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Nickel tolerance is channeled through C-4 methyl sterol oxidase Erg25 in the sterol biosynthesis pathway [1]

['Amber R. Matha', 'Department Of Microbiology', 'University Of Georgia', 'Athens', 'Georgia', 'United States Of America', 'Xiaofeng Xie', 'Robert J. Maier', 'Xiaorong Lin']

Date: 2024-09

Nickel (Ni) is an abundant element on Earth and it can be toxic to all forms of life. Unlike our knowledge of other metals, little is known about the biochemical response to Ni overload. Previous studies in mammals have shown that Ni induces various physiological changes including redox stress, hypoxic responses, as well as cancer progression pathways. However, the primary cellular targets of nickel toxicity are unknown. Here, we used the environmental fungus Cryptococcus neoformans as a model organism to elucidate the cellular response to exogenous Ni. We discovered that Ni causes alterations in ergosterol (the fungal equivalent of mammalian cholesterol) and lipid biosynthesis, and that the Sterol Regulatory Element-Binding transcription factor Sre1 is required for Ni tolerance. Interestingly, overexpression of the C-4 methyl sterol oxidase gene ERG25, but not other genes in the ergosterol biosynthesis pathway tested, increases Ni tolerance in both the wild type and the sre1Δ mutant. Overexpression of ERG25 with mutations in the predicted binding pocket to a metal cation cofactor sensitizes Cryptococcus to nickel and abolishes its ability to rescue the Ni-induced growth defect of sre1Δ. As overexpression of a known nickel-binding protein Ure7 or Erg3 with a metal binding pocket similar to Erg7 does not impact on nickel tolerance, Erg25 does not appear to simply act as a nickel sink. Furthermore, nickel induces more profound and specific transcriptome changes in ergosterol biosynthetic genes compared to hypoxia. We conclude that Ni targets the sterol biosynthesis pathway primarily through Erg25 in fungi. Similar to the observation in C. neoformans, Ni exposure reduces sterols in human A549 lung epithelial cells, indicating that nickel toxicity on sterol biosynthesis is conserved.

Nickel is commonly known as an allergen and toxin for humans, but the way in which nickel causes adverse effects is unknown. We sought to use C. neoformans as a model to investigate the primary targets of nickel and how cells tolerate this commonly occurring metal. We found that in both mammalian cells and fungal cells, exposure to nickel causes sterol deficiency. We discovered that Erg25, an essential enzyme key to the production of ergosterol (fungal equivalent of cholesterol), was critical for cryptococcal cells to tolerate nickel. Cells unable to increase production of this enzyme in response to nickel exposure, such as the sre1Δ mutant with the Sterol Regulatory Element-Binding regulator disrupted, were incapable of growing in the presence of nickel. Therefore, it appears that both cells react to nickel through upregulating a conserved biochemical pathway and particularly the Erg25 enzyme. This work could guide future investigations into novel approaches to manage nickel toxicity.

Funding: This work was supported by National Institutes of Allergy and Infectious Diseases ( http://www.niaid.nih.gov ) (R01AI140719 to XL) and University of Georgia Gene E. Michaels endowment fund (to XL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Given that animals and fungi are closely related in the eukaryotic domain, understanding the effect of nickel on fungi and how fungi tolerate Ni could be informative. This study aims to identify pathways and factors critical for cryptococcal tolerance to Ni. We found that Ni exposure reduces ergosterol levels and altered lipid profiles in C. neoformans. We screened the transcription factor deletion set and identified Sre1 as an essential factor for cryptococcal growth on Ni-supplemented medium. Sre1 is highly conserved across Eukarya, and it is known to regulate the ergosterol biosynthesis pathway (EBP) in response to hypoxia and hypoxia-mimicking conditions [ 37 – 41 ]. We found that nickel, in contrast to hypoxia that exerts a broad impact on cryptococcal transcriptome, more narrowly but profoundly alters expression of EBP genes. Erg25, a conserved C-4 methyl sterol oxidase, but not other Erg enzymes in the sterol biosynthetic pathway or the known nickel-binding protein Ure7, is specifically required for cryptococcal tolerance to Ni. Increasing the levels of the C-4 methyl sterol oxidase effectively mitigates Ni toxicity, suggesting that Erg25 is a primary target of Ni toxicity. We further demonstrated that exposure to Ni in mammalian cells also reduced the sterol levels, mimicking what we observed in the fungus. Thus, nickel might exert its toxicity effects by primarily targeting the conserved sterol biosynthetic pathway in both fungi and animals.

