics of microtubule assembly and disassembly acting as a toxic agent inhibiting chromosome movement. It binds to tubulin and disrupts microtubule assembly/disassembly inhibiting mitotic spindle formation and chromosome segregation44,62. Therefore, we treated M. pachydermatis with nocodazole by adding it to the growing culture and allowed the cells to go through two doubling times. S. cerevisiae was taken as a positive control, to compare with M. pachydermatis (test). Upon treatment with nocodazole, the treated cells were stained with PI stain and observed under a microscope. The cells that responded to Nocodazole treatment arrest at the G2/M phase displayed distinctive characteristics, notably large buds with stained nuclei observed under a microscope Arrest of the S. cerevisiae cells happened in 2–3 h (Fig. 4a) but it was delayed in Malassezia to 5–6 h (Fig. 4b) because of its increased doubling time of approximately 3 h. Additional confirmation of cell cycle arrest was obtained through Fluorescence-activated cell sorting (FACS) analysis, revealing a prominent peak corresponding to the G2/M phase (Fig. 4 a,b).

Figure 4.

Figure 4

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Cell cycle analysis of Malassezia. Microscopic images and FACS profiles illustrating cell cycle arrest induced by nocodazole in (a) Saccharomyces cerevisiae (asynchronous cells without nocodazole and cells treated with nocodazole for 0 h, 1.5 h, and 3 h) and (b) Malassezia pachydermatis (asynchronous cells without nocodazole and cells treated with nocodazole for 0 h, 3 h, and 6 h). Black arrows indicate large budded cells resulting from nocodazole-induced arrest.

While established methods for budding yeasts like Saccharomyces and Candida staining rely on their “permeable” cell walls, Malassezia demands a different approach since it has a lipid-rich thick cell wall (15–20%)4 compared to S. cerevisiae having ∼3% lipid content63. This novel method specifically targets the unique structure of the Malassezia cell wall and specifically stains the nuclear DNA, which remains unaffected by washing and quenching procedures (Supplementary Fig. 1b). By directly visualizing nuclear morphology and DNA content, researchers can now paint a detailed picture of the Malassezia cell cycle, unveiling the distribution of cells across different phases and identifying any potential disruptions or bottlenecks. Additionally, the ability to combine this method with DNA-damaging agents opens doors to studying the response of Malassezia to genotoxic stress and associated DNA repair mechanisms, potentially paving the way for novel antifungal strategies. Especially, by shedding light on how Malassezia regulates its cell cycle, this research provides a sturdy groundwork for unravelling the intricate molecular events that drive cell division processes. Researchers can now delve into the molecular pathways and signalling cascades that orchestrate cell cycle transitions in Malassezia. The methodology has advantage of using minimal consumables, rendering them cost-effective and accessible for laboratories with limited resources. Most importantly this methodology provides improved specificity and clarity, enhancing the reliability of research outcomes. This reliability is crucial for scientific advancements, potentially accelerating discoveries and innovations that address health challenges, improve diagnostics, and inform therapeutic interventions.

Conclusion

In summary, we have provided a DNA staining methodology specifically tailored for fungi of the Malassezia genus, and similar fungi having thick cell walls, which is a challenging task due to their unique structural barriers. To overcome these obstacles, we employed a combination of enzymatic and chemical treatments to partially permeabilize their thick cell walls without significantly compromising their viability. This allowed us to successfully introduce DAPI and Hoechst dyes into the cells, enabling precise staining of the nucleus- an achievement not previously attained. In addition to this novel staining methodology, we undertook a cell cycle analysis of Malassezia. A brief timing steps associated with the protocol is presented in Supplementary Fig. 4 and we have also provided a troubleshooting guide for the users in Supplementary Table-1. Utilizing the cell cycle arresting agent Nocodazole, we were able to halt cells at the G2/M phase, as confirmed by both microscopy and FACS analysis. This pioneering work provides researchers with a direct visualization of the Malassezia nucleus and its cell cycle, facilitating the identification of its various phases. By combining this method with DNA damage-inducing agents, we can gain valuable insights into how Malassezia responds to genotoxic stress and repairs DNA, thereby opening avenues for novel antifungal strategies. Understanding the regulatory mechanisms governing the Malassezia cell cycle would lay the groundwork for unravelling the molecular events that drive cell division. This knowledge offers opportunities to explore molecular pathways and signalling cascades involved in cell cycle transitions, potentially revealing therapeutic targets for the regulation of Malassezia and related fungi.

Supplementary Information

Supplementary Information 1. (1.6MB, jpg)
Supplementary Information 2. (3.4MB, jpg)
Supplementary Information 3. (2MB, jpg)
Supplementary Information 4. (1.6MB, jpg)
Supplementary Information 5. (16.7KB, docx)
Supplementary Information 6. (14.2KB, docx)

Author contributions

SPD conceptualized, reviewed, and approved the manuscript. JS contributed towards writing, revision, and providing critical suggestions and artwork. SL helped in FACS, revising the manuscript. BN helped in the initial standardization and review of the manuscript.

Funding

The authors would like to acknowledge funding from the Indian Council of Medical Research (ICMR) in New Delhi, INDIA for funding our research program. SPD is supported by the DHR Grant GIA/2019/000620/PRCGIA/2020-ECD II. JS is supported by a junior research fellowship from Yenepoya (Deemed to be University). The authors would like to thank Yenepoya Research Centre for the laboratory facilities and the Department of Oncopathology, Yenepoya (Deemed to be University) for the FACS.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-69024-z.

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