Introduction

Candida albicans, a polymorphic opportunistic pathogen associated with superficial to life-threatening systemic infections among immunocompromised individuals is included in the list of organisms with potential antibiotic resistance threat, recently (CDC report 2013; Kullberg and Arendrup 2015). Morphogenic plasticity is considered as a survival strategy that enables C. albicans to colonize and invade host tissues by evading host defense mechanisms under a wide range of extreme micro-environments (Brown et al. 2014; Cutler 1991; Ernst 2000; Lim et al. 2012; Ruhnke 2006). Different morphological forms viz. yeast, hyphae, pseudohyphe, chlamydospore, opaque cells and biofilms exhibit differential responses towards host defense mechanisms as well as antifungal agents (Cutler 1991; Lim et al. 2012; Ruhnke 2006; Tyc et al. 2014). Among these, the hyphal form is a prerequisite for tissue invasion and invasive candidiasis and biofilms on indwelling medical devices are considered as difficult-to-treat infections with very high mortality (Mc Manus and Coleman 2014; Mun et al. 2016; Neville et al. 2015; Williams 2011). Considering the significance in virulence, these morphological and growth forms were studied exhaustively in recent years while chlamydospore, considered as a non-virulent form is neglected by scientific community (Bottcher et al. 2016; Citiulo et al. 2009).

Chlamydospores are thick-walled spherical cells (6–10 µm) considered as dormant form induced under unfavorable environmental conditions like oxygen limitation and embedded growth in matrix (Giosa et al. 2017; Nobile et al. 2003; Sonneborn et al.1999). The presence of chlamydospores in clinical specimens is reported but it may be unlikely to play any role in pathogenesis (Citiulo et al. 2009). Chlamydospores are more specialized and relatively rare morphological states formed to survive under harsh conditions (Nobile et al. 2003; Sosinska 2012). It could be an adaptive response towards exposure to reactive oxygen species of host cells or towards co-existing microorganisms (Berman and Sudbery 2002; Douglas et al. 2005). Chlamydospores are metabolically active, can germinate under favorable condition and produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae (Citiulo et al. 2009; Staib and Morschhäuser 2007). Chlamydospores thick wall is providing protection against the adverse micro-environments. However, not much study is available on structure and composition of chlamydospore cell wall (Jansons and Nickerson 1970).

Though various environmental, nutritional and genetic factors are implicated, regulation and significance of chlamydospore formation are not very clear (Bottcher et al. 2016). In present study, we have made an attempt to identify chlamydospore specific proteins using LC–MS/MS analysis. This is the first attempt at identifying chlamydospore specific proteins. Proteomic analysis revealed morphophysiological modulations responsible for altering cellular architecture that enables C. albicans to survive under extreme micro-environments.

Material and methodology

Candida albicans strain and growth condition

Candida albicans ATCC 10231 a quality control strain was procured from Institute of Microbial Technology (IMTECH), Chandigarh, India and maintained on yeast extract peptone dextrose (YPD) agar at 4 °C. Rice extract agar with 1% Tween 80 was used for chlamydospore induction. Yeast Extract Peptone Dextrose (YEPD) broth and Rice extract agar were purchased from Hi-media Laboratories, Pvt. Ltd. Mumbai (India). All the chemicals used in this study were purchased from Sigma-Aldrich Pvt. Ltd., Bangalore (India) and solvents from Qualigens and SD Fine Chemicals Ltd, Mumbai (India).

Induction of chlamydospores

Inoculums were prepared using C. albicans cells grown for 48 h at 30 °C in YEPD broth. Cells were harvested by centrifugation at 448 g for 2 min, washed thrice with sterile distilled water and re-suspended in 1 ml of sterile distilled water (Holmes and Shepherd 1987; Odds 1988).

Chlamydosporulation was induced using rice extract agar (Rice extract 1.3 g, Tween 1%, pH 7.1). In brief, sterile rice extract with low melting agar plates were inoculated with 1 × 103 cells per plate (Kelly and Funigiello 1959). Plates were overlaid with sterile, polyethylene sheets of 7.5 cm diameter for anaerobiosis and invasive growth necessary for chlamydospores induction. Plates were wrapped with parafilm and incubated at 30 °C for 14 days (Miller et al. 1974). Chlamydospore formation was monitored by observing the plates under dissecting microscope. Chlamydospores were stained using Lactophenol cotton blue, observed microscopically (OLYMPUS CX21i) and photographed using microscope attached camera (OLYMPUS digital camera E-PL1) (Kim et al. 2002).

Harvesting of chlamydospores

Chlamydospores were harvested from 12-day-old plates grown at 30 °C. In brief, chlamydospores plates were washed with sterile distilled water to remove colonies of yeast phase cells growing on the agar surface. Rice extract agar containing chlamydospores was molten using warm (45 °C) sterile distilled water, centrifugation at 112 g for 1 min. Chlamydospore pellets were washed thrice with sterile distilled water and used for protein extraction. However, chlamydospores as harvested from semisolid agar, traces of hyphae producing chlamydospores were present along with chlamydospores in the sample. Cells from the colonies growing on the rice extract agar surface were harvested by centrifugation, washed with sterile distilled water and used as a control.

Cell surface hydrophobicity

Cell surface hydrophobicity of yeast phase cells and chlamydospores of C. albicans was analyzed using a method developed by Rosenberg, et al. (1980) and Hazen and Hazen (1987). Briefly, yeast cells and chlamydospores were re-suspended in PBS until the OD (at 620 nm) reaches to 0.5. 1.3 ml from each of these suspensions was distributed in three test tubes. 100 μl from each test tube were again added into the wells of 96 well micro titer plates and initial OD (at 620 nm) was recorded using Thermoscan-Ex micro plate reader (Thermo Fisher Scientific Inc., 168 3rd Ave, Waltham, MA 02451, USA). 0.3 ml of octane was added to remaining cell suspension (1.2 ml) separately, mixed vigorously (3 min), and allowed to separate for 15 min. From these suspensions 100 μl were cautiously transferred to the wells of 96-well micro-titer plates and final OD was recorded. Triplicates were used for each samples and experiment was repeated thrice. Percentage CSH was calculated using following formula and compared with control. Results were presented as percentage of CSH ± SD (standard deviation) (Hazen and Hazen 1987; Rosenberg et al. 1980).

$$\left[ {{\text{CSH}}\,{\text{percentage}} = \left( {1 - {\text{final}}\,{\text{OD}}\,{\text{of}}\,{\text{aqueous}}\,{\text{phase}}/{\text{initial}}\,{\text{OD}}\,{\text{of}}\,{\text{cell}}\,{\text{suspension}}} \right)\, \times \,100} \right].$$

Estimation of adhesion

Adhesion of yeast phase cells and chlamydospores of C. albicans was determined using method described by Panagoda et al. (2001) and He et al. (2006). Briefly, 100 μl of cell suspension (1 × 107 cells/chlamydospores/ml) was inoculated in 96 wells micro titer plates and incubated at 30 °C for 90 min with moderate shaking (50 rpm) on orbital shaker for adhesion. After incubation, wells were washed thrice with PBS to remove un-adhered ones and the numbers of adhered cells/chlamydospores in each well were counted, microscopically (Metzer Make inverted Microscope) and compared. The average number of yeast phase cells adhered is considered as 100% adhesion. Experiment was repeated thrice and triplicates were used for each sample and results were shown as adhesion percentage ± SD (standard deviation) (He et al. 2006; Panagoda et al. 2001).

Ergosterol extraction

Ergosterol content of yeast phase cells and chlamydospores of C. albicans was estimated as per Arthington-Skaggs et al. (1999). Briefly, 0.1 g of washed cell/chlamydospore pellets were suspended into 300 μl of ethanolic KOH (25%) and incubated at 85 °C for 1 h and 2 h, respectively. Samples were cooled to room temperature and ergosterol was extracted using n-heptane [75% (v/v)] with vortexing. Layers were allowed to separate and n-heptane layer was transferred to new vial, cautiously. 200 μl of n-heptane layer was diluted to fivefold in ethanol (100%) and spectrum was recorded in the wavelength range of 230–300 nm using a UV–Visible spectrophotometer (Shimadzu Analytical (India) Pvt. Ltd. Mumbai- 400 059, India).

Ergosterol estimation

Ergosterol content was determined using the values of absorbance at 230 nm and 281.5 nm and the formula by Arthington-Skaggs et al. (1999):

$$\begin{aligned} & {\text{Percentage}}\,{\text{ergosterol}} + \% 24\left( {28} \right){\text{DHE}} = \left[ {\left( {{\text{A}}281.5/290} \right) \times F} \right]/{\text{pellet}}\,{\text{weight}} \\ & \% 24\left( {28} \right){\text{DHE}} = \left[ {\left( {{\text{A}}230/518} \right) \times F} \right]/{\text{pellet}}\,{\text{weight}}, \\ & {\text{So}},{\text{ percentage}}\,{\text{ergosterol}}\, = \,\left[ {{\text{percentage}}\,{\text{ergosterol}} + 24\left( {28} \right){\text{DHE}}} \right] - \% 24\left( {28} \right){\text{DHE}}] \\ \end{aligned}$$

where F is the dilution factor in ethanol and 290 and 518 are the E values (percent/centimeter) determined for crystalline ergosterol and 24(28) DHE, respectively. Results were showed as ergosterol percentage ± SD (standard deviation).

Cell lysis and protein extraction

Proteins (chlamydospore and yeast phase cells) were extracted using the protocol optimized by Haar (2007). Briefly, chlamydospores and yeast phase cells (1x108 equivalent cells) were resuspended in 200 µl of freshly prepared lysis buffer (0.1 M NaOH, 0.5 M EDTA, 2% SDS and 2% β-mercaptoethanol) containing (10 µl/ml) PIC (protease inhibitor cocktail) and vortexed. Samples were incubated at 90 °C for 15 min and neutralized using 5 µl of 4 M acetic acid after incubation. Samples were further incubated at 90 °C for 15 min and centrifuged at 2800 g for 5 min. Supernatants were transferred to new vials containing 5 µl of PMSF (Phenyl methane sulphonyl fluoride). Proteins were precipitated using 4 volumes of methanol, 1 volume of chloroform and 3 volumes of sterile distilled water with vortexing. Precipitated proteins were centrifuged at 2800g for 5 min, pellets were washed using 3 volumes of methanol, centrifuged at 2800 g for 5 min and air dried. Air dried pellets were re-suspended in rehydration buffer (6 M urea, 2 M Thiourea, 2% CHAPS, 1% DTT, pH 8.75) (Haar 2007) and protein concentrations were determined as per Bradford method (Bradford 1976).

Sample preparation

Proteins (50 µg) were dissolved in ammonium bicarbonate buffer (50 mM) containing rapigest (0.1%). Proteins were reduced with 3 µl of DTT (100 mM) at 60° C for 15 min, alkylated using 3 µl of Idoacetamide (200 mM) at room temperature for 30 min, and digested at alkaline pH using 2 µg of trypsin per 50 µg of proteins (i.e. 2:50). Digestion was stopped by adding 2 µl of concentrated HCL after 18 h of digestion. Peptides were separated by spinning at high speed (1500 rcf) for 15 min at 4 °C, washed several times with 0.1% TFA, size fractionated (̴̴ 3 kDa) using Zip tip C18 chromatography columns (Millipore; Billerica, MA) and eluted in 100% Acetonitrile. Samples were reconstituted in 15 µl of Acetonitrile (3%) and formic acid (0.1%) with continuous vortexing and used for further analysis (Gillet et al. 2012).

Liquid chromatography and mass spectrometry analysis

Peptides (4 µg) were separated and mass was determined using Micro LC 200 (Eksigent; Dublin, CA) coupled with Triple- TOF 5600 (AB Sciex; Concord, Canada) mass spectrometer in high-sensitivity mode. Equal amounts of samples of chlamydospores and control were spiked to generate the SWATH (sequential window acquisition of all theoretical fragment ion spectra) spectral library of fragment ions and analyzed using Information Dependant acquisition (IDA) (Collins et al. 2013; Gillet et al. 2012; Liu et al. 2006, 2013).

SWATH MS analysis

Swath MS analysis was carried out using the instrument setting as described in Ingle et al. (2017), Collins et al. (2013); Gillet et al. (2012); Liu et al.(2006); Liu et al. (2013). The mass spectral data acquired in triplicates was searched against Candida databases, Uniprot ids were searched using Protein Pilot software and differentially expressed proteins were identified using markerview software. Subsequently SWATH-MS was performed for relative quantification of differentially expressed proteins as mentioned in Ingle et al. (2017).

Statistical analysis

The student t test and probability were performed for statistical analysis. Samples with probability (p) value ≤ 0.05, number of matching peptides ≥ 2 and fold change ≥ 2, were considered for further analysis.

Validation of proteomic data using real-time qPCR analysis of selected genes

Expression of selected genes during chlamydosporulation was evaluated at mRNA level using real-time qPCR analysis. Gene-specific primers were designed using primer3 plus software (Tm 58–60 °C, product size 120–150 bp preferred for primer pairs) (Table 5). Total RNAs were prepared using RNeasy Mini kit (50 reactions) (Cat. No. 74104, Qiagen Pvt. Ltd) by lysing Chlamydospores using lyticase and purified using RNA Sure Mini Kit, Nucleo-pore (Genetix) according to the manufacturer’s instructions. cDNAs were prepared using purified RNA (2 µg) as a template and High-Capacity cDNA Reverse Transcription Kit as per the manufacturer’s instructions (Green et al. 2004). RNA level was measured using KAPA SYBR® FAST qPCR Kit as per manufacturer’s instructions and parameters and CFX96 Touch TM Real-Time PCR Detection System (Biorad Pvt. Ltd). Samples were analyzed in triplicates using biological replicates and data are reported as mean ± SD. Using ANOVA, statistical significance was calculated and p-values less than 0.05 were considered significant. Gene expression was normalized with GAPDH levels and with control cells.

Results

Induction of chlamydosporulation

Embedded and microaerophilic growth on rice extract agar medium at 30 °C induced invasive growth in Candida albicans, initially. The invading hyphae later developed suspensor cells that produced thick-walled chlamydospores, terminally within 4 days of incubation. Further incubation up to 12 days leads to increase in the number of chlamydospores (Fig. 1).

Fig. 1
figure 1

Light microscopy images of Chlamydospore (with hyphae) formation of C. albicans, ATCC 10231. Numbers indicates the days of incubation, white arrow indicates developing chlamydospores, and black arrow indicates mature chlamydospores

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Modulation in cell surface hydrophobicity (CSH), adhesion and ergosterol during chlamydosporulation

In present study, in response to diverse morphology cell surface hydrophobicity, adhesion as well as ergosterol significantly modulated. Cell surface hydrophobicity of C. albicans yeast phase cells and chlamydospores (with filaments) at 30° C were found (37.92 ± 4.98, 31.70 ± 3.64, 27.61 ± 3.12) and (9.12 ± 1.29, 11 ± 3.33, 10 ± 4.05) respectively (Fig. 2a). This showed that CSH of yeast phase cells more as compare to chlamydospores (with filaments). However, adhesion of yeast phase cells and chlamydospore (with filaments) at 30° were found (82 ± 3.61, 79.67 ± 4.51, 77.67 ± 3.79) and (2.33 ± 0.58, 3 ± 2, 2.33 ± 2.08), respectively (Fig. 2b). Yeast pahse cells are more adhesive than chlamydospores (with filaments). In addition to this, ergosterol content was analyzed through spectrophotomer, which showed that chlamydospore (with filaments) contain more ergosterol as compare to yeast phase cells (Fig. 2c). Percent ergosterol content of yeast phase cells and chlamydospores (with filaments) was found (0.0073 ± 0.00076, 0.0087 ± 0.0013, 0.0076 ± 0.001) and (0.124 ± 0.06, 0.0207 ± 0.002, 0.0196 ± 0.0018) respectively (Fig. 2c).

Fig. 2
figure 2

Determination of a cell surface hydrophobicity b adhesion and c ergosterol content of yeast phase cells and Chlamydospore (with filaments) of C. albicans (ATCC 10231)

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Identification of chlamydosporulation specific proteins using LC–MS/MS analysis

LC–MS/MS analysis identified 1177 proteins out of which, 319 were modulated significantly (Ingle et al. 2017). MS/MS data is submitted to Peptide Atlas and data set is publically available with the data set identifier PASS01061 at http://www.peptideatlas.org/PASS/PASS01061. Differentially expressed proteins were identified; functionally annotated and grouped into different categories according to their functions using databases like CGD, SGD, KEGG and Uniprot etc., using David software (Functional annotation Bioinformatics Microarray). Among these differentially expressed proteins, 137 were up-regulated while 182 were Down-regulated under chlamydospore inducing condition (Ingle et al. 2017). The number of up-regulated and down-regulated proteins in different biological processes viz. metabolism, cell wall and membrane composition, stress response and signaling, gene expression, transport etc. were shown in Tables 1, 2, 3 and 4, Fig. 2a, b.

Table 1 Modulation of proteins involved in metabolism during chlamydosporulation in Candida albicans (ATCC 10231)
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Table 2 Modulation of proteins involved in cell wall/membrane biosynthesis, cell signaling, stress response and transport during chlamydosporulation in Candida albicans (ATCC 10231)
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Table 3 Modulation proteins involved in cell cycle, genome organization, replication, gene expression during chlamydosporulation in Candida albicans (ATCC 10231)
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Table 4 Modulation of un-characterized proteins during chlamydosporulation in Candida albicans (ATCC 10231)
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Modulation of proteins involved in metabolism

A total of 108 proteins involved in metabolism were modulated significantly, out of which 51 were involved in carbohydrate metabolism and energy generation (Table 1). The maximum up regulation of 259.37-fold was observed in case of Pgk1 followed by 64.83 (CAWG_04860/Mal32), 35.99 (Ald4), 17.93 (CAWG_01786/Hxk2), 13.85 (LELG_01826/Icl1) fold while maximum down-regulation of 46.59-fold was observed in case of (CaO19.285/Ctn1) followed by 32.83 (Ams1) and 10.48 (Glo2) fold etc. (Table 1). Five proteins involved in fermentation were up-regulated and maximum up regulation of 12.10-fold was observed in case of Ife2 (Table 1).

Eleven out of the thirteen proteins involved in lipid, sterol and fatty acid biosynthesis were up-regulated and maximum up-regulation of 36.85 was observed in case of Scs7 (Sphingolipid alpha-hydroxylase), while two were down-regulated (Table 1). Seventeen proteins expressed differentially indicating significant modulation in amino acid metabolism, wherein Gcv1 was up-regulated by 10.66-fold while maximum down-regulation of 14.55-fold was observed in case of Gln1 (Table 1). Similarly, differential expression of fourteen proteins indicates significant modulation in nucleotide metabolism i.e. Ysa1 was down-regulated by 18.19-fold while Ura4 was up-regulated by 6.25-fold (Table 1). Heme (Hem13, Hem15) and Vitamin (Snz1) biosynthesis and fatty acid degradation were enhanced under chlamydospore inducing condition (Table 1).

Modulation of proteins involved in cell wall and membrane biosynthesis

A total of eighteen proteins involved in biosynthesis of cell wall components viz. mannan (2), beta 1, 3-glucan (1), beta 1, 6-glucan (2), chitin (1), cell wall proteins (7) and membrane (5) were modulated significantly during chlamydosporulation (Table 2). The cell wall proteins viz. Hyr1, Als3 (hyphae specific) and Ldg1 were up-regulated maximally by 218.24, 38.16 and 38.43-fold respectively while maximum down-regulation of 49.15-fold was observed in case of Kre9 involved in biosynthesis of beta 1,6-glucan (Table 2). Among the proteins involved in membrane structure and biosynthesis like Fmp52, Scs7, Ino1 etc., were up-regulated by 82.65 36.85 and 12.82-fold respectively (Table 2). It indicates that cell wall of chlamydospore is rich in mannan, chitin and beta 1, 3-glucan while membrane is enriched with sphingolipids, phospholipids, sterols etc.

Modulation of proteins involved in stress response and signal transduction

Twenty out of the twenty-six proteins modulated during chlamydospore growth were involved in oxidative stress response while four in heat stress and one each in metal ion and drug-induced stress (Table 2). Among these, ten were up-regulated while sixteen were down-regulated and maximum up-regulation of 242.48-fold was observed in case of Yhb1 (Nitric oxide dioxygenase), and down-regulation in case of Whs11 (176.48-fold) (Table 2).

Among the six proteins involved in signal transduction, maximum up-regulation of 8.83-fold and down-regulation of 16.7-fold was observed in case of Rsr1 (CLUG_ 03767) and Asr2 (CAWG_02167), respectively (Table 2).

Modulation of proteins involved in transport

Twenty-eight proteins involved in transport were significantly modulated (23 down-regulated, 5 up-regulated) during chlamydospore form growth (Table 2). Among the twenty-one proteins involved in cytoplasmic transport, six were up-regulated and fifteen were down-regulated (Table 2). Maximum up-regulation of 35.08-folds was observed in case of Vma5 (Putative vacuolar H (+)-ATPase) and down-regulation of 23.44-fold in Sbe22 (CaO19.10437), respectively (Table 2).

Modulation of proteins involved in regulation of cell cycle, cytoskeleton, genome organization, replication and repair

Among the sixteen proteins modulated, two, six, five and three were involved in cell cycle, cytoskeleton, genome organization and replication and repair, respectively (Table 3). One of the two involved in cell cycle regulation, four out of the six in cytoskeleton, all the five in genome organization and one of the three involved in replication and repair were up-regulated respectively (Table 3). Among these, maximum up- regulation of 8.15-fold and down-regulation of 12.5-fold was observed in case of Arp7 (genome organization) and Ssp2 (cell cycle) respectively (Table 3).