Despite the fact that Ni is abundant in the environment, little research has been done to identify how environmental microbes tolerate this metal. Cryptococcus neoformans, a ubiquitous environmental fungus, is a model organism for studying fungal cellular biology due to the abundance of tools available to study and manipulate the fungus [ 18 – 21 ]. C. neoformans has been used as a model to better understand cellular mechanisms conserved in mammals such as uniparental mitochondrial inheritance [ 22 , 23 ], meiosis [ 24 – 27 ], epigenetic regulation [ 28 , 29 ], and intercellular communication [ 30 , 31 ]. Here, we chose this fungus to investigate the molecular mechanism for Ni tolerance. In C. neoformans, urease is the only known protein that requires Ni for its function, although there are nine known Ni-dependent enzymes in other microbes [ 32 ]. Despite the fact that the role of urease in cryptococcal pathogenesis is well defined [ 33 – 36 ], its role in nickel tolerance is unknown.

It is hypothesized that Ni is toxic to mammalian cells due to its ability to catalyze Fenton chemistry, which culminates in oxidative stress to the cells [ 7 , 8 ], including lipid peroxidation [ 9 – 11 ]. Ni is also capable of inducing calcium signaling pathways and activating HIF1-α [ 12 , 13 ], which plays a central role in the progression of some cancers [ 14 ]. HIF1-α is typically activated when cells experience hypoxia in the tumor microenvironment. This activation causes transcriptional increases in genes associated with angiogenesis, growth factors, pH regulation, and apoptosis [ 15 ]. Thus, Ni has been characterized as a carcinogen in mammalian systems. Additionally, Ni has been shown to induce a disturbance in testosterone synthesis [ 16 ]. The main therapy to mitigate Ni toxicity is administration of antioxidants, such as glutathione, which reduces lipid peroxidation in human lymphocytes [ 17 ]. Because of the pleiotropic effects of Ni on cell structures and metabolism, it is difficult to define the primary mechanism of nickel toxicity.

Ni is an abundant natural element ubiquitously found in soil and through industrial pollution [ 1 , 2 ]. The concentrations of this metal vary widely in different environments and organisms cope in various ways [ 2 ]. Unlike metals such as copper (Cu) or iron (Fe), no mammalian enzymes require Ni as a cofactor [ 3 ]. Indeed, Ni is generally characterized as a toxic heavy metal for humans. Exposure to Ni primarily occurs by inhalation or ingestion but also through interaction with everyday items that contain Ni, such as jewelry, zippers, paper clips, and stainless steel dining flatware [ 4 ]. Additionally, corrosion of Ni-containing implants used in joint and hip prostheses may lead to elevated Ni levels in the body [ 5 ]. Occupational exposure to nickel is the highest for those involved in producing, processing, and using nickel [ 6 ]. A National Occupational Exposure Survey conducted by the NIOSH agency from 1981 to 1983 estimated that 727,240 workers in the US were exposed to toxic levels of Ni (NIOSH 1990).

