Adaptive responses of marine-derived Aspergillus terreus to salinity and chromium stress analysed by peptide mass fingerprinting

Industrialisation and infrastructure development require extensive extraction and use of heavy metals, generating large volumes of waste that often enter the environment. Heavy metal pollution remains a pressing concern due to its severe effects on human health and the lack of sustainable remediation solutions. Among these, chromium is one of the most carcinogenic metals, widely used in tanneries, steel/electroplating industries, and the textile industry^[1,2]. Over the past 30 years, nearly 30,000 tons has been released into the environment globally^[3]. In its hexavalent form, chromium mimics the sulphate oxyanion, allowing easy transmembrane transport into prokaryotic and eukaryotic cells. Once inside, it is reduced to the trivalent form, generating reactive oxygen species (ROS) that induce lipid peroxidation, DNA damage, and protein degradation^[2,4,5]. Despite extensive documentation of chromium contamination in industrial effluents, the persistent toxicity of hexavalent chromium (Cr⁺⁶) remains a major unresolved environmental and public health challenge. While conventional physical and chemical methods can partially mitigate heavy metal contamination, these technologies fail to remove residual Cr⁺⁶, especially in saline wastewaters. This gap underscores the urgent need for eco-friendly biological strategies that can function under high ionic stress.

Marine-derived fungi have emerged as promising candidates for bioremediation in saline and metal-polluted environments. Unlike many terrestrial microbes, they exhibit inherent halotolerance, osmotic adjustment via compatible solutes (e.g., glycerol and trehalose), halophilic enzymes, and robust ion-transport systems^[6,7]. These adaptations allow survival in hypersaline and alkaline habitats where conventional remediation agents fail. Several genera, including Aspergillus, Penicillium, and Fusarium, have demonstrated the ability to take up heavy metals, biosorb them, and enzymatically detoxify them under saline conditions^[6,8]. Their ability to couple extracellular mechanisms (biosorption, bioleaching, precipitation) with intracellular strategies (enzymatic Cr⁺⁶ reduction, vacuolar sequestration, metallothionein production) makes them ideal candidates for sustainable wastewater treatment^[7−9].

Salinity levels in contaminated habitats can vary widely, and hypersaline conditions (> 100 PSU) are encountered in evaporation ponds, tannery discharge sites, and industrial brines^[9,10]. In this study, a salinity of 100 PSU was used to represent such extreme ionic environments, thereby enabling assessment of fungal performance in scenarios where conventional bioremediation agents would be ineffective. Cr⁺⁶ was chosen as the test metal because of its toxicity challenge posed for detoxification studies. The isolate Aspergillus terreus (NCBI accession no. KT956259) was selected based on preliminary screening that revealed exceptional Cr⁺⁶ tolerance (up to 750 mg/L) and removal efficiency (13 mg Cr g⁻¹ biomass at 300 mg/L). Its marine origin suggests inherent halotolerance, and prior scanning electron microscopy (SEM) imaging and diphenylcarbazide assays indicated that both biosorption and enzymatic reduction are primary survival strategies. These features make it an ideal candidate for exploring molecular adaptations under simultaneous salt and metal stresses. Aspergillus niger and Aspergillus terreus have been reported to have potential mycoremediative capabilities for Cd²⁺ removal^[11]. ESI-LCMS analysis of the crude extract of A. terreus revealed the expression of two new lumazine-containing peptides with pharmacological activities, thereby promoting this species for multiple applications^[12]. These studies collectively strengthen the ecological and biotechnological significance of A. terreus and support it as a candidate organism for remediation-driven research. At the cellular level, tolerance to heavy metals and salinity is mediated by a suite of stress-responsive proteins. Transporter families such as ATP-binding cassette (ABC) transporters, cation diffusion facilitators (CDF), and P-type ATPases play central roles in metal efflux and compartmentation^[13,14]. Redox regulators, including thioredoxin and glutathione reductase, along with antioxidant enzymes such as catalases and peroxidases mitigate ROS toxicity^[15,16]. Heat shock proteins and T-complex proteins further assist in protein folding and stabilization under stress^[17].

Despite increasing recognition of fungal tolerance to heavy metals and salinity, most studies have examined these stressors in isolation, primarily using physiological assays or targeted biochemical measurements^[15−18]. In contrast, saline industrial effluents typically impose concurrent ionic and metal stress, creating synergistic cellular pressures that cannot be predicted from single-factor experiments^[9,10]. Although proteomic analyses have been conducted in selected terrestrial or clinical fungi^[19,20], the integrated molecular responses of marine-derived filamentous fungi under combined stress remain poorly understood. In particular, Aspergillus terreus, a species noted for environmental resilience and biotechnological potential^[21−23], has not been systematically investigated at the global proteome level under simultaneous salinity and chromium exposure. To address this gap, we employed LC-MS/MS-based peptide mass fingerprinting to profile whole-cell protein expression across nine salt-metal regimes. This systems-level strategy reveals coordinated stress-response pathways, metabolic prioritisation, and adaptive survival mechanisms that cannot be inferred from conventional growth or enzymatic assays, thereby providing a new mechanistic insight into fungal adaptation to complex environmental stressors. Accordingly, this study aimed to: (1) characterise the proteomic response of A. terreus under individual and combined Cr⁺⁶ and salinity stresses, (2) identify key tolerance pathways involving ROS detoxification, ion homeostasis, and metal reduction, and (3) evaluate the expression of housekeeping proteins under multi-stress exposure to gain insights into metabolic prioritisation.

Aspergillus terreus, #NIOSN-SK56-S52, is a previously isolated culture from Arabian Sea sediments, identified using molecular techniques (NCBI accession no. KT956259), and has been previously reported for chromium tolerance^[8].

Extraction of proteins

Non-sporulated crushed mycelia of previously grown culture #NIOSN-SK56-S52 were inoculated in Czapek Dox Broth (CDB) with varying concentrations of Cr⁺⁶ (0, 100, and 500 mg/L), each prepared with salt solutions of 0, 35, and 100 PSU, in triplicate. The nine different metal-salt combinations were incubated at 80 rpm at 28 °C for 1 week, following which the contents were filtered, lyophilised, weighed, and processed for protein extraction. A previously standardised 3-buffer extraction method^[24] was used to extract proteins from a fixed biomass quantity (30 mg). Briefly, the biomass was subjected to buffer I (Tris–MgCl₂, pH 8.3, 0.5 M Tris–HCl, 2% CHAPS, 20 mM MgCl₂, 2% DTT); buffer II (9 M urea, 4% CHAPS, 100 mM DTT—prepared in 40 mM Tris–HCl, pH 8.0); and buffer 3 (6 M urea, 3 M thiourea, 4% CHAPS, 100 mM DTT—prepared in 40 mM Tris–HCl, pH 8.0). The samples were homogenised at 6.5 m/s for 45 s after each buffer addition and centrifuged to separate the fractions. All three fractions were mixed, and the extracted proteins were quantified using the standard Folin's method and analyzed by SDS-PAGE to assess quality before proceeding with MS analysis.

Tryptic digestion and LC-MS separation

Prior to MS analysis, the protein extracts were processed for In-solution digestion using trypsin (protocol modified from Kinter & Sherman^[25]). The extract was initially precipitated with HPLC-grade methanol, vacuum-dried, and suspended in urea to facilitate dissolution. Subsequently, the protein samples were reduced using dithiothreitol (DTT) and further alkylated with Iodoacetamide (IAA). The samples were digested using trypsin and analysed using the LC-MS QToF facility (6538 UHD Accurate Mass QTOF LC/MS, Agilent Technologies, USA). Digested protein samples (8 µL, four replicates) were injected in a 150 × 300 A C18 150 mm column protein chip via an autosampler. A gradient of 3%–97% across HPLC-grade acetonitrile as an organic phase, and 0.22 µm filtered and autoclaved deionised water as an aqueous phase was used to carry out LC separations over a time period of 100 min. Formic acid (0.1%) was added as an additive in both phases. An accuracy rate of 2 mg/L was maintained for the separated ions in positive mode over the range of 50–2,000 m/z. The spectral data were acquired using Mass Hunter software v5.0 (Agilent Technologies, USA) after MS/MS, and were searched in NCBI using Spectrum Mill MS Proteomics Workbench vB.04.01.141 (Agilent Technologies, USA) at 50–100 mg/L accuracy against the organism-specific database (NCBI txid 33178). Protein sequences corresponding to the differentially expressed proteins (DEPs) were retrieved from the NCBI Aspergillus terreus genome. The MS searches were auto-validated (false discovery rate of 1.2%), and protein summary and MPP files were generated. These files were processed in Mass Profiler Professional v14.09 (MPP13, Agilent Technologies, USA) for further analysis. Mapping and annotation steps were carried out to assign GO terms under the three ontologies: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). Differential protein abundance was calculated from MS intensity values using log 2-transformed fold changes. Multivariate differences among treatment groups were examined using Principal Component Analysis based on Euclidean distances. Statistical significance was set at p < 0.05.

Protein profiling of A. terreus, #NIOSN-SK56-S52, identified 659 proteins expressed across the nine different metal-salt combinations. Based on their functional role as elucidated from KEGG pathways and metabolic processes, they were divided into 17 different categories (Fig. 1). Amino acid metabolism (41), biosynthesis of secondary metabolites (6), carbohydrate metabolism (63), cellular processes (30), energy metabolism (30), environmental information processing (38), genetic information processing (146), glycan biosynthesis and metabolism (3), lipid metabolism (12), metabolism of cofactors and vitamins (10), metabolism of other amino acids (5), metabolism of terpenoids and polyketides (4), nucleotide metabolism (16), stress response (32), xenobiotics biodegradation and metabolism (4), hypothetical proteins (213), and uncharacterised proteins (6). To further understand the contribution of proteins under each functional process under salinity stress, proteins expressed only under 0, 35, and 100 PSU were analysed (Fig. 2). Most proteins expressed at 100 PSU belonged to carbohydrate metabolism, genetic information processing, and hypothetical proteins.

Figure 1. 

Graphical representation of all proteins expressed by A. terreus (#NIOSN-SK56-S52) %.

Figure 2. 

Distribution of proteins expressed by A. terreus (#NIOSN-SK56-S52) under 0, 35, and 100 PSU salinity.

Amongst the expressed proteins, 53 were commonly expressed across all nine conditions, suggesting they are involved in housekeeping metabolic activities (Table 1). Amongst these, 16 proteins (cytochrome c oxidase polypeptide IV (NCBI gi–114188084), cytochrome c oxidase polypeptide VI (NCBI gi−114188679), enolase (NCBI gi–114194476), eukaryotic initiation factor 4A (NCBI gi–114196711), Glucose-6-phosphate isomerase (NCBI gi–114193156), glyceraldehyde-3-phosphate dehydrogenase (NCBI gi–540849772), heat shock 70 kDa protein (NCBI gi–114187927), histone H2A (NCBI gi–114189434), histone H2B (NCBI gi–114189435), malate dehydrogenase (NCBI gi–114191202), phosphoglycerate kinase (NCBI gi–114197170), pyruvate kinase (NCBI gi–114193787), thioredoxin (NCBI gi–114190288), transaldolase (NCBI gi–114191388), transketolase 1 (NCBI gi–114197498), and tubulin beta chain (NCBI gi–114197233) have been previously reported as housekeeping genes in humans. In a separate study, reference genes in filamentous fungi suitable for RT-qPCR were studied and revealed nine such novel genes, namely, the β-actin, 18S rRNA, cyclophilin, histone H3, glyceraldehyde-3-phosphate dehydrogenase, α, β1, β2-tubulin, and ubiquitin.

A group of unique proteins expressed under specific conditions was also studied (Table 2). At 0 PSU, expression of proteins involved in biosynthetic pathways and genetic information processing was dominant, indicating a relatively stress-free physiological state consistent with the facultative marine nature of the isolate. In the presence of 500 mg/L of Cr⁺⁶, proteins such as WD-repeat protein mip1 (NCBI gi–114191519), involved in cell senescence, and nonhistone chromosomal protein 6A (NCBI gi–114193442), which is involved in replication and repair were expressed. In addition to Cr⁺⁶ stress in the presence of 35 PSU, magnesium transporter MRS2 (NCBI gi–114190882) and T-Complex protein one subunit delta (NCBI gi–114193665) were also expressed.

The protein regulation of A. terreus subjected to salinity and chromium stresses was analysed using MPP software (v14.09), and the up-/downregulated proteins are shown in Table 3. It was observed that proteins commonly expressed across all conditions were generally downregulated under metal stress at all salinities, except for a few, suggesting that stress affects the expression of housekeeping genes. The priority of organisms is survival, rather than growth and reproduction, leading to downregulation of these proteins. Oxidoreductive proteins, such as cytochrome c oxidase (NCBI gi–114188084), were found to be upregulated at both 35 and 100 PSU in the presence of Cr^+6, along with heat shock protein 82 (NCBI gi–114187927). The DNA damage checkpoint protein Rad24 (NCBI gi–114188055), involved in checkpoint response and DNA double-strand break repair was also found to be upregulated. Thioredoxin (NCBI gi–114190288), which is known to scavenge ROS actively, was upregulated at all salinities with chromium stress, with a fold change value of 5.011 and 5.532 at 100 and 500 mg/L Cr⁺⁶ under 35 PSU salinity, respectively. The catalase B precursor (NCBI gi–114190039) was downregulated at 35 PSU under Cr⁺⁶ stress. Antioxidant enzymes can be affected by free radical regeneration, and the expression of common housekeeping genes may also be altered. The downregulation of carbohydrate metabolism proteins indicated this: pyruvate kinase (NCBI gi−114193787) and transketolase 1 (NCBI gi–114197498) at 0 and 100 PSU, respectively, when subjected to Cr⁺⁶.

This study provides an in-depth mechanistic insight into how a marine-derived Aspergillus terreus isolate responds to simultaneous hypersaline (100 PSU) and Cr⁺⁶ stress—conditions that mimic industrial effluents from tanneries, electroplating, and salt-production sites where heavy metals and high ionic strength co-occur. Cr⁺⁶ is recognised for its high mobility, toxicity, and ability to induce oxidative and genotoxic stress, while hypersalinity creates osmotic and ionic stress that impairs many traditional bioremediation microbes.

Metal tolerance in A. terreus has been extensively explored and well cited^[26,27]. When subjected to multi-metal stress, Aspergillus fumigatus PD-18, isolated from polluted banks of the Yamuna River, India expressed about 2,190 proteins, of which 387 were exclusively present under stressed conditions^[19]. In another study, 2,646 proteins were identified in Aspergillus ochraceus grown on 20 and 70 g/L NaCl substrates, resulting in elevated ergosterol production^[9]. In our study, we have shown a synergistic effect of hypersalinity and heavy metal stress on A. terreus.

Survival mechanisms in fungi are broadly categorised as extracellular mechanisms involving biosorption, bioleaching, biotransformation, and bioprecipitation, and intracellular mechanisms where enzymatic transformation, chelation, and multiple signalling pathways play a very important role, along with transporter proteins^[27]. The most prominent transporters identified in fungi are reported to be ATP-binding cassette (ABC) transporters, conjugated ABC transporters (CT), cation diffusion transporters (CDF), MIT family (Cor A metal ion transporters), SIT (siderophore ion transport) subfamily under major facilitator superfamily (MFS), mitochondrial carriers, P-type ATPases, and Nramp family transporters^[9,28]. Some of these were identified in the present study too (Table 2, Fig. 2). MacDiarmid & Gardner^[14] reported aluminium tolerance in Saccharomyces cerevisiae due to overexpression of Mg transporters (ALR1/ALR2). In another study, Mg transporters conferred resistance to Co⁺² growth inhibition in Salmonella typhimurium^[29].

Fungi in the marine environment initially need to adapt to high osmotic pressure and then devise mechanisms to tolerate any metal stress. Salinity stress alone (100 PSU) caused the expression of some of the very important proteins, such as the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key enzyme in the mevalonate pathway and sterol biosynthesis^[17], as reported in this study. Aspergillus terreus (NTOU4989) isolated from a Kueishan Island hydrothermal vent showed upregulation of glycerol lipid metabolism, mitogen-activated protein kinase, and pyrroline-5-carboxylate dehydrogenase genes in the presence of salt^[21]. Another study reported overexpression of specific alkaloids in A. terreus, an endophyte isolated from Porites pukoensis, Xuwen coral reef, China, in the presence of 10% salt in PDB, known to modulate cell membranes and regulate membrane fluidity^[22]. Interestingly, the endophytic fungus A. terreus isolated from the roots of Chenopodium album (wheat) under saline conditions showed low activities of ascorbate, catalase, and peroxidase^[23]. Collectively, these three studies demonstrated that diverse A. terreus strains modulate both primary and secondary metabolism under salinity stress, regardless of ecological origin. Transcriptional activation of osmoadaptive pathways and alkaloid biosynthesis demonstrates the multi-faceted influence of salinity on metabolic output and stress physiology in fungi. The synergistic effect of salinity and Cr⁺⁶ stress led to four major stress response-related mechanisms in the current study (Fig. 3).

Figure 3. 

Mechanism of metal tolerance by A. terreus (#NIOSN-SK56-S52) under salinity and chromium stress.

Sodium ions, which are themselves toxic, act as signalling molecules to open up non-selective cation channels within the cell membrane, causing activation of Ca⁺² transporters, MAPK pathways, ROS generation, and activation of subsequent repair mechanisms such as SOS repair and production of NADPH oxidase^[30] HMG-CoA, also identified as the biochemical signature of halophily, is reportedly involved in post-translational modification by prenylation in H. werneckii and catering to metabolic demands under hypo-, hypersaline conditions^[31]. Protein kinases, a group of silent stress busters, are widely reported and cited in eukaryotic systems for regulatory roles in managing stress response^[32]. Expression of proteins involved in signalling, regulation, or the promotion of protein kinase production is indicative of stressful conditions. Almeida-Dalmet et al.^[33] reported upregulated expression of the protein kinase gene and phenyl acetyl CoA ligase gene under salinity and temperature stresses in the Haloarcula strain.

ROS-generated stress leads to the production of proteins such as kinases and phosphatases, which may be grouped as salt stress proteins that further enhance the production of proteins involved in cell wall building, nutrient metabolism, carbohydrate metabolism, and cell enlargement and division^[34]. Both enzymatic (catalase, superoxide dismutase, peroxidases, glutathione reductases) and non-enzymatic (ascorbic acid, glutathione, phenolics, proline) antioxidants have immense ability to scavenge ROS^[18]. Ankyrin repeats have also been reported to participate in ROS under salt stress^[35]. Some of these proteins were expressed under salinity stress in the current study (Tables 1, 2). Proteins such as thioredoxin, catalase, superoxide dismutase, and glutathione S-transferase, along with heat shock proteins have been previously reported in the opportunistic pathogen, A. terreus, when subjected to 37 °C for 48 h and exposed to amphotericin B^[20]. Toxicity of metal, once within the cell, can be curbed using multiple measures, one of which is organelle compartmentation or vacuolar accumulation after being chelated using glutathione, metallothioneins, or phytochelatins, as in the case of hyperaccumulating plants^[36,37]. Expression of these T-complex proteins (TCPs) has been reported to be dominant under salt stress, along with heat shock proteins^[38], and was detected under 35 and 100 PSU salt stress in the current study. Elevated salt concentrations may cause physiological changes in subjected organisms and trigger molecular, cellular, and metabolic responses. Expression of stress proteins, proteins involved in oxidative phosphorylation, and transporter proteins was observed under Cr⁺⁶ stress and increasing salinity, indicating a synergistic effect of both stresses on the culture.

Housekeeping proteins are vital for survival and are expressed in organisms at all times, irrespective of favourable or stressful conditions^[39]. However, their expression may be altered or become irregular under certain conditions^[40]. We found changes in the expressions of some housekeeping proteins in response to the stress imparted. Glyceraldehyde-3-phosphate dehydrogenase expression was downregulated at 0 PSU salt concentration in the presence of Cr⁺⁶, but was upregulated at 100 PSU (Table 3). Similarly, the expression of the Tubulin beta chain varied under different salt concentrations in the presence of metal. Differential expression of glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes was also reported during the course of hepatitis C virus-induced hepatocellular carcinoma (HCV-HCC)^[41]. Further, this differential expression property has previously been used to diagnose lung cancer^[42]. Therefore, any drastic variations in the expression levels of such genes may shed light on an organism's adaptive strategies in the presence of stress. On the contrary, relying solely on the expression of housekeeping genes as an internal reference may not always generate dependable results. A study by Irie et al.^[43] reported that atorvastatin altered the expression of housekeeping genes, thereby misleading the data obtained for the target gene expression set, and emphasised caution when using internal reference gene expression data, especially in cancer-related studies. Such caution during experimental setup has also been reported previously^[44]. Although RT-qPCR analysis of this gene expression in the current study would shed more light on this conclusion, the preliminary experiments also provide reliable proof.

This study shows that the marine-derived A. terreus employs a coordinated and multi-layered defense network to survive the combined effects of Cr⁺⁶ stress and hypersaline (100 PSU) conditions. Based on proteomics results, the isolate primarily activates DNA damage control and repair systems, in addition to robust ROS detoxification mechanisms, when exposed to salinity alone, thereby protecting cellular components and genomic integrity. The addition of Cr⁺⁶ induces further adaptive responses, such as upregulation of membrane transporters to mediate metal and ion flux and the conversion of toxic Cr⁺⁶ to the less harmful Cr⁺³ via enzymatic reduction pathways. Together, these systems lessen the cumulative harm and enable the cell to redistribute resources from growth to essential survival functions. The ecological adaptability of A. terreus and its potential for bioremediation of saline, metal-contaminated wastewaters are highlighted by its ability to continue functioning under extreme ionic and oxidative pressures, where conventional microorganisms are ineffective. Together, these abilities contribute to the strategic application of marine fungi for high-salinity sustainable heavy-metal remediation technologies, and enhance our knowledge of fungal stress adaptation.