Modulation of proteins involved in genome function (transcription, RNA processing, translation and proteolysis)

Twelve proteins involved in transcription, six in RNA processing, 71 in translation and fifteen in proteolysis were significantly modulated under chlamydospore form growth (Table 3). Among the twelve proteins involved, eight were involved in transcription initiation and four in elongation (Table 3). All the eight involved in transcription initiation were down-regulated while three of the five involved in elongation were unregulated (Table 3). Similarly, six proteins involved in RNA processing were down-regulated (Table 3).

Out of the seventy-one proteins involved, thirty-eight (21 up-regulated, 17 down-regulated) were involved in ribosomal assembly, nine in translation initiation (2 up-regulated, 7 down-regulated), three in elongation (2 up-regulated, 1 down-regulated), fifteen in post translational modification (2 up-regulated, 13 down-regulated) and six were involved in mitochondrial ribosome assembly (3) and mitochondrial PTM (3) (Table 3). Maximum up-regulation of 16.23-fold and down-regulation of 16.35-fold was observed in case of Rbp4 and Rtg1 respectively (Table 3). On the other hand, Snu13 (CAWG_02750) was down-regulated maximally by 7.24-fold amongst the six down-regulated proteins involved in RNA processing (Table 3). Similarly amongst the 71 proteins involved in translation and post-translational modifications, maximum up regulation of 11.15-fold observed in case of Rps23 while Tif3 was down-regulated maximally i.e. 24.28-fold (Table 3). Among the fifteen proteins involved in proteolysis (2 up-regulated and 13 down-regulated), maximum up-regulation of 66.85-fold and down-regulation of 84.5-fold was observed in case of Pre7 (AGOS_AGL324 W) and Pbi2 (CaO19.10285), respectively (Table 3). Thirteen modulated proteins were uncharacterized (Table 4).

Discussion

Chlamydospore, the thick-walled resting spores induced in response to stresses (like the nutrient limitation, microaerophilic and osmotic stress etc.), is considered as a survival strategy of C. albicans (Citiulo et al. 2009; Staib and Morschhäuser 2005; Nobile et al. 2003). C. albicans cells experience most of these stresses (except temperature 37 °C) during growth on rice extract agar containing Tween 80 in our study, citing the biological significance of our data (Berman and Sudbery 2002; Douglas et al. 2005). Though it was found in some of the tissue samples recently, chlamydospore is considered as a non-virulent form of C. albicans and thus neglected by the scientific community (Palige et al. 2013; Citiulo et al. 2009; Lim et al. 2012). However, few recent studies have provided some insights into the regulation of chlamydospore formation and survival strategies (Bottcher et al. 2016; Giosa et al. 2017; Palige et al. 2013; Lim et al. 2012). Mutant analysis by Navarathna et al. (2016) revealed significance of a component of chromatin remodeling complex (ISW2) in chlamydosporulation (Navarathna et al. 2016). Our study showed that nutrient limiting and microaerophilic condition enhances activity of Ras related protein Rsr1, the upstream regulator while down regulate Bcy1, a negative regulator of cAMP-PKA pathway (Table 2) (Biswas et al. 2007). It suggests that chlamydospore formation is induced through cAMP-PKA pathway (Table 2) (Biswas et al. 2007). Earlier studies have shown that nutrient starvation activate cAMP-PKA pathway through Ras1 and induce chlamydospore formation through EFG1 in presence of minimal cAMP i.e. surplus cAMP is inhibitory (Bottcher et al. 2016). It was further supported by down regulation of adenyl cyclases (to avoid surplus cAMP concentration) and Tfs1, a negative regulator of Ras protein in our study (Bottcher et al. 2016). It confirms that involvement of cAMP-PKA pathway is essential for chlamydospore formation and thus osmotic, heat shock and cell wall integrity damage induced stress tolerance (Bottcher et al. 2016; Chautard et al. 2004; Harcus et al. 2004). However, Hog1 mitogen-activated protein (MAP) kinase and quorum sensing molecule farnesol were also implicated in chlamydosporulation (Eisman et al. 2006; Martin et al. 2005). Chlamydosporulation need reprogramming of the transcriptional program through chromatin remodeling complexes (ISW2) that leads to chlamydospore specific transcripts including components of cytoskeleton (actin, tubulin and cofilin) (Cao et al. 2005; Navarathna et al. 2016). Components of cytoskeleton (actin, tubulin and cofilin) and chromatin remodeling complex (Arp7) were up regulated in our study. Similarly, enhanced levels of components (Yke2 and Cct2) involved in proper folding of alpha, beta tubulin and actin indicate increased strain on PTM machinery, as reported earlier in response to GlcNAc (García-Sánchez et al. 2005; Kamthan et al. 2012). Enhanced Phb12 level in our study contributes in stabilizing newly synthesized proteins (CGD 2010; Schleit et al. 2013). Up-regulation of Pre7 (66.85-fold) a proteasome component indicates that ubiquitin-dependent proteasome activity was enhanced during chlamydospore growth (Table 3) (CGD 2010; Liu et al. 2016; Verma et al. 2000). Up-regulation of Tpd3 in our study confirms enhanced cytokinesis) during chlamydosporulation as reported earlier (Liu et al. 2016; Lorenz et al. 2004; Sarkar et al. 2002).