Results

Overexpression of ERG25, but not other four ERG genes tested, confers Ni tolerance Sre1 is known to regulate ergosterol biosynthesis, and our RNA-seq data showed that Ni causes an upregulation of multiple EBP genes. We postulated that ergosterol deficiency due to the SRE1 deletion may have contributed to sre1Δ Ni hypersensitivity. If this is true, then perturbations of the EBP pathway may also alter cryptococcal tolerance to Ni. So, we tested the deletion mutants of the non-essential ERG3 and ERG4 genes. Previous studies have shown that mutation of these genes perturbs ergosterol biosynthesis. For example, deletion of ERG3 causes increased resistance to azoles (target Erg11) and Amphotericin B (bind to ergosterol) in C. neoformans and in Candida species [55–57]. Deletion of ERG4 causes an increased sensitivity to caspofungin that targets β1–3 glucan synthase in the membrane [58]. erg3Δ and erg4Δ mutants grew like the wild type on RPMI+Ni (S6 Fig). Thus, it appears that perturbation of the EBP pathway in general does not alter cryptococcal tolerance to Ni. To further interrogate our hypothesis, we decided to examine the impact of overexpression of ERG genes on Ni tolerance. We chose to overexpress ERG genes because most ERG genes are essential and cannot be deleted. Overexpression of ERG genes has been adopted as an effective approach to study the EBP pathway in S. cerevisiae [59]. To that end, we selected and overexpressed five EBP genes −ERG2, ERG11, ERG25, ERG26, and ERG27 − in the wildtype and the sre1Δ backgrounds. To determine if these overexpressed ERG genes are functional, we first tested the susceptibility of these ERG gene overexpression strains in H99 background to fluconazole [60]. Fluconazole is an antifungal drug that inhibits Erg11, causing a reduction of ergosterol and a buildup of methylated sterols, collectively disrupting membrane stability [61]. We found that overexpression of any of the five EBP genes enhanced resistance to fluconazole, albeit at varied degrees (Fig 3A). As expected, overexpression of ERG11, the direct target of azole drugs, offered the highest level of resistance to fluconazole relative to the other ERG genes (Fig 3A). This result indicates that the overexpressed ERG genes are functional. However, when introduced to the sre1Δ mutant, none of the ERG overexpression was able to restore tolerance to fluconazole, with ERG11 being the only exception (Fig 3B). This result reaffirms that Erg11 is the direct target of fluconazole and that overexpression of ERG11 confers resistance to fluconazole regardless of the strain background. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Overexpression of ERG25, but not other ERG genes tested, drastically increases Ni tolerance in both wild type and sre1Δ. (A) H99 strains with overexpression of the indicated ERG genes (ERG2, 11, 25, 26, and 27) were serially diluted and plated on RPMI plates with fluconazole (Flu) at 4 or 8 μg/ml. (B) The sre1Δ strains with overexpression of the indicated ERG genes (ERG2, 11, 25, 26, and 27) were serially diluted and plated on RPMI plates with Flu at the indicated concentrations. (C) The same strains as in Panel A were spotted onto RPMI media with the indicated concentrations of Ni. (D) The same strains as in Panel B were spotted onto RPMI media with the indicated concentrations of Ni. All plates were incubated for two days prior to imaging. https://doi.org/10.1371/journal.pgen.1011413.g003 After confirmation that these overexpressed EBP genes are functional, we sought to determine the effect of overexpression of these EBP genes on Ni tolerance. In contrast to what we observed in fluconazole resistance, the ERG25OE strain in wildtype background was exceedingly tolerant of Ni. The ERG25OE strain grew much better than the wild type on RPMI+500μM Ni, a condition that all other strains, including WT, were unable to tolerate (Fig 3C). Overexpression of ERG25, a known direct target of Sre1 [39], conferred marked Ni tolerance to the sre1Δ mutant as well. The ERG25OE sre1Δ strain was much more tolerant to Ni than even the wildtype strain (Fig 3D). Interestingly, overexpression of any of the other ergosterol biosynthesis genes, including ERG11 and ERG2 that lie upstream and downstream of ERG25 respectively, did not confer Ni tolerance to either the wild type or the sre1Δ mutant. The overexpression of ERG26 and ERG27, which encode enzymes that complex with Erg25, did not impact Ni tolerance in either strain backgrounds. This suggests that Erg25 specifically, not the EBP in general, plays a major role in mediating cryptococcal tolerance to Ni. As we noted previously, hypoxia and Ni elicit both shared and distinct transcriptome changes in Cryptococcus, with Ni eliciting more profound and specific changes in the EBP pathway. ERG25 is the shared gene upregulated by both stressors. A previous study identified ERG25 as a multicopy suppressor of scp1Δ and sre1Δ sensitivity on CoCl 2 , a known hypoxia-mimicking agent [39]. We found that overexpression of ERG25 conferred tolerance to CoCl 2 in both wildtype and sre1Δ backgrounds (S7A and S7B Fig), in agreement with the previous study. Overexpression of other ERG genes tested failed to confer significant tolerance to CoCl 2 in either the wildtype or the sