C. neoformans strains

All C. neoformans strains included in this study were derived from C. neoformans wild-type reference strain H99 (MAT⍺) and are listed in Table 1. The strain expressing Cdc10-mCherry (LK60) and the strain expressing Cdc3-mCherry (LK140) were generated as previously described [8]. The wild-type H99 strain was used as a negative control.

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Table 1. List of strains and plasmids utilized in this study.

https://doi.org/10.1371/journal.pone.0313444.t001

All strains expressing N-terminally tagged green fluorescent protein (GFP) were generated as previously described [8]. Strains expressing GFP-profilin and GFP-Afr1 were made by ectopic integration of plasmids, based on pLKB90 encoding GFP expressed from the GPD1 promoter. Plasmid pLK90 is identical to previously described pLK55 [40], except it has replaced mCherry-encoding sequence by GFP-encoding sequence. Genes of interest were amplified via PCR and restriction cloning was used to generate cloned plasmids. To amplify profilin ORF (CNAG_00584) to generate pSM1, the following primers were used: forward primer with NheI restriction site added at 5’ end (ACAGCTAGCCatgtcctggcaaggtac), and reverse primer with PacI restriction site added at 3’ end (CAGTGTTAATTAAgtgaagacggaactgg). To amplify AFR1 ORF (CNAG_00730) to generate pSM2, the following primers were used: forward primer with NheI restriction site added at 5’ end (TCAGCTAGCaatgtcagctgcaggcgt), and reverse primer with PacI restriction site added at 3’ end (AGTCTTAATTAAcatcgcttgagacgatcagg). The plasmids generated for this study were confirmed via sequencing (S1 and S2 Data). The integrations were conducted via biolistic transformation, as described previously [41, 42]. The positive clones were screened based on growth media containing Hygromycin 300 μg/mL. In addition, the clones were confirmed by examining the fluorescent signal.

Media and growth conditions

Unless otherwise stated, C. neoformans strains were routinely maintained in YPD medium (2% yeast extract, 1% bacto-peptone, 2% dextrose, 2% bacto-agar) at ∼25°C.

To collect samples for the MS analysis, strains LK60, LK140, and H99(⍺), were initially cultured for 24 h in 1000 mL YPD media at ∼25°C (cells grown to mid-exponential phase; OD₆₀₀ ≤ 1). Following this, each 1000 mL culture was divided into two 500 mL cultures. For analysis of cells approaching the stationary phase of growth, one of the divided cultures continued incubation in the same medium for an additional 48 h at ∼25°C. For analysis of cells at exponential phase of growth, the other 500 mL cultures were refreshed in YPD media (cell density ∼ 10⁵ cells/mL) and grown for 3 hours at ∼25°C. Next, the 500 ml culture was divided into two 250 ml cultures (cell densities ∼ 10⁷ cells/ml). One of the divided cultures was incubated at ∼25°C and the other at 37°C for 2 h. All cultures were then used to prepare cell lysates, as described in the next section.

Preparation of the cell lysates

After the final incubations (described in the previous section), the cultures representing each experimental condition were rapidly chilled using dry ice and independently centrifuged at 5,000 x g for 20 mins at 4°C. Cells were then re-suspended in 30 ml of ice-cold lysis buffer (10 mM Tris/Cl pH  =  7.5, 150 mM NaCl, 13% (v/v) glycerol, 0.5 mM EDTA) supplemented with protease inhibitor tablets (Roche cOmplete™, Mini, EDTA-free, Protease Inhibitor Cocktail, Millipore Sigma, Sigma-Aldrich) and 1 mM PMSF. The re-suspended cell pellets were aliquoted in 1 ml microcentrifuge tubes with screw caps and disrupted using ∼500 μl of 0.5 mm glass beads (Sigma-Aldrich) in a Mini-Beadbeater (Biospec Products). Samples were homogenized at 4°C for 20 sec, followed by 1 min incubations on ice. This process was repeated 8 times to ensure that a sufficient level of cell disruption had been achieved (initially confirmed by direct microscopic observation). Afterward, the lysate of disrupted cells from each condition was centrifuged at 1200 × g for 10 min at 4°C to remove large cell debris. The supernatant was subsequently collected and stored at −80°C until use.

Anti-mCherry immunoprecipitation & GeLC-MS/MS (Orbitrap Fusion)

The RFP-TRAP affinity resin coupled with RFP Nanobody/VHH from ChromoTek® agarose beads were employed for immunoprecipitation of the fluorescently tagged bait protein, as these beads are well-suited for mCherry tagged fluorescent bait proteins with no contamination concerns from heavy or light chain polypeptide chains in the elution fractions (https://www.ptglab.com/products/pictures/pdf/rfp-trap_brochure.pdf).

The lysate from each experimental condition was thawed on ice. The relative protein expression levels in the lysate were ascertained as a control as previously described [44]. For analysis of proteins associated with Cdc10-mCherry and Cdc3-mCherry, respectively, in every experimental condition, ∼5 ml of lysate was incubated for 4 h with slow end-over-end mixing at 4°C with 50 μl of ChromoTek RFP-Trap® agarose bead slurry, prepared according to manufacturer instruction. After the 4 h incubation, the ChromoTek RFP-Trap® agarose beads were washed 3× with 500 μl of wash buffer (TBS with 2 M urea, pH 7.5). Next, the wash buffer was removed and the ChromoTek RFP-Trap® agarose beads were resuspended in 100 μl of 2x electrophoresis sample buffer. The samples were then boiled for 10 min at 95°C to dissociate the immunocomplexes from the beads. Then, the eluted fraction was removed and collected via centrifugation at 2,500 × g for 2 min at 4°C. The eluted sample (supernatant) was then resolved on SDS-PAGE gel and stained with Coomassie blue. Each SDS-PAGE gel lane was sectioned into 12 segments of equal volume. Each segment was subjected to in-gel trypsin digestion as described previously [44]. Of note, the in-gel digestion included the entire gel lane and therefore did not omit the bait protein or other hypothetical proteins that might have migrated like the bait protein. Gel slices were destained in 50% methanol (Fisher), and 50 mM ammonium bicarbonate (Sigma-Aldrich), followed by a reduction in 10 mM Tris[2-carboxyethyl] phosphine (Pierce) and alkylation in 50 mM iodoacetamide (Sigma-Aldrich). Gel slices were then dehydrated in acetonitrile (Fisher), followed by the addition of 100 ng porcine sequencing grade modified trypsin (Promega) in 50 mM ammonium bicarbonate (Sigma-Aldrich) and incubation at 37°C for 12–16 h. Peptide products were then acidified in 0.1% formic acid (Pierce). Tryptic peptides were separated by reverse phase XSelect CSH C18 2.5 μm resin (Waters) on an in-line 120 x 0.075 mm column using a nanoAcquity UPLC system (Waters). Peptides were eluted using a 45 min gradient from 98:2 to 67:33 buffer A: B ratio. [Buffer A = 0.1% formic acid, 0.5% acetonitrile; buffer B = 0.1% formic acid, 99.9% acetonitrile.] Eluted peptides were ionized by electrospray (2.2 kV) followed by MS/MS analysis using higher-energy collisional dissociation (HCD) on an Orbitrap Fusion Tribrid mass spectrometer (Thermo) in top-speed data-dependent mode. MS data was acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375 to 1500 m/z. Following HCD activation, MS/MS data was acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0 [45, 46].

Data analysis

Proteins were identified using EncyclopeDIA [47] against the C. neoformans H99 UniProtKB database (Proteome ID: UP000010091) (Database last modified: February 15, 2023) [48]. Verification of MS/MS-based peptide and protein identifications was done using Scaffold DIA (Proteome Software), adhering to stringent false discovery rate criteria of 1% at both the protein and peptide levels.

The exclusive MS2 intensity data in Scaffold DIA was imported to RStudio to be analyzed by the R package proteoDA for quantitative proteomics [49]. Protein MS2 exclusive intensity values were assessed for quality using ProteiNorm [52]. The data were normalized using cyclic loess [50] and analyzed using proteoDA to perform statistical analysis using Linear Models for Microarray Data (limma) with empirical Bayes (eBayes) smoothing to the standard errors [49, 50]. Proteins with an FDR adjusted p-value < 0.05 and a fold change > 2 were considered significant. Using this method, quantitative analysis between the ∼25°C and 37°C experimental conditions was performed. This approach was employed to determine whether or not there is differential binding between these two experimental conditions for each binding category.

Heat map generation

The exclusive dataset derived from each of the five biological replicates from the bulk protein cell lysate of the wild type H99 control under three varying conditions: ∼25°C (exponential growth phase), ∼25°C (stationary growth phase), and 37°C, was subjected to analysis. A heat map was generated utilizing Heatmapper [51] to visually illustrate protein expression variations across these conditions. The heatmap was constructed employing average clustering, coupled with Kendall’s Tau distance measurement method, to accurately discern and represent the hierarchical relationships in protein expression.

Gene Set Enrichment Analysis (GSEA)

The differentially expressed proteins across the defined conditions were subjected to Gene Set Enrichment Analysis (GSEA) using the Blast2GO function within OmicsBox software to pinpoint significant gene sets correlating with various biological processes, molecular functionalities, and cellular components. This method of analysis follows the knowledge-based approach for interpreting genome-wide expression profiles as delineated by Subramanian et al. [52]. For this analysis, a False Discovery Rate (FDR) of 0.25 was employed instead of the more traditional 0.05. The rationale behind this choice lies in the exploratory nature of this study, where the primary aim is to formulate hypotheses for subsequent validation in future research. Given the intrinsic lack of coherence in most expression datasets and the relatively compact number of gene sets being examined, a more stringent FDR cutoff might lead to the omission of potentially significant results. The utilization of an FDR of 0.25 ensures a reasonable balance, allowing for the identification of valid results 3 out of 4 times, thereby broadening the scope of uncovering relevant findings. The discernment of significant gene sets elucidates the underlying biological narratives amidst the protein expression alterations, enabling a holistic understanding of the systemic impacts upon C. neoformans H99 (wild type) under differing environmental conditions.

Functional Enrichment Analysis

The identified binding partners of septins Cdc3 and Cdc10, obtained via mass spectrometry, were subjected to Functional Enrichment Analysis using the Blast2GO function within OmicsBox software. For gene enrichment analysis, Fisher’s Exact Test was employed as per the methodology elucidated by Al-Shahrour et al. [53]. This analysis facilitated the demarcation of predominant biological processes, molecular functions, and cellular components associated with these binding partners, thereby further elucidating the cellular roles and implications of septins Cdc3 and Cdc10. By exploring the enriched functional annotations and gene ontology terms, a comprehensive understanding of the functional landscape in which septins Cdc3 and Cdc10 operate was achieved, granting insights into their potential biological significance and interactions within the cellular milieu.

In Vivo co-immunoprecipitation

Cells were cultured for 24 h in 500 mL YPD media at ∼25°C. Subsequently, cell cultures were refreshed in YPD media (cell density ∼ 10⁵ cells/mL) and grown for 3 hours at ∼25°C. Next, each 500 ml culture was divided into two 250 ml cultures (cell densities ∼ 10⁷ cells/ml). One culture from each strain was incubated at ∼25°C and the other at 37°C. After a 2 h incubation, cell cultures from each experimental condition were rapidly chilled using dry ice and independently centrifuged at 5,000 x g for 20 mins at 4°C. The centrifuged cell pellets from each experimental condition were then subject to cell lysis according to the protocol utilized for the MS analysis (described above). For analysis of proteins associated with Cdc10-mCherry and Cdc3-mCherry, respectively, in every experimental condition, ∼5 ml of lysate was incubated for 4 h with slow end-over-end mixing at 4°C with 25 μl of either ChromoTek RFP-Trap®, or ChromoTek GFP-Trap® agarose bead slurry, prepared according to manufacturer manual. After the 4 h incubation, the agarose beads were washed 3× with 500 μl of wash buffer (TBS with 2 M urea, pH 7.5). Next, the wash buffer was removed, and the agarose beads were resuspended in 80 μl of 2x electrophoresis sample buffer. The samples were then boiled for 10 min at 95°C to dissociate the immunocomplexes from the beads. Then, the eluted fraction was collected via centrifugation at 2,500 × g for 2 min at 4°C. The eluted sample (supernatant) was then resolved on SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes, which were then blocked with 5% nonfat dry milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS/Tween). To detect GFP chimeras via western blotting, an anti-GFP monoclonal antibody (3H9, Chromotek®) was used at a 1:1,000 dilution and incubated overnight at 4°C, followed by HRP-conjugated Affinipure Goat Anti-Rat IgG(H+L) (SA00001-15, Proteintech®) at a dilution of 1:2000, incubated at room temperature for 1 h. To detect mCherry chimeras, an anti-RFP monoclonal antibody (6G6, Chromotek®) was used at a 1:1,000 dilution and incubated overnight at 4°C, followed by HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (SA00001-1, Proteintech®) at a dilution of 1:10000, incubated at room temperature for 1 h. Subsequently, blots were developed with Pierce™ ECL Plus Western Blotting Substrate (32132, Thermo Scientific™) and imaged using the Analytik Jena UVP Chem Studio. Raw data of western blot images related to Fig 10 are available as a supplementary file (S1 Raw images).

Microscopy

For microscopy, cells were grown overnight in YPD liquid medium at ∼25°C and refreshed the following morning using a 1:10 dilution. Cells were then counted and diluted to achieve a concentration of 1×10⁶ cells/ml. Subsequently, 200–400 μl of cell suspension was aliquoted into a microslide glass chamber (μ-Slide 2 Well Glass Bottom; Ibidi-cells in focus) for imaging. Brightfield, differential interface microscopy (DIC), and fluorescence images were captured with Leica GDSF/TIRF inverted microscope. Images were processed through ImageJ; Fiji software [54, 55].

For cells imaged at 37°C, cultures were initially grown overnight as described above. After refreshing the cultures the following morning using a 1:10 dilution, cells were grown at 37°C for two hours. Subsequently, the cultures were diluted to achieve a concentration of 1×10⁶ cells/ml. Next, 200–400 μl of cell suspension was aliquoted into a pre-heated (37°C) microslide glass chamber (μ-Slide 2 Well Glass Bottom; Ibidi-cells in focus) for imaging. Brightfield, differential interface microscopy (DIC), and fluorescence images were captured with Leica GDSF/TIRF inverted microscope with the incubator chamber set to 37°C during the entire imaging session. Images were processed as described above.

Experimental setup

We had initially hypothesized that cell cycle-related proteins would be among the candidate septin-binding partners identified in this study, given the conserved localization of septins in C. neoformans to the site of cell division [8]. We also anticipated our analysis revealing differences in the septin binding partners between S. cerevisiae and C. neoformans due to the following reasons: [1] septin proteins are essential for growth in S. cerevisiae at non-stress conditions, while they are dispensable for growth in C. neoformans only at non-stress conditions; [2] there is significant phylogenetic divergence between these two species.

According to literature based on the S. cerevisiae model and a study performed in C. neoformans, septins in C. neoformans function as a complex that consists of all four septin proteins present in C. neoformans genome (Cdc3, Cdc10, Cdc11, and Cdc12). While Cdc10 is dispensable for growth at 37°C, the remaining three septins are essential for growth at host temperature [8]. Furthermore, the septin complex does not form in the absence of Cdc3 or Cdc12 [8]. Since Cdc10 is presumably dispensable for septin complex formation, it was chosen as one of the “baits” for the pull down, as tagging this protein should have a minimal impact on septin complex functionality. Furthermore, Cdc10 was chosen since it has the highest homology to its S. cerevisiae septin counterpart.

As part of the initial study design, we planned to determine the binding partners of septin Cdc10 in the absence of Cdc3, conditions at which functional higher order septin complex does not form [8]. This approach was intended to identify Cdc10 interactions that could be independent of the formation of the whole septin complex. However, we could not successfully pull-down septin Cdc10 in the absence of Cdc3 and therefore did not follow this initial plan.

Due to septin Cdc3’s presumed critical role in septin ring homeostasis, it was chosen as a second “bait” for the tandem mass spectrometry. Identifying the interacting proteins of two septin proteins provided better insight into their functional roles and involvement in cellular pathways.

Studies in other organisms point to roles for septins that are independent of their established function during cytokinesis [34, 39, 56], and we anticipated that our findings may reveal septin interactions supporting this possibility. Specifically, we initially planned to utilize cultures grown to the stationary phase of growth, predicted to consist of mostly unbudded cells. We reasoned that unbudded cells would represent interactions with septins that are not relevant to cytokinesis. Unfortunately, we could not obtain a sufficient quantity of pulled-down fluorescently tagged septins when cells were in stationary phase. However, as the cells in the stationary phase cultures are expected to be undergoing starvation due to limited nutrient availability, we nonetheless utilized those samples to test the impact of nutrient deprivation stress on the whole C. neoformans proteome.

The bulk protein lysate from each sample was analyzed via mass spectrometry for the following two reasons: 1. To compare the whole proteome of C. neoformans grown at no-stress conditions with the proteome of cultures grown under high temperature stress or nutrient deprivation stress, 2. To provide a background control of protein levels for differential analysis of proteins associated with the septins. Consequently, label-free proteomic profiling of the differentially expressed proteins in cells exposed to heat stress (37°C) and nutrient starvation stress (stationary phase) as compared to no-stress control was performed and those data served also as a control for the analysis of the septin-associated proteome.

A schematic representation of the the GeLC-MS/MS workflow is depicted in Fig 1. A total of 9 experimental samples (Table 2), and 9 control samples, were collected for mass spectrometry analysis (Fig 2). The control samples were the bulk protein lysate (input) of each experimental pulldown condition. The following two comparisons were performed to gain information regarding the differentially expressed proteome of C. neoformans strain H99 (WT) during the exposure to two stress conditions: [1] Comparison of the bulk protein cell lysate obtained from cells grown exponentially at ∼25°C vs. the lysate obtained from cells incubated at 37°C (Heat Stress); [2] Comparison of the bulk protein cell lysate obtained from cells grown exponentially at ∼25°C vs. the lysate obtained from stationary growth phase culture grown at ∼25°C (Nutrient deprivation stress).

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Fig 1. Schematic representation of the GeLC-MS/MS workflow.

(A) C. neoformans cells were grown for 24 h in YPD rich media at ∼25°C. Subsequently, the cell culture was split, and half of the culture was further grown in the same media for an additional 48 h at ∼25°C (to ensure it approached the stationary phase of growth (SP)), while the other half was refreshed in YPD media and grown for 3 h at ∼25°C (to assure it was at an exponential phase of growth (EP)). Each EP culture was then divided into two cultures and grown either at ∼25 or at 37°C for 2 h before harvesting the cells. (B) Cell lysis and immunoprecipitation of bait protein using RFP-TRAP resin. Bound proteins were eluted and resolved on SDS-PAGE gel. (C) Each lane of the SDS-PAGE gel was excised into twelve fragments and subjected to in-gel trypsin digestion and analyzed by in-gel liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer (Thermo). Raw data was processed with Scaffold DIA.

https://doi.org/10.1371/journal.pone.0313444.g001

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Fig 2. Schematic representation of samples that were analyzed via label-free quantitative mass spectrometry.

The bulk protein cell lysate and subsequent co-immunoprecipitation elution fractions from Cdc3-mCherry and Cdc10-mCherry pulldowns of the following strains were analyzed via mass spec: H99 (WT), Cdc10-mCherry, and Cdc3-mCherry. The experiment was conducted under three independent conditions (N = 5): [1] exponential growth phase at ∼25°C (Ambient Temperature), [2] stationary growth phase at ∼25°C (Nutrient deprivation stress), and [3] exponential growth phase at 37°C (Heat Stress).

https://doi.org/10.1371/journal.pone.0313444.g002

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Table 2. Samples collected for the subsequent MS analysis (N = 5).

https://doi.org/10.1371/journal.pone.0313444.t002

Global proteomic profiling: Changes to C. neoformans protein levels due to low nutrient availability

The analysis of the bulk protein lysates from all the samples served as a background protein content in the input fractions that was critical in evaluating the septin interactome. The analysis of the bulk protein cell lysates from the wildtype strain H99 allowed to compare C. neoformans proteome between no stress and two stress conditions, nutrient deprivation, and high temperature. This analysis revealed that the proteome is differentially expressed both during heat stress at 37°C and during nutrient starvation associated with the stationary phase growth (Fig 3A). Changes to protein abundance with a log₂FC >2 or <-2 (with adjusted p-value of 0.001) were considered significantly upregulated or downregulated, respectively.

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Fig 3. Heat stress and starvation stress associated proteome changes in C. neoformans revealed by label-free quantitative proteomics.

(A) Heat map representation of differentially expressed proteins across the following conditions: ∼25°C during the exponential growth phase (control), ∼25°C during the stationary growth phase (starvation stress), and 37°C during the exponential growth phase (heat stress). The Log2 Cyclic Loess Normalized Exclusive Intensities of each protein per sample were transformed to Z-scores. Clustering was performed both on proteins and samples using an average linkage method and Kendall’s Tau distance measurement method. Upregulated and downregulated proteins are represented with red and blue colors, respectively. (B) Top: Volcano plot displaying results of differential expression analysis comparing proteome of control (∼25°C during exponential growth phase), and nutrient starvation stress (∼25°C during stationary growth phase) proteome. Bottom: Volcano plot displaying results of differential expression analysis comparing proteome of control (∼25°C during exponential growth phase), and heat stress (37°C during exponential growth phase) proteome. The dashed vertical lines differentiate the upregulated and downregulated proteins (absolute value FC > 2). The proteins above the dashed horizontal line represent the statistically significant proteins (BH-adjusted p < 0.001). Proteins highlighted in red are significantly upregulated, and proteins highlighted in blue are significantly downregulated.

https://doi.org/10.1371/journal.pone.0313444.g003

Differential expression analysis revealed 413 significantly upregulated and 480 significantly downregulated proteins during the stationary versus exponential growth phase (Fig 3B) (Table 3). Gene set enrichment analysis (GSEA) demonstrated that glucose homeostasis, antibiotic catabolic process, fermentation, response to host defense, fatty acid catabolic process, lysosomal autophagy, and phosphatidylcholine biosynthetic process were among the biological processes that were positively enriched during stationary phase growth when compared to exponential culture (Fig 4A and 4B). In contrast, the biological process of tRNA modification had a negative enrichment score indicating that protein synthesis might be decreased during the stationary growth phase. Consistent with this possibility, the pre-ribosome was one of the cellular compartments with a negative enrichment score during stationary phase growth. In contrast, the extracellular region and ascospore wall compartments were among the cellular compartments with a positive enrichment score. This might be indicative of the importance of cell wall integrity during stress response (Fig 4A and 4B). Likewise, the claim that protein synthesis-related pathways might be suppressed during stationary phase growth is further supported by the significant negative enrichment score for the molecular function of catalytic activity on RNA and nucleic acid. Nonetheless, the catalytic activity on proteins, specifically exopeptidase activity was a positively enriched molecular function. This could indicate that protein synthesis is not completely compromised due to downregulation in tRNA modification proteins. Additionally, antioxidant activity, oxidoreductase activity, and transporter activity were also among the molecular functions with positive enrichment in stationary phase stressed cells (Fig 4A and 4B).

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Fig 4. Gene set enrichment analysis (GSEA) of differentially expressed proteome in stationary growth phase versus exponential growth phase.

(A) Bar plot for significantly enriched gene ontology (GO) categories with p-value < 0.05 and FDR < 0.25 from GSEA. To avoid overlooking potentially significant gene ontology terms, an FDR of 0.25 rather than 0.05 was used for GSEA. (B) Directed acyclic graph of enriched GO terms with GSEA. The GO terms with positive enrichment are highlighted in red; while the GO terms with negative enrichment are highlighted in blue.

https://doi.org/10.1371/journal.pone.0313444.g004

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Table 3. Differentially expressed proteins in C. neoformans WT (H99) cultures exposed to either heat stress or nutrient deprivation.

(The top 30 candidates of each condition are summarized in this table, the complete dataset can be found in the S3 Data).

https://doi.org/10.1371/journal.pone.0313444.t003

Network analysis via STRING [57] on upregulated and downregulated proteins during the stationary phase identified significant enrichment in metabolic pathways such as biosynthesis of secondary metabolites, pentose phosphate pathway, oxidative phosphorylation, fatty acid degradation, fructose and mannose metabolism, glycolysis/gluconeogenesis, methane metabolism, and citrate cycle (TCA cycle) among upregulated proteins. No significantly enriched KEGG pathways were found for downregulated proteins, though the septin ring and septin complex cellular components were notably downregulated (S1 Fig in S1 File).

Global proteomic profiling: Changes to C. neoformans protein levels due to heat stress

The differential expression analysis comparing the proteomes identified 396 proteins as significantly upregulated and 478 proteins as significantly downregulated after wild type exponential growth culture, initially grown at ∼25°C, was exposed to 37°C heat stress for two hours (Fig 3B) (Table 3). Gene Set Enrichment Analysis (GSEA) was conducted to identify the gene ontology terms significantly enriched during heat stress in C. neoformans. The GSEA revealed significant positive enrichment in the following biological processes: oxidative phosphorylation, mitochondrial ATP synthesis coupled transport, purine nucleobase metabolic process, and respiratory electron transport chain (Fig 5A and 5B). In contrast, biological processes such as autophagosome assembly/organization and cell growth exhibited significantly negative enrichment (Fig 5A and 5B). Additionally, cellular components like the mitochondria and respirasome demonstrated a positive enrichment score, whereas the Golgi apparatus was the only cellular compartment with a negative enrichment score. Among molecular functions, ATP-dependent activity, ATP hydrolysis, and pyrophosphatase activity were negatively enriched, while isomerase activity and oxidoreductase activity were positively enriched (Fig 5A and 5B).

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Fig 5. Gene set enrichment analysis (GSEA) of differentially expressed proteome at 37°C (heat stress) vs. ∼25°C (ambient temperature).

(A) Bar plot illustrating significantly enriched gene ontology (GO) categories with p-value < 0.05 and FDR < 0.25 from GSEA. To account for the potential overlook of significant gene ontology terms, an FDR of 0.25 rather than 0.05 was used for GSEA. (B) Directed acyclic graph of enriched GO terms from GSEA, with positive enrichment are highlighted in red, while the GO terms with negative enrichment are highlighted in blue.

https://doi.org/10.1371/journal.pone.0313444.g005

A STRING network analysis was performed on the significantly upregulated and downregulated proteins during heat stress, independently. This analysis revealed that upregulated proteins were enriched in metabolic pathways including purine metabolism, carbon metabolism, glycolysis/gluconeogenesis, and biosynthesis of amino acids. Conversely, downregulated proteins were significantly enriched in the pathway of protein processing in the endoplasmic reticulum (S2 Fig in S1 File). Notably, the STRING analysis also suggested that septin complex is significantly downregulated at 37°C (S2 Fig in S1 File).

Comparing the heat stress and stationary growth phase (nutrient starvation) proteomes unveiled 162 overlapping upregulated proteins (S3A Fig in S1 File) and 255 overlapping downregulated proteins (S4A Fig in S1 File). A subsequent STRING network analysis on both sets of shared proteins revealed significant enrichment for RNA processing, ribosome biogenesis, cellular nitrogen compound metabolic process, mating projection, septin complex, and septin ring among the shared downregulated proteins (S4B Fig in S1 File). In contrast, shared upregulated proteins were significantly enriched for the biosynthesis of amino acids, citrate cycle (TCA), glycolysis/gluconeogenesis, and 2-Oxocarboxylic acid metabolism (S3B Fig in S1 File). Furthermore, the STRING-generated protein-protein interaction network for the shared upregulated proteins was significantly enriched (p-value: 0.000168), indicating a higher degree of interactions than expected from a random set of proteins. Collectively, these data outline the significantly upregulated and downregulated pathways during stress response in C. neoformans. Given the objective of this study to profile the protein interactome of Cdc3 and Cdc10, the significant enrichment of the septin complex & septin ring among the shared downregulated proteins warrants further attention.

Septin interactome analysis: Identification of septin Cdc10 protein interactome via co-IP-MS

In this study, the interacting partners of septin proteins Cdc10 and Cdc3 were identified. Utilizing bait proteins with the mCherry fluorescent tag, immunoprecipitation was carried out using RFP-Trap agarose beads (Chromotek). The proteins extracted were then eluted and subjected to GeLC-MS/MS analysis, facilitating the identification of the bait protein (in this case, Cdc10 and Cdc3) and potential interacting partners via mass spectrometry. However, the co-IP-MS approach using a tagged protein as bait could potentially yield false positives. To mitigate this risk, an established practice is to employ a protein extract derived from a wild type strain that does not express the tagged bait protein, serving as a negative control [58, 59]. Only proteins exhibiting a logFC >2 and an adjusted p-value of <0.05 were considered sufficiently enriched when comparing the protein extract containing the tagged bait septin protein against the control. However, only those with a logFC >2 and an adjusted p-value of <0.001 were regarded as top candidates.

At 25°C, 51 proteins were identified as significantly enriched in Cdc10-mCherry-expressing strain over the negative control (Table 4) (Fig 6A). Additionally, 22 proteins were deemed significantly enriched in Cdc10-mCherry-expressing strain over the negative control at 37°C (heat stress) (Table 5) (Fig 6A). However, significant enrichment of septin Cdc10 was unattainable in stationary growth phase samples. Although the protein Cdc10 was among the peptides identified in the pulldown (S3 Data), statistical analysis via a t-test did not validate Cdc10 as significantly enriched when compared to the negative control (S5 Fig in S1 File). In contrast, the entire septin complex was identified in the Cdc10 pulldown at both 25 and 37°C from exponentially grown cultures, which provided a strong validation for our approach (Fig 6A).

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Fig 6. Characterization of Cdc10-mCherry interactome at ∼25°C and 37°C (heat stress).

(A) Volcano plots of protein interactome results show proteins that are significantly enriched by Cdc10-mCherry co-immunoprecipitation from samples grown at ∼25 and 37°C, respectively. Significant interacting partners were determined by statistical t-test using logFC >2 and FDR of 0.05. The most significant candidates (highlighted) were considered those with a logFC >2 and FDR of 0.001. (B) Dot plots of functional enrichment analysis of septin Cdc10 protein interactome obtained from both temperature conditions using Fisher’s Exact Test. Gene count refers to the number of genes enriched in a GO term, and gene ratio is the percentage of total differential expressed genes in the given GO term.

https://doi.org/10.1371/journal.pone.0313444.g006

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Table 4. List of significantly enriched (logFC >2 and adjusted p-value of <0.001) septin Cdc10 interacting partners based on samples incubated at ∼25°C (The significant binding partners list with logFC >2 and adjusted p-value of <0.05 can be found in S1 Table).

https://doi.org/10.1371/journal.pone.0313444.t004

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Table 5. List of significantly enriched (logFC >2 and adjusted p-value of <0.001) septin Cdc10 interacting partners based on samples incubated at 37°C (The significant binding partners list with logFC >2 and adjusted p-value of <0.05 can be found in S2 Table).

https://doi.org/10.1371/journal.pone.0313444.t005

Functional enrichment analysis of Cdc10’s interactome (candidates with logFC >2 and FDR <0.001) spotlighted septin filament array, septin assembly, and molecular adaptor activity as some of the GO terms with the highest gene ratio and gene count (Fig 6B). Other notable GO terms identified at ∼25°C for the Cdc10 interactome included structural constituent of the cytoskeleton, Gin4 complex, phosphatidylinositol-4,5-phosphate binding, and cyclin-dependent protein serine/threonine kinase activator activity (Fig 6B). Expectedly, functional enrichment of Cdc10’s interactome during heat stress exhibited common elements with the ambient temperature interactome such as septin ring assembly, septin filament array, Gin4 complex, phosphatidylinositol-4,5-phosphate binding, and molecular adaptor activity (Fig 6B). However, the analysis also uncovered GO terms specific to Cdc10’s interactome during heat stress: negative regulation of TORC1 signaling, negative regulation of organelle organization, positive regulation of glutamate-cysteine ligase activity, establishment or maintenance of microtubule cytoskeleton polarity, and regulation of nitrogen compound metabolic process (Fig 6B).

A comparison of Cdc10’s interactome between ambient temperature and heat stress (candidates with logFC >2 and FDR <0.001) identified 11 proteins common to both conditions (S6A Fig in S1 File). Further, the STRING protein-protein interaction (PPI) network analysis of the shared interacting proteins of Cdc10 during ambient temperature and heat stress displayed a high enrichment score (p-value: 0.00035), indicating more interactions than expected from a random protein set (S6B Fig in S1 File). Among the enriched gene ontology terms in the STRING network were septin ring organization, cytoskeleton-dependent cytokinesis, molecular adaptor activity, cell division site, and cell cortex.

Septin interactome analysis: Identification of septin Cdc3 protein interactome via co-IP-MS

Mass spectrometry (MS) analysis identified 135 proteins significantly enriched in the Cdc3-mCherry pulldown from samples grown at ∼25°C (Table 6) (Fig 7A), and 29 significantly enriched proteins during heat stress (37°C) over the negative control (Table 7) (Fig 7A). Similar to the Cdc10 pulldown during stationary phase, the interactome of Cdc3 was not significantly identifiable in the stationary growth phase. The peptide corresponding to Cdc3 was identified in the Cdc3-mCherry co-immunoprecipitation (S3 Data); however, a statistical t-test analysis did not show significant enrichment of septin Cdc3 in the sample (S7 Fig in S1 File). Reassuringly, the entire septin complex was identified in the Cdc3-mCherry pulldown and subsequent MS analysis from exponentially grown cultures from both ∼25 and 37°C (Fig 7A) (Table 7).

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Fig 7. Characterization of Cdc3-mCherry interactome at ∼25°C and 37°C (heat stress).