Nutrient limitation, microaerophilic and osmotic stress modulate metabolism during chlamydospore growth that leads to acquire unique morphological and architectural characteristics in addition to survival using nonconventional and complex carbohydrates (viz. maltose, xylitol etc.) (Fig. 3) (Jamai et al. 2007; Bruno et al. 2006). Significant up-regulation of maltase (64-fold) followed by enhanced glycolysis and tricarboxylic acid cycle in our study confirms earlier hypothesis that states, “Enhanced glycolysis and tricarboxylic acid cycle under glucose limiting condition contribute to link the regulation of chlamydospores production in Candida” (Table 1 and Fig. 3) (Brown et al. 2014; Bottcher et al. 2016; CGD 2010; Han et al. 2011). Enhanced glyoxylate cycle reported to facilitate survival of C. albicans under glucose limiting condition is enhanced in our study (Fig. 3) (Barelle et al. 2006; Ene et al. 2012). Microaerophilic condition triggers alcohol production leading to hyphae induction required for chlamydospore formation (Fig. 3) (Chauhan et al. 2011; Smith et al. 2004). In addition to energy generation, glycolysis provides precursors for the biosynthesis of lipids (storage molecules) and trehalose required for stress (oxidative, heat, desiccation, osmotic etc.) tolerance (Fig. 3) (Brown et al. 2014; Pereira et al. 2001; Yoda et al.2000). Inhibition of NAD+ synthesis increases oxidative stress tolerance and extend lifespan as NAD is an essential cofactor for cellular redox reactions (Table 1) (Pereira et al. 2001; Bedalov et al. 2003; Kato and Lin 2014). Enhanced glutathione biosynthesis, up-regulation of Osm1, Yhb1 and down-regulation of Ysa1 could be the compensatory responses to enhanced oxidative, nitrossative and osmotic stresses during chlamydospore growth (Table 2) (Chen et al. 2008; Cottier et al. 2012; Liu et al. 2000; Michán and Pueyo 2009; Nett et al. 2009; Yadav et al. 2011). Enhanced level of Yhb1 was also validated at RNA level using qRT-PCR analysis (Tables 2, 5 and Figs. 4, 5). Chlamydospore inducing condition further potentiates antioxidant machinery through the maturation of Sod1 and trehalose homeostasis (through up-regulation of Lys7 and Hsp21 respectively) (Dong et al. 2013; Gleason et al. 2014; Mayer et al. 2012). Morpho-physiological and cellular architectural modulations seems to affect cellular and mitochondrial membrane functions like transport and osmotic stability as proteins involved in maintaining these functions like Gdi1, Cof1, Por1 were up-regulated (Cederquist et al. 2012; Curwin et al. 2012; Kamthan et al. 2012; Lin et al. 2010; Cao et al. 2005). However vacuolar transport and vacuolar (H)-ATPase activities (viz. endocytosis, tagging of lysosomal enzymes) were modulated in our study (Cabezon et al. 2009; Cabrera et al. 2013; Johnston et al. 2013).

Fig. 3
figure 3

Chlamydospore specific proteins of Candida albicans (ATCC 10231). a Up-regulated b down-regulated proteins

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Table 5 Primers used in real time-qPCR analysis
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Fig. 4
figure 4

Hypothetical model based on proteomic analysis showing modulation of metabolism in C. albicans ATCC 10231 during chlamydosporulation

