Heavy Metal -Induced Oxidative Stress And Alteration In Secretory Proteins In Yeast Isolates

In the recent years, yeasts have evolved as potent bioremediative candidates for the detoxication of xenobiotic compounds found in the natural environment. Candida sp. are well studied apart from Saccharomyces in heavy metal detoxication mechanisms. In the current study, Candida parapsilosis strain ODBG2, Candida sp. strain BANG3 and Candida viswanathii strain ODBG4 were isolated from industrial euents and contaminated ground water were studied for their metal tolerance. Among these three isolates, the metal tolerance was found to be more towards Lead (Pb 2mM), followed by Cadmium (Cd 1.5mM) and Chromium (Cr(VI), 1mM). On further exploring the involvement of primary defensive enzymes in these isolates exhibited towards metal tolerance, the anti-oxidative enzyme Superoxide dismutase (SOD) was found to be prominently high as 25% during rst 24h of metal-isolate interaction. In the Catalase (CAT) enzyme assay, it was observed that, the increased enzyme activity at 48h also triggered the activity of peroxidases (PO), which lead to the increase in reduced glutathione (GSH) in the organism by 0.87-1.9 folds, as a metal chelator and also as a second line of defensive molecule. The exoproteome prole showed the early involvement (exponential growth phase) of secreted proteins (low molecular weight) of about ~40-45kDa under Cd and Pb stress (0.5mM). The exoproteome proling under heavy metal stress in Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 is the rst report.


Introduction
Metals play an important role in homeostasis n the biological systems. Among the metals few are considered as hazardous to living biota and characterized as heavy metals (HM)s and metalloids. The In the global scenario, the scarcity of portable water is on an higher end due contaminants in fresh water system and as well as ground water due to geogenic origin as well as percolating water from industries, that too especially with arsenic (As). The HMs like mercury (Hg), lead (Pb), cadmium (Cd), uoride (F), accounts as major contaminants (Mohankumar et al. 2016; Shaji et al. 2021). Certain microbes like bacteria and fungi form habitat naturally in these contaminated sites by developing strong biochemical defense mechanisms to thrive under harsh conditions (Bhavya et al. 2015;Geetha et al. 2020). Among the microbes, lamentous fungi and yeasts have evolved strongly and potraying has potent bioremeditative candidates for xenobiotic compounds (Dey et al. 2020). The recent studies, have reported on Candida spp. especially Candida tropicalis, Candida albicans, Candida parapsilopsis, Candida etchellsi, Candida lipolytica, as heavy metal resistant yeasts (Kachiprath et al. 2019). The fungi/yeast possess a versatile group of mechanisms such as biosorption, bioaccumulation, sequestration which aids in the detoxi cation of heavy metals. Apart from these mechanisms, primary way to combat the metal stress is through the action of antioxidant enzymes such as catalase (CAT), peroxidases (PO), glutathione peroxidases (GPx), superoxide dismutase (SOD), glutathione s transferase (GSTs), etc.,. The primary targets of these enzymes are the reactive oxygen species (ROS) generated through biotic and abiotic stresses (Bandyopadhyay et al. 1999) thus reducing the toxicity as well as activation of series of enzymatic pathway and leading to production of low molecular compound like GSH, Metallothiones, oxalic acid, secondary metabolites, etc. These molecules interact with the heavy metals through -SH groups, rendering the inactivating heavy metals and rendering the homeostasis maintenance of the cell. The detoxi cation process is a combitorial strategy during which, involvements of various extracellular enzymes like lipase, DNAase, laccase are identi ed or observed (Santos et al. 2017). During stress and infectious state, yeast secretes various kinds of metabolites to its surrounding environment to regulate its growth and maintenance. The secretory molecules are not only encompassed of primary and secondary metabolites, exopolysaccharides, but also possess various sets of proteins known as exoproteome/extracellular proteins. These metabolites and exoproteins help the fungi to adapt for the harsh changes in its living habitat. The pro ling of exoproteins in fungi under heavy metal stress helps in understanding the stress response systems (Selvam et al. 2015).
Based on this knowledge, the current work was targeted to study the role of SOD and CAT, a antioxidant enzymes and reduced GSH levels at different time intervals and metal concentrations. Further, as there is a lacuna in the eld of exoproteome stress biology; preliminary work was carried out to know the secretory proteome pro les on SDS-PAGE. This part of work has been reported for the rst time in Candida parapsilosis and Candida viswanathii strains isolated from heavy metal contaminated water source from the prevailing study.

Isolation of yeasts from industrial e uents.
The textile e uent samples and heavy metal contaminated ground water samples were collected from Bengaluru and Odisha industrial areas respectively and brought to the laboratory, stored at ambient temperature till further utilization. Isolation of yeasts was carried out by spread plate technique using 100µl of each sample plated separately on yeast extract dextrose agar media (YPDA: yeast extract 1%, peptone and dextrose 2% each, agar 1.5%; pH 6.2) and incubated at 35 0 C for 48hr. The colonies were selected and pure cultures were used for further studies.

Preparation of metal stock solutions
The metal salts as a source of heavy metal (HM) ions used for the experiments were cadmium chloride (CdCl 2 ), lead nitrate [Pb(NO 3 ) 2 ], and potassium dichromate (K 2 Cr 2 O 7 ). Each HM stock solution (50mM) was prepared in deionized water and stored at room temperature.

Determination of MIC (Minimum inhibitory concentration) /Metal Tolerance Test.
The MIC towards the heavy metals was determined at different metal ion concentrations for the yeast isolates at a constant temperature of 35 0 C in YPD broth media. The actively growing cells (optical density 1.5 at 600nm) were transferred into YPD broth amended with heavy metal salts and the growth was recorded at 24hr interval till 120hr. The isolate growing in non-heavy metal-amended media was considered as a control. The concentration at which the cultures exhibited minimal growth was considered as MIC.
2.4 Molecular Identi cation of heavy meal tolerant yeast isolates/strains. Colony PCR technique was chosen for the ampli cation of 5.8s rRNA gene by utilizing the universal primers, the forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the reverse primer ITS4 (5' TCCTCCGCTTATTGATATGC-3') (White et al. 1990). In brief, the log phase growing colonies were selected and dispensed in 50µl of deionized water. From this, 2 µl was used for ampli cation. The PCR reaction mixture consisted of ready to use master mix 20 µl (TaKaRa Taq HS perfect Mix), forward primer (2 µl -ITS 1), reverse primer (2 µl -ITS 4), 2 µl cells as template and 14 µl of sterile water. PCR was carried out at 95 0 C for 5 min 30sec for initial denaturation followed by 30 cycles of denaturation at 94 0 C for 45sec, annealing at 56 0 C for 55sec, and extension at 72 0 C for 60sec followed by nal extension for 10min at 72 0 C. PCR products (amplicons) were analyzed on 1.2% agarose gel using 1X Tris borate EDTA buffer (1X TBE). The PCR amplicons were sent for Sanger sequencing to Juniper Life science Pvt Ltd, Bengaluru for sequencing using ITS 1 (forward primer). The obtained sequences were identi ed by using nBLAST from NBCI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were deposited in Gene Bank and accession numbers were obtained. The sequence alignment and phylogenetic tree was carried out among the identi ed stains using MEGA X (Kumar et al. 2018).

5.1. Enzyme extraction:
The protein extraction was carried out according to Ilyas et al. (2016) with slight modi cation. In brief, cells were harvested by centrifuging at 12000rpm, washed thrice in phosphate buffer of pH 7. Cells were suspended in 1.5ml of extraction buffer containing 50mM Tris-HCl buffer of pH 7.4, 5mM EDTA, 0.5M sucrose, 100mM KCl and 1mM PMSF. Cells were sonicated for 20s with 50s interval at 25Hz for 3 cycles, and centrifuged at 10000rpm for 12min (4 0 C). Protein concentration in the sample was determined by Bradford method and BSA as a standard (Bradford, 1976). The extracts were aliquot and stored at 4 0 C until further use. 200mM EDTA, 0.1% quercetin and crude enzyme extract. The reaction was monitored at 406nm for 1 min. Percentage inhibition of autoxidation of quercetin was determined.

Catalase (CAT) assay
The CAT activity was carried out according to Zeng et al. (2012). The reaction mixture contained of 50mM potassium phosphate buffer of pH 7, 8mM hydrogen peroxide (H 2 O 2 ) (30%), and the crude enzyme extract. The decrease in the absorbance at 240nm was recorded for 2min. The activity was calculated and expressed as µmoles of H 2 O 2 decomposed/min/mg of protein.

Anti-oxidant biomarker Assay
Reduced Glutathione (GSH) assay The reduced GSH level in the samples was determined according to Ellman's method (Ellman, 1959) with slight modi cation. The cells were homogenized in extraction buffer consisting of 0.6% sulfosalicyclic acid, 0.1% Tritonx100 in 0.1M potassium phosphate buffer pH 7.5 with 5mM EDTA. The cell lysate was collected by centrifuging at 8000rpm for 10min at 4 0 C. For the determination of reduced GSH content, to 100µl of about extract, Tris-HCl buffer of pH 8.2, 10mM DTNB was added and incubated for 10min at ambient temperature. The reaction concoction was spinned at 3,000g for 5min. The absorbance of clear supernatant was measured at 415nm. The amount of reduced GSH in the sample was determined by using standard reduced GSH curve. The amount of reduced GSH was expressed in the terms of µM/mg of protein.

Secretory protein analysis by SDS-PAGE -A preliminary study
The cells were separated from the culture broth by centrifuging at 12,000rpm for 20min at 4 0 C. The ltrate was ltered through 0.22µm membrane to remove the cell debris. The proteins were precipitated by TCA-acetone precipitation method with modi cation (Wu et al. 2014 andChen et al. 2015). Brie y, the ltrate was added with 10% TCA followed by overnight incubation at 4 0 C. The precipitated proteins were separated by centrifuging at 12,000rpm, 15min at 4 0 C. Then the proteins were suspended in 80% ice-cold acetone encompassed of 0.07% β-mercaptoethanol and incubated for 2hrs at 4 0 C. The contents were centrifuged; pellet was washed thrice with methanol and acetone respectively. Finally, the protein pellets were air-dried and solubilized in buffer containing urea (7M), thiourea (2M), CHAPS (1%) and 65mM DTT.
The Bradford method (Bradford, 1976) was adapted for determination of protein concentration in the samples. The 60µg of protein was suspended in SDS-sample buffer (Tris-Hcl buffer of pH 6.8, SDS, βmercaptoethanol, glycerol and bromophenol blue), heated at 95 0 C for 5min. An equal amount of protein (60µg/sample) was loaded along with the marker; the proteins were separated on 10% SDSpolyacrylamide mini gel on constant voltage of 70V for 30min, followed by 80V till the complete run. The gel was stained by CBB staining technique and differences in the banding pattern was noted (Laemmli, 1970).
Results And Discussion 3.1. Isolation of yeasts from industrial e uents and contaminated ground water.
The industrial e uent collected from the Bengaluru and contaminated ground water from Gajapathi area in Odisha, lead to the isolation, growth and axenic culturing of one and two yeast colonies respectively. The isolates were coded as BANG3, ODBG2, and ODBG4 for further studies and molecular identi cation.
The microscopic observation exhibited the single cell structure ovoid in shapes with various budding stages along with clear and distinguished cell wall structure. Figure 01 represents the macroscopic and microscopic views of the yeast isolates. There were not much changes in the morphological and microscopic observations, thus the exact identi cation the isolates was not revealed, and indicated the necessity of molecular based identi cation. The PCR amplicons of the isolates with the codes ODBG2, BANG3, and ODBG4 resulted in the product of 500bp size (Fig. 3). Further the sequencing of the region ITS1, 5.8rRNA and ITS2 using ITS 1 (forward primer) of the amplicons ODBG2, BANG3, and ODBG4 resulted in 493bp, 438bp, and 447bp product. BLAST analysis showed > 98% identity with sequences of Candida parapsilosis (KX652405, JN989529, MNT33072) with respect to our isolate code ODBG2, Candida sp. (MH802509, MN124747, KJ706734) with respective isolate code BANG3 and Candida vishwanathii (KU729067, MK394124,KC608220) with respect isolate code ODBG4. The obtained and identi ed sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and Accession numbers were obtained. The code of isolates and accession no. has been represented in the table 02.
The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei, 1993). Evolutionary analyses was conducted in MEGA X (Felsenstein, 1985;Kumar et al., 2018), Fig. 4 represence the phylogenetic tree.

Effect of heavy metal-induced oxidative stress on SOD activity
The primary defense antioxidant enzymes in any biological system are attributed towards the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidases (PO). The activity of SOD was detected based on the percentage inhibition of quercertin autoxidation in the control and treated samples. The SOD activity in Candida parapsilosis strain ODBG2 was found to be high in the range of 16% (Cd 0.5mM), 27.5% (Cd 1.0mM), 22.5% (Pb 0.5mM) and 25% (Pb 1.0mM) during rst 24hr of stress, however the gradual decrease in the abundance at 48hr and thereafter (Fig. 5A). The SOD activity in Candida viswanathii strain ODBG4 was relatively high for Cd (0.5mM concentration) at 24hr, for Pb (1mM) it slightly increased at 48 and 72hr (Fig. 5B), indicating minimal effect on SOD. SOD is the primary defense enzyme found to be active during abiotic stress-induced intracellular ROS in biological systems, which interacts and converts into H 2 O 2 and water molecule, leading to the sequential activation of other primary defense enzymes such as CAT and PO, subsequently the non-enzymatic small compounds like GSH (Bandyopadhyay et al., 1999). The varied response of SOD with respective different time intervals and concentrations has been observed in fungi such as Pleurotus ostretus HAU-2 upon Pb (Zhang et al., 2016), Trichosporon asahii upon different heavy metal and metalloid treatments (Ilyas et al., 2014). SOD was found to be not effective at the higher concentration rate and incubation time, which is indicating the role of CAT and PO in the system.

Effect of heavy metals induced oxidative stress enzyme -CAT
The CAT showed increased activity of 38 and 40 µmoles/mg of protein/min upon Pb stress at 05mM and 1.0mM concentration during at 48hr incubation, subsequently decreased on 72hr of incubation with 20 and 28 µmoles mg of protein/min in Candida parapsilosis strain ODBG2 (Fig. 6A). Similarly, CAT activity was higher at 48hr under Pb stress at both the concentrations with 15 and 16 µmoles/mg of protein/min in Candida viswanathii strain ODBG4, whereas the Cd stress at 0.5mM induced an steady increase with activity of about 16 µmoles and 18 µmoles/mg of protein/min at 48hr and 72hr respectively (Fig. 6 B).
The activity of CAT was found to vary based on the metal ion concentration, time and the isolate. The high CAT activity in the basal cells might trigger other relative enzymes such as PO, which might be strategic in mitigating the toxicity of heavy metal ion such as Cd, thus exhibiting steady increase under Cd stress (Pradhan et al., 2017).

Effect of heavy metal-induced oxidative stress on non-enzymatic antioxidant -Reduced GSH
The intracellular GSH plays a key role for maintaining cell homeostasis under normal and stress conditions. In the current study, the levels of reduced GSH in the cells under heavy metal stress was found to be time and concentration-dependent with respect to the treated yeast isolates. In Candida parapsilosis strain ODBG2, the levels of reduced GSH under Cd and Pb stress at 24hr was found to be lesser to that of untreated cells, whereas at 48hr there was an increase by 1.9 fold (Cd 1.0mM) followed by 0.87 fold in Pb (1.0mM) at 48hr. In Candida viswananthii strain ODBG4, a maximum of 1.6 and 1.7 fold increased reduced GSH was found at Cd and Pb (1.0mM) concentration. Increased GSH content at 48hr under Pb stress is similar to the studies by Rehaman and Anjum (2011), with Cd stress in Saccharomyces spp. (Fauchon et al., 2002). Further, decrease in reduced GSH at 72h under metal treatment might be due to the interaction of SH-group of GSH with that of heavy metal, the protective ability towards the toxicity and detoxi cation of heavy metals (Gharieb and Gadd, 2004;Ilyas et al., 2017) or due to the increased intracellular ions induced activation of other pathways, which leads to imbalance riot in homeostasis (Zhang et al., 2016). The reduced GSH content under different metal stress and at different time intervals is represented in the Fig. 7(A) and Fig. 7(B) for the Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 respectively.

Alterations in Secretory proteins upon different heavy metal stress in Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 by SDS-PAGE.
Candida parapsilosis strain ODBG2 and Candida viswanantii strain ODBG4 extracted at 48hr of postheavy metal stress (0.5mM concentration) of Pb(II), Cr(IV) and Cd (II) showed similar secretory protein pro les on 10% SDS-PAGE (Fig: 8.A and Fig. 8.B). In the present study, the SDS-PAGE pro le showed two (2) over-expressed and intense protein bands in the molecular range of ~ 40-45kDa under Cd stress and faint polymorphic bands in the range of ~ 66-116kDa. The Pb stress also signposted the secretory protein prominently expressed approximately at − 43-44kDa. The similar pro ling under Cd and Pb in these isolates might be attributed to similar kind of signal perception, gene activation, protein and 1 0 and 2 0 metabolite production at the species level. Since this work is been carried out for the rst time, further studies are required for specifying these expressed protein bands at various molecular ranges.

Conclusion
The current study is emphasized on the differential ability of heavy metal tolerance among Candida sp. i.e Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG2 isolated from industrial e uent-mediated contaminated ground water system. These isolates have an ability to be used as potent heavy metal detoxi ers in contaminated environment. It can be presumed that the potentiality of heavy metal tolerance towards Cd and Pb is due to communication in intra and extra-cellular secretory molecules modulated cell signaling during log phase growth under heavy metal stress. This contemporary work, has paved a way for further investigations on exploring exoproteome strategies in Candida sp. upon heavy metal stress.