(A) Volcano plots of protein interactome results showing proteins that are significantly enriched by Cdc3-mCherry co-immunoprecipitation during ambient temperature and heat stress, respectively. Significant interacting partners were determined by statistical t-test using logFC >2 and FDR of 0.05. The most significant candidates (highlighted) were considered those with a logFC >2 and FDR of 0.001. (B) Dot plots of functional enrichment analysis of septin Cdc3 protein interactome during ambient temperature and heat stress, respectively, using Fisher’s Exact Test. Gene count refers to the number of genes enriched in a GO term, and gene ratio is the percentage of total differential expressed genes in the given GO term.

https://doi.org/10.1371/journal.pone.0313444.g007

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Table 6. List of significantly enriched (logFC >2 and adjusted p-value of <0.001) septin Cdc3 interacting partners based on samples incubated at ∼25°C.

(The significant binding partners list with logFC >2 and adjusted p-value of <0.05 can be found in S3 Table).

https://doi.org/10.1371/journal.pone.0313444.t006

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Table 7. List of significantly enriched (logFC >2 and adjusted p-value of <0.001) septin Cdc3 interacting partners based on samples incubated at 37°C.

(The significant binding partners list with logFC >2 and adjusted p-value of <0.05 can be found in S4 Table).

https://doi.org/10.1371/journal.pone.0313444.t007

Functional enrichment of Cdc3’s interactome (candidates with logFC >2 and FDR <0.001) at ∼25°C revealed the following GO terms with the highest gene ratio and gene count: septin complex, septin cytoskeleton, cell cortex, microtubule cytoskeleton, and cell division (Fig 7B). Additionally, GO terms such as phosphatidylinositol phosphate binding, heterocycle catabolic process, and proton-transporting V-type ATPase, V1 domain, were also identified for Cdc3’s interactome at ∼25°C (Fig 7B).

During heat stress (37°C), functional enrichment for Cdc3’s proteome (candidates with logFC >2 and FDR <0.001) uncovered GO terms with the highest gene count and gene ratio including septin complex, Gin4 complex, molecular adaptor activity, GTP binding, GTPase activity, DASH complex, microtubule, and phosphatidylinositol-5-phosphate binding (Fig 7B). Noteworthy among the identified GO terms for Cdc3’s proteome during heat stress were the following: negative regulation of TORC1 signaling, negative regulation of organelle organization, positive regulation of glutamate-cysteine ligase activity, and mRNA processing (Fig 7B).

Comparison of Cdc3’s interactome (candidates with logFC >2 and FDR <0.001) from samples incubated at ∼25°C and those from 37°C identified 18 proteins common to both (S8A Fig in S1 File). These overlapping proteins reassuringly included the remaining 3 septins. A STRING PPI network analysis was conducted on these eighteen candidates, revealing gene ontology enrichments in the categories of cytoskeleton-dependent cytokinesis, mitotic cytokinesis, cell division, GTPase activity, septin complex, cell cortex, and microtubule cytoskeleton. Moreover, the interaction network showcased a significant enrichment (p-value: 0.00459), indicating a higher interaction level within the network than what would be expected from a random protein set (S8B Fig in S1 File).

Characterizing the septin-complex interactome in C. neoformans: Comparison of Cdc3’s and Cdc10’s binding partners

To elucidate the common proteins between the Cdc10 and Cdc3 septin proteins interactome, a comparative analysis was performed on the significantly enriched proteins for both Cdc10 and Cdc3 at ambient temperature (∼25°C) and heat stress conditions (37°C), with a log2 fold change (logFC) greater than 2 and an adjusted p-value less than 0.05. The adjusted p-value cutoff was relaxed from 0.001 to 0.05 to ensure potential significant candidates were not overlooked. At ambient temperature (∼25°C), 112 proteins were identified as significantly enriched for septin Cdc10 (S1 Table), while 46 proteins were significantly enriched during heat stress (37°C) (S2 Table). The gene set functional enrichment analysis on the STRING PPI network for the 112 identified candidate binding partners for Cdc10 at ambient temperature highlighted several significantly enriched cellular components: septin complex, septin ring, cytoplasm, and intracellular anatomical structure (S9 Fig in S1 File).

Although the list of significantly enriched candidate binding partners for Cdc10 during heat stress is smaller in quantity than at ambient temperature (S1 and S2 Tables), the STRING PPI network’s gene set functional enrichment analysis for the 46 proteins that interact with Cdc10 during heat stress revealed a broader range of identified gene ontology terms (S10 Fig in S1 File). The biological processes significantly enriched for Cdc10’s interactome during heat stress include cytoskeleton-dependent cytokinesis and mitotic cytokinesis, with microtubule plus-end binding being the enriched molecular function. The enriched cellular components were septin complex, septin ring, spindle, microtubule cytoskeleton, cell cortex, and intracellular anatomical structure (S10 Fig in S1 File).

For septin Cdc3’s interactome (logFC >2 and adjusted p-value of <0.05), 196 proteins were identified as significantly enriched at ambient temperature (S3 Table), and 40 proteins during heat stress (S4 Table). The gene set functional enrichment analysis for the 196 proteins associated with Cdc3 at ambient temperature revealed septin complex, cytoplasm, intracellular anatomical structure, and cellular anatomical entity as the enriched cellular compartments (S11 Fig in S1 File). Furthermore, enzyme and protein binding were among the molecular functions enriched for Cdc3’s interactome at ambient temperature (S11 Fig in S1 File).

Similar to Cdc10’s interactome at 37°C (heat stress), the number of candidate binding partners of Cdc3 was smaller for heat stress than ambient temperature (S3 and S4 Tables). However, the STRING PPI network’ gene set functional enrichment analysis for Cdc3’s heat stress interactome showcased a wider range of gene ontology terms. The enriched cellular components for Cdc3’s interactome at 37°C include septin complex, septin ring, microtubule cytoskeleton, cell cortex, and ribonucleoprotein complex (S12 Fig in S1 File). Additionally, mRNA processing, cytoskeleton-dependent cytokinesis, and mitotic cytokinesis were significantly enriched biological processes for Cdc3’s interactome during heat stress (S12 Fig in S1 File).

The functional enrichment analyses of Cdc10’s and Cdc3’s independent interactomes during both ambient temperature and heat stress conditions offered further insights into the common pathways both septins might engage in. A total of 14 proteins were consistently identified for both septins at both temperature conditions (Fig 8).

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Fig 8. Overlap between Cdc10 and Cdc3 protein interactome.

Significant interacting partners were determined by statistical t-test using logFC >2 and adjusted p-value of 0.05. Venn Diagram showing the overlap of significantly enriched proteins for co-immunoprecipitation of both Cdc3-mCherry and Cdc10-mCherry between ambient temperature (∼25°C) and heat stress (37°C).

https://doi.org/10.1371/journal.pone.0313444.g008

Furthermore, 68 proteins were identified as interacting with both septin Cdc3 and Cdc10 exclusively at 25°C (Fig 8), whereas 9 proteins were found to interact with Cdc10 and Cdc3 exclusively at 37°C (Fig 8). To delve into the interactions of both Cdc3 and Cdc10 in the context of the septin complex, a functional enrichment analysis employing Fisher’s exact test was conducted on all proteins identified as consistently interacting with both septins at 25°C. Hence, the 68 proteins (exclusive to ambient temperature), and the 14 proteins identified across all ambient temperature and heat stress conditions for both septins were regarded as part of the ambient temperature septin complex interactome (Fig 8). The inclusion of the set of 14 proteins in this analysis is justified as these peptides have indeed been identified during ambient temperature growth for both septin Cdc10 and Cdc3. Functional enrichment analysis utilizing Fisher’s exact test for the both Cdc3 and Cdc10 associated proteome at ambient temperature revealed the septin complex, septin ring, cell division site, microtubule cytoskeleton, cytoskeleton-dependent cytokinesis, mRNA polyadenylation, and double-strand break repair as the enriched GO terms for ambient temperature (Fig 9A). Furthermore, the following enriched KEGG pathways for the septin interactome at ambient temperature were the following: cell cycle, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, longevity regulating pathway, base excision repair, and the MPAK signaling pathway (Fig 9B).

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Fig 9. Septin Cdc3 and Cdc10 shared protein enrichment analysis.