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Metabolic modulation enhances biosynthesis of components of cell wall and membrane during chlamydospore growth. The thick cell wall is reported to provide protection against adverse microenvironment, however, structure and composition of cell wall of chlamydospore is not very clear (Hazel and Williams 1990; Citiulo et al. 2009; Jansons and Nickerson 1970). Different environmental stresses/factors reported to modulate cell surface chemistry viz. cell surface hydrophobicity (CSH) and adhesion, the two important virulence factors facilitating survival of C. albicans (Odds and Bernaerts 1994). These morphophysiological modulations are results of modulation in central metabolic pathway that leads to modulations in cellular architecture and thus cell surface properties (Ingle et al. 2017). Cell surface properties are defined by cell surface chemistry, cell surface hydrophobicity (CSH) and adhesion etc. (Brown et al. 2014; Hazen et al. 1986; Hazen 1990; Klotz and Penn 1987). Cell surface properties are extremely important in survival and virulence of C. albicans (Odds and Bernaerts 1994). CSH determines host-Candida cell interaction, i.e. adhesion and colonization followed by tissue invasion at different tissue sites with varied microenvironments (Ener and Douglas 1992). In general, adhesion increases with increase in CSH i.e. CSH and adhesion is directly proportional (Ener and Douglas 1992; Klotz et al. 1985). More numbers of adhesins in C. albicans compared to non-virulent yeast S. cerevisiae cites the importance of cell surface molecules in pathogenicity (Guthrie and Fink 2002). Csh1 (Cell surface hydrophobicity protein 1) was down regulated in our study (Table 5). Down regulation of Csh1 during chlamydosporulation correlates positively with adhesion (Klotz 1990; Samaranayake et al. 2003). Decreased cell surface hydrophobicity is indicative of decreased virulence in chlamydospores (Fig. 2a) (Guthrie and Fink 2002; Samaranayake et al. 2003).

Our result suggests that enhanced mannan, β1, 3-glucan and chitin biosynthesis thicken and strengthen the cell wall of chlamydospore (Table 2) (CGD 2010). Increased components viz. β-glucan (β 1, 3-glucan) and chitin are reported to strengthen cell wall under hypo-osmotic and hypoxic stress (Table 2) (Ene et al. 2012; Hall 2015; Smits et al. 2001). Interestingly, down regulation of Kre9 (at protein and RNA level) and Phr2 indicates lack of β 1,6-glucan leading to abnormal cross-linking, however enhanced chitin content could be a compensatory response to maintain cell wall strength (Tables 2, 5 and Figs. 3, 4, 5) (Aimanianda et al. 2009; Lee et al. 2012; Smits et al. 2001). Over expression of two hyphae specific GPI (glycosylphosphatidylinositol) anchored proteins (Als3, Hyr1) involved in adhesion, indicates that sample exhibit hyphae producing chlamydospores and those were highly adhesive (Table 2, Fig. 5) (Heilmann et al. 2011; Klis et al. 2009; Richard et al. 2002). However, reduced hydrophobicity (down-regulation of Csh1) as well as down regulation of potential virulence factors (Pst3 and Ttr1) known to affect redox state of target proteins of host cells confirms attenuated virulence in chlamydospores (Table 2) (Collinson et al. 2002; Karababa et al. 2004; Seneviratne et al. 2008). Enhanced biosynthesis confirms that lipids (ergosterol, sphingolipid, phospholipids and fatty acids) are the storage molecules of chlamydospores in addition to providing more strength and rigidity to membranes (Table 2) (Fu et al. 2012; Walther et al. 2006; Young et al. 2002).

Fig. 5
figure 5

Real Time qPCR analysis of selected genes. Data is shown as mRNA copies in cells, where significance refers to the difference between chlamydospores and control (yeast form cells), (n = 3), ****P < 0.001, **P < 0.01, Bar indicates mean and error bars indicates SD

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Our proteomic data confirms that nutrient limiting and microaerophilic microenvironment is sensed by C. albicans through RAS mediated cAMP-PKA pathway that modulate metabolism to use complex carbohydrates like maltose (Fig. 6). Enhanced degradation of complex carbohydrates releases simple sugars and enhances glycolysis and TCA cycle, significantly. In addition to energy, enhanced glycolysis and TCA cycle provides precursors for biosynthesis of lipids and glycogen (energy reserve), sterol and fatty acids (strengthen membrane), chitin, mannan, Beta, 1–3, glucan (strengthen cell wall architecture) and glutathione and trehalose (potentiate stress tolerance) (Fig. 6). In general, we conclude that chlamydosporulation confer tolerance towards hostile microenvironment by strengthening cellular architecture and stress responses through modulations in metabolic pathways and thus survival of C. albicans.

Fig. 6
figure 6

Hypothetical model based on proteomic analysis showing modulation of cellular processes during chlamydosporulation

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