(A) Dot plots of functional enrichment analysis of septin Cdc10 and Cdc3 shared protein interactome during ambient temperature (∼25°C) and heat stress (37°C) using Fisher’s Exact Test. (B) Dot plots of KEGG pathway functional analysis of septin Cdc10 and Cdc3 shared protein interactome during ambient temperature and heat stress. Gene count refers to the number of genes enriched in a GO term, and gene ratio is the percentage of total differential expressed genes in the given GO term.

https://doi.org/10.1371/journal.pone.0313444.g009

A total of 25 proteins were identified as part of the septin interactome at 37°C (Fig 8). Out of the 25 proteins, 14 are consistently expressed across both temperature conditions (as described above) and 9 are exclusively enriched at 37°C. Consequently, the 9 proteins (exclusive to 37°C), and the 14 proteins identified across all ambient temperature and heat stress conditions for both septins were regarded as part of the heat stress septin complex interactome (Fig 8). In addition, functional enrichment analysis utilizing Fisher’s exact test for both Cdc3 and Cdc10 associated proteome during heat stress revealed the following enriched GO terms: cell division, microtubule cytoskeleton, cell cycle, septin complex, septin ring, nuclear protein-containing complex, negative regulation of TORC1 signaling, and negative regulation of organelle organization (Fig 9A). Furthermore, the following KEGG pathway were among the significantly enriched in the septin Cdc3 and Cdc10 shared interactome during heat stress: regulation of actin cytoskeleton, nucleocytoplasmic transport, thiamine metabolism, cell cycle, purine metabolism, Rap1 signaling, and MAPK signaling pathway (Fig 9B).

The STRING PPI network for the significantly enriched binding partners of septins across all conditions displayed a significant enrichment value (S13B Fig in S1 File), indicating a higher level of interactions than expected from a random protein set. The two identified local STRING clusters for Cdc3 and Cdc10 during growth at ∼25°C and heat stress were the septin complex & cell cortex, and MAPK signaling pathway-yeast (S13B Fig in S1 File).

Cdc10 associates with the actin-binding protein profilin

One of the most significant binding partners identified in this study is profilin (CNAG_00584). profilin was identified as associated with both Cdc3 and Cdc10 at both ∼25 and 37°C (Figs 6 and 7) (Tables 4–7). Profilin binds to actin, a key component of the cytoskeleton, and regulates its polymerization and depolymerization. In higher eukaryotes, this allows cells to undergo shape changes and move in response to external signals [60].

To validate the mass spectrometry data, and confirm the association of Cdc10 with profilin, GFP-profilin was co-expressed with Cdc10-mCherry in C. neoformans, and the Cdc10-mCherry was precipitated with RFP-Trap resin. GFP-profilin was efficiently co-precipitated from cells that also expressed Cdc10-mCherry; importantly, the GFP-profilin did not show affinity to the RFP-Trap resin (Fig 10A and 10B). In addition, GFP-profilin was precipitated with GFP-Trap resin from cells expressing Cdc10-mCherry, and Cdc10-mCherry was successfully co-precipitated (Fig 10B).

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Fig 10. Co-immunoprecipitation and fluorescence microscopy of septins Cdc3 and Cdc10 and their interacting partners profilin and AFR1.

A co-IP of the Cdc10-mCherry using the GFP-Trap resin and a co-IP of the GFP-profilin using the RFP-Trap resin, at (A) 25°C and (B) heat stress (37°C). A co-IP of the Cdc3-mCherry using the GFP-Trap resin and a co-IP of the GFP-AFR1 using the RFP-Trap resin, at (C) 25°C and (D) heat stress (37°C). Strain that expresses only Cdc10-mCherry or Cdc3-mcherry, and strains that express only GFP-tagged interacting proteins were used as negative controls. The membranes were initially probed with an anti-GFP antibody and imaged and subsequently membranes were stripped and probed with an anti-RFP antibody to detect the precipitated GFP-tagged and mCherry-tagged proteins, respectively. (E) Cells expressing GFP-profilin and Cdc10-mCherry were visualized via live fluorescent microscopy from a culture grown at ∼25°C and after 2 hours of incubation at 37°C. GFP-profilin doesn’t show co-localization with septin Cdc10-mCherry. (F) Cells expressing GFP-Afr1 and Cdc3-mCherry were visualized via live fluorescent microscopy from a culture grown at ∼25°C and after 2 hours of incubation at 37°C. GFP-AFR1 does not colocalize with Cdc3-mCherry at 25°C; however, it co-localizes partially during high temperature stress.

https://doi.org/10.1371/journal.pone.0313444.g010

Live fluorescent microscopy revealed that at ∼25°C, Cdc10-mCherry localized to the mother-bud neck, as expected, whereas GFP-profilin fluorescence was mostly cytoplasmic (Fig 10E). At 37°C, while GFP-profilin remained cytoplasmic, the Cdc10-mCherry formed punctate localization (in addition to its localization at the mother-bud neck) that likely corresponded to the plasma membrane (Fig 10E). Although there was no evident co-localization observed for GFP-profilin and Cdc10-mCherry, the association of Cdc10 with profilin detected via co-immunoprecipitation was independent of growth temperature, which is in accord with the mass spectrometry data (Fig 10A, 10B and 10E). It is also worth noting that profilin (CNAG_00584) was among the proteins that were identified as significantly upregulated with a LogFC of 3.446 during heat stress when compared to ∼25°C (S3 Data). Consequently, we hypothesize that septins possibly contribute to heat stress response via their association with profilin and the actin-cytoskeleton. Furthermore, the microtubule cytoskeleton was one of the enriched gene ontology terms in the functional enrichment conducted on the shared interactome of Cdc3 and Cdc10 both at ambient temperature (∼25°C), and during heat stress (37°C) (Figs 6 and 7).

Cdc3 associates with the ATP-binding cassette transporter Afr1, and partially colocalizes with Afr1 during heat stress

The mass spectrometry data revealed both septin proteins Cdc3 and Cdc10 associate with Afr1 (CNAG_00730) exclusively during heat stress (37°C) (Figs 6 and 7) (Tables 4–7). AFR1, which stands for Antifungal Resistance 1, encodes an ATP-binding cassette (ABC) transporter protein that is likely associated with the plasma membrane in C. neoformans [61, 62].

To validate the mass spectrometry data and confirm the association of Cdc3 with Afr1, GFP-Afr1 was co-expressed with Cdc3-mCherry, and the Cdc3-mCherry was precipitated with RFP-Trap resin. GFP-Afr1 was successfully co-precipitated from cells that also expressed Cdc3-mCherry when cells were incubated at 37°C but not from cultures incubated at ∼25°C. In addition, the GFP chimera did not show affinity to the RFP-Trap resin (Fig 10C and 10D). Reassuringly, GFP-Afr1 was precipitated from cells expressing Cdc3-mCherry with GFP-Trap resin, and Cdc3-mCherry was successfully co-precipitated when cells were grown at 37°C but no association was detected when cells were grown at ∼25°C. (Fig 10C and 10D).

Live fluorescent microscopy revealed that at ∼25°C, Cdc3-mCherry localized to the mother-bud neck, as expected, whereas GFP-Afr1 fluorescence was mostly visible as a rim of the cells likely corresponding to the predicted plasma membrane localization (Fig 10F). At ∼25°C, there was no evident co-localization observed for GFP-Afr1 and Cdc3-mCherry. At 37°C, while GFP-Afr1 remained localized to the rim of the cell, likely corresponding to the plasma membrane, the Cdc3-mCherry formed punctate localization that partially overlapped with localization of GFP-Afr1 (Fig 10F). Thus, the partial co-localization between GFP-Afr1 and Cdc3-mCherry detected at 37°C is consistent with the association of Cdc3-mCherry with GFP-Afr1 that was detected both via co-immunoprecipitation and mass spectrometry exclusively at 37°C (Fig 10C, 10D and 10F).

Uncovering septin interactions in C. neoformans

The elution fractions based on the strain H99 provided an avenue to control for potential false positives in the analysis of septin-binding partners; the proteins that bind to the ChromoTek® agarose beads RFP-TRAP (affinity resin coupled with RFP Nanobody/VHH) in these control pulldowns were considered contaminants. Thus, a comparison of the peptides detected in the elution fraction of the strain H99 and the elution fraction that was based on the strains expressing mCherry tagged septin proteins provided a comprehensive list of proteins that represented candidate binding partners of septins. Arguably, the more stringent control could have been a strain that expresses the mCherry protein, which would have accounted for those proteins that have affinity to this fluorescent tag. In that regard, proteins identified as binding partners of only one of the septins (either Cdc3-mCherry or Cdc10-mCherry) may be considered most likely physiologically relevant. Nonetheless, the inclusion of 5 biological replicates and the correction of the results by the relative abundance of the candidate proteins in the cell lysate (the enrichment in the pulldown versus abundance in the protein lysate) provided a robust list of bona fide septin interactors with high confidence level.

Our investigation into septins protein interactome elucidates their fundamental role as scaffolding entities, orchestrating a plethora of cellular processes essential for maintaining cellular homeostasis. These processes include cell division, membrane trafficking, and cellular signaling [12]. A notable validation of our findings was attained through the successful elution of the entire septin complex during both Cdc3-mCherry and Cdc10-mCherry pulldown assays at both ambient temperature (∼25°C) and heat stress (37°) conditions. However, we were not able to effectively pull down the bait proteins from the stationary phase cultures, suggesting a phase-specific modulation of septin complex architecture and dynamics. This deviation underscores the necessity for further examination into the spatiotemporal dynamics governing septin assembly and function across various environmental conditions.

Association of septins with profilin

Profilin was a significant and confirmed interacting partner for the shared interactome of Cdc3 and Cdc10 during both ambient temperature (∼25°C) and heat stress (37°C). The role of profilin during thermal stress is well documented. Polymerized actin (F-actin) is inherently unstable at high temperatures, and profilin helps maintain actin in its filamentous state during heat stress, possibly by stimulating actin polymerization in a proper spatiotemporal manner [66].

The association of septin Cdc3 and Cdc10 with profilin during both ambient temperature (∼25°C) and heat stress (37°C) conditions implicates the septin complex might have a role in heat stress response in C. neoformans by contributing to F-actin stability. This is further supported by our finding that profilin was among the proteins identified as significantly upregulated, with a LogFC of 3.446, at 37°C (S3 Data). Moreover, Cdc3 and Cdc10’s association with profilin might link the septin complex to more than one stress response pathway, as discussed below.

Profilin helps protect cells from thermal damage by stabilizing the cytoskeleton and preventing protein denaturation. In S. cerevisiae, profilin plays a significant role in maintaining normal levels of F-actin, especially in response to shifts to high temperature [66]. However, profilin is not essential for maintaining F-actin concentrations during steady-state growth at room temperature [66]. These findings may provide an explanation as to why in C. neoformans Cdc3 and Cdc12 are only essential during stress, including growth at 37°C.

In addition to actin, profilin interacts with other proteins, such as integrins and growth factors, to coordinate cell signaling and behavior [67]. Profilin also binds to membrane phosphoinositides such as P­I(3,4,5)P­3 and P­I(4,5)P­2, which inhibits profilin’s interactions with G-actin and proline-rich stretches [68–72]. Association of profilin with membrane phosphoinositides has been proposed to regulate the temporal and spatial levels of profilin-actin by two possible mechanisms [67, 73]. One possibility is that external signal-mediated phosphorylation of phospholipase C hydrolyzes P­I(4,5)P­2, releasing membrane-bound profilin to presumably facilitate actin assembly by the proteins formin and Ena/VASP. Second, sequestration of profilin to membrane regions with high concentrations of P­I(4,5)P­2 could increase the level of free G-actin, unbound to profilin, which might preferentially incorporate into branched actin filament networks generated by the Arp2/3 complex [73, 74].

Profilin has also been identified as having a role in stress granules in yeast and mammalian cells [75]. Stress granules are formed in response to stress conditions such as heat shock or oxidative stress [76–78]. Profilin 1 localizes to stress granules upon exposure to diverse environmental stressors. This links profilin 1, the newest amyotrophic lateral sclerosis (ALS) disease protein, to stress granules. The study by Figley, et al. also found that ALS-linked profilin 1 mutations alter stress granule dynamics, providing a potential mechanism to explain their role in disease [75]. Notably, in our study, one of the pathways with highest gene count and gene ratio that was identified in the KEGG functional enrichment for the shared proteome of Cdc3 and Cdc10 during heat stress (37°C) is ALS disease protein. Strikingly, a recent study in C. neoformans identified septin Cdc3 as one of the targets (direct or indirect) of stress related phosphatase calcineurin, in addition to other targets including proteins involved in RNA processing, stability, and translation, which colocalize together with calcineurin in stress granules/P-bodies upon thermal stress [79]. Moreover, C. neoformans strains lacking Cdc3 or Cdc12 are inviable in the absence of calcineurin activity at 25°C [64]. These findings and data presented here collectively suggest that septins Cdc3 and Cdc10 are involved actin dynamics and stress granule formation and/or dynamics during heat stress through their association with profilin.

Given the above findings, we predicted the GFP-profilin would form fluorescent puncta at the plasma membrane and/or at the stress granules/P-bodies when C. neoformans is shifted to 37°C. Interestingly, we observed Cdc3-mCherry and Cdc10-mCherry forming puncta at the plasma membrane and in the cytoplasm (which may correspond to stress granules) specifically at 37°C. However, GFP-profilin exhibited an overall cytoplasmic signal at both ∼25 and 37°C. One explanation for this inconsistency is that the plasma membrane signal of GFP-profilin is not discernible due to the overexpression of the chimera, in contrast to the native level of expression of Cdc3-mCherry and Cdc10-mCherry. A non-exclusive possibility is that the GFP-profilin may not be fully functional while still retaining its capacity to associate with the septins.

The connection between profilin, septins, and the MAPK pathway

The highly conserved Mitogen-Activated Protein Kinase (MAPK) pathway facilitates responses to changes in environmental conditions and various other stimuli; It is a three-tiered cascade of protein kinases [80]. The components of these pathways and the mechanisms by which they operate were first identified and characterized in S. cerevisiae and are now known to have been conserved during the evolution of the entire eukaryotic kingdom [81]. Furthermore, these pathways regulate downstream molecular and cellular processes and are controlled by regulatory factors both internal and external to the pathways [82, 83].

S. cerevisiae’s profilin deficient mutants exhibit various abnormalities such as sensitivity to caffeine and NaCl, growth defects at altered temperatures, accumulation of intracellular vesicular structures, and a random budding pattern. Nonetheless, the profilin-deficient phenotype can be suppressed by the Rho2 signaling pathway [84]. Rho2 is a small GTPase of the Rho/Rac family of Ras-like proteins, which is involved in the establishment of cell polarity and in microtubule assembly [85, 86]. The only suppressor that can correct the phenotype of the profilin-deficient S. cerevisiae strain is Rho2p, which suggests that the suppressor acts through the Rho2p signaling pathway to repolarize cortical actin patches [84]. The C. neoformans homologue for Rho2, Rho family GTPase–Rho104 (CNAG_06606), was identified here as a significant interacting protein of Cdc10 during heat stress (37°) (S2 Table). This implicates septin Cdc10 in cytoskeleton stability via its association with profilin and Rho2’s homologue. Furthermore, Rho2 has been identified as a component of the MAPK signaling pathway in S. pombe [87–89].

Rho2 is a target of the farnesyltransferase Cpp1 and functions upstream of the Pck2–Pmk1 MAPK signaling pathway [90]. Moreover, Rho2 is involved in the regulation of cell wall integrity [90]. The palmitoylation of Rho2 enhances its plasma membrane localization and function, which is essential for signaling to the cell integrity pathway during vegetative growth and in response to stress. In the context of the MAPK pathway, Rho2 and Rho3 regulate the cell integrity MAPK pathway in an antagonistic fashion. The palmitoylation of Rho2 by Erf2 is essential for the signaling to the cell integrity pathway [91]. It is plausible that Cdc10’s interaction with the Rho2 homologue in C. neoformans during heat stress is important for Rho2’s function at the plasma membrane and subsequent MAPK signaling. Furthermore, the MAPK signaling was among the significantly enriched KEGG pathways identified here by the shared interactome functional enrichment analysis (Fig 9B).

In our study, Cdc10 and Cdc3 also associated with other crucial elements of the MAPK pathway including a Rab GTPase and a Rho GTPase activator [92, 93]. Cdc3 associated significantly with the Rab family protein (CNAG_04771) during both ambient temperature (∼25°C), and heat stress (37°C) (Fig 7 and S8 Fig in S1 File). In S. cerevisiae, a Rab escort protein has been identified as a regulator of the MAPK pathway that controls filamentous growth [93].

During ambient temperature (∼25°C), both septin Cdc10 and Cdc3 were found to interact with the Rho GTPase activator, Lrg1 (CNAG_05703) (Tables 4, 5 and Fig 8). The homologue of this protein in S. cerevisiae is a GTPase-activating protein (GAP) that contains a Rho1p-specific GAP activity. Furthermore, it is a negative regulator of the Pkc1 mediated cell wall integrity signaling pathway, and a negative regulator of cell wall 1,3-β-glucan biosynthesis [94–96]. This further implicates a functional connection between septins and components of the MAPK signaling pathway involved in cell wall integrity in C. neoformans. Moreover, Rho1 is involved in septum formation in fission yeast [86]. Remarkably, the Rho GTPase activator Lrg1 also controls small GTPase Cdc42 in S. pombe and Candida albicans [97–99]. Therefore, Lrg1 is not only involved in maintaining cell wall integrity in these fungi but it also interacts with Cdc42, which is a key component in the regulation of septin filaments [100]. In C. neoformans, loss of Cdc42 function results in cytokinesis defects due to loss of the organization of septin proteins [101, 102]. Thus, the putative interaction of Cdc3 and Cdc10 with the Rho GTPase activator Lrg1 suggests a link between septins and cell wall integrity via the MAPK pathway.

In C. neoformans, the MAPK pathway is involved in regulating virulence and environmental adaptation. Activation of the pathway in response to host signals and environmental cues leads to the regulation of genes involved in capsule formation, melanin production, and stress responses [103, 104]. Thus, its presence in the septin interactome suggests potential involvement in cell wall maintenance and repair, which are pivotal for fungal survival. Additionally, given the interactions between septins and the plasma membrane, the MAPK pathway’s association may signify a regulatory mechanism that ensures plasma membrane stability and lipid homeostasis under various environmental conditions. In C. albicans, septin proteins have been found to scaffold cell-wall proteins via the cell wall integrity (CWI) MAPK pathway [12, 14, 105, 106]. Additionally, septin proteins in Aspergillus nidulans promote cell wall integrity and homeostasis in lipid metabolism through crosstalk with the MAPK CWI pathway [107]. Thus, the putative cross-regulatory relationship between septin proteins and the MAPK pathway in C. neoformans warrants further investigation and may provide insights into cryptococcal adaptability and pathogenicity.

Association of septins with Afr1

Our findings suggest that both Cdc3 and Cdc10 associate with Afr1 and these interactions are more pronounced at 37°C. Importantly, GFP-Afr1 exhibited localization in C. neoformans that is consistent with the predicted association with the plasma membrane (Fig 10). ABC transporters are a large family of membrane-associated proteins that use ATP to pump substrates across cellular membranes [108–110]. The Afr1 (Antifungal Resistance 1) protein, a member of the ABC family, functions as an efflux pump that exports antifungal drugs like fluconazole out of fungal cells, thereby reducing their intracellular accumulation and conferring resistance [111]. Overexpression of AFR1 leads to upregulation of this efflux activity, resulting in high-level fluconazole resistance in C. neoformans both in vitro and in vivo [112–114]. Interestingly, beyond mediating drug resistance, the Afr1 protein also appears to contribute directly to the virulence of C. neoformans. Strains overexpressing AFR1 were found to be significantly more virulent than wild-type in mouse models of cryptococcosis, regardless of the infection route (intravenous or inhalational). This enhanced virulence was linked to the increased intracellular survival of these strains inside macrophages, which are key immune cells that normally kill and clear C. neoformans [62, 112, 113]. Intriguingly, our microscopy data suggest that at 37°C, septins accumulate at the plasma membrane where they may interact with Afr1 and other stress related proteins and contribute to stress response (Fig 10). Therefore, the association of septin proteins with Afr1 during heat stress suggests that septins might contribute to virulence via a pathway independent of their role in cytokinesis that may involve localization to the plasma membrane, a possibility that should be addressed in future studies.

Other notable potential septin-interacting proteins

Another important candidate protein for the shared interactome of Cdc10 and Cdc3 during both ambient temperature (∼25°C) and heat stress (37°C) is importin (CNAG_04973) (Fig 8). Notably, importins have been recently identified as regulators during meiosis and cytokinesis in a study by Beaudet, et al. [115]. In mammalian cells, the C-terminal nuclear localization sequence (NLS) of anillin is required for its localization and function during cytokinesis [115]. Furthermore, binding to active RhoA facilitates a conformational change in anillin that increases accessibility to the C-terminus, and importin binding stabilizes this conformation to facilitate anillin’s recruitment to the equatorial cortex. The study found that mutating the NLS alters anillin’s cortical dynamics. It also found that increasing active RhoA facilitates importin binding, while inactive RhoA or mutating the RhoA-binding domain (RBD) decreases importin binding. Anillin’s localization and function are abolished when NLS mutations are combined with additional point mutations in the C-terminus domain that weaken its interface with the RBD [115]. Thus, the relative position of the RBD and C-terminus is essential for anillin’s cortical recruitment, but also for ensuring that importin can be outcompeted by phospholipids and/or other cortical components [115]. In addition, septin recruitment in mammalian cells depends on anillin [116]. However, septins can also interact with F-actin and myosin II, which gives rise to multiple possibilities for how septins might be recruited to the contractile ring (CR) in an anillin-dependent manner. Septin recruitment to the CR appears to require the anillin C-terminus uniquely and specifically, which must be both competent to bind active RhoA-GTP and contain its PH domain. Thus, Rho1 GTPase controls anillo-septin assembly and facilitates contractile ring closure during cytokinesis [116]. Therefore, the link between importin and septin proteins suggests a potential mechanism for their coordinated regulation during cytokinesis. Importin binding to anillin stabilizes its conformation, facilitating its recruitment to the equatorial cortex [115]. This recruitment is crucial for anillin’s cortical dynamics and function [115]. Meanwhile, Drosophila melanogaster’s septin recruitment to the contractile ring also depends on anillin, specifically its C-terminus, which interacts with active RhoA-GTP and contains a PH domain [117, 118]. The Rho1 GTPase further controls anillo-septin assembly and facilitates contractile ring closure [119]. Thus, the existing literature supports the claim that importin is part of the septin complex interactome. The functional enrichment of the shared interactome of Cdc10 and Cdc3 revealed cytokinesis and cytoskeleton-dependent cytokinesis as GO terms with significant gene count and gene ratio (Fig 9A). Furthermore, cell cycle was among the significantly enriched KEGG pathways (Fig 9B). In C. neoformans anillin-like protein, a homologue of the S. cerevisiae Bud4, is crucial for proper organization and dynamics of the septin ring and the loss of Bud4 results in a phenotype strikingly similar to that of the septin deletion mutants [120]. The role of anillin-like proteins in septin organization during cytokinesis appears to be a conserved feature in fungi [16, 121–123]. Further studies investigating the interplay between importin, Bud4 homologue, and septins in C. neoformans will provide insights into the regulatory network governing cytokinesis in this species that may be conserved in other eukaryotes.

There are other noteworthy physical protein interactors of the septin proteins Cdc10 and Cdc3 that are also informative regarding septin function in C. neoformans. Several proteins of the DASH complex (Fig 8), which is involved with microtubules when the kinetochore attaches to the spindle, and plays a role in spindle attachment, chromosome segregation and spindle stability [124–126]. This might be an indication that septin proteins interact with the spindle stability and/or positioning during cytokinesis, similar to the roles implicated in S. cerevisiae [21]. In addition, several PH domain-containing proteins were identified as binding partners of both septin Cdc10 and Cdc3 (Fig 8) (Tables 6, 7), consistent with the association of septins with the plasma membrane in C. neoformans.

Concluding remarks

This study constitutes the first comprehensive evaluation of the protein interactome of the septins Cdc3 and Cdc10 in C. neoformans, which should facilitate future investigations to determine the roles of septins in cell division and stress response in this fungal pathogen. In addition, this study has also provided the differentially expressed proteomic landscape of C. neoformans during heats tress and stationary growth phase compared to the exponential growth at ambient temperature control. Consequently, we have not only provided candidates for the proteins that belong to the septin complex interactome in C. neoformans, but we have also provided the context in which they may be interacting.

Fungal pathogens like C. neoformans encounter diverse environmental stressors during host colonization and infection. It is plausible that the putative association of the MAPK pathway with septins facilitates adaptive responses to these stressors. Septins are known to be involved in cytokinesis and morphogenesis, which are essential for pathogenicity. The MAPK pathway may modulate septin functions to aid in the fungus’s ability to adapt and thrive within the host. These interactions underscore the pivotal roles of septin proteins in mediating cellular responses to environmental stressors, potentially forming a regulatory nexus essential for C. neoformans’ environmental adaptation and offering a framework for deeper insights at the mechanistic level. Furthermore, understanding the role of the MAPK pathway in the septin interactome has potential therapeutic implications, as disrupting this pathway could compromise cell wall integrity and plasma membrane stability, both essential for pathogenicity. The presence of the MAPK pathway within the septin interactome adds a layer of complexity to our understanding of fungal biology, particularly in the context of stress responses, cell wall integrity, and plasma membrane stability. Deciphering these intricate molecular relationships could deepen our understanding of fungal pathogenesis and offer new strategies for combating cryptococcal and other fungal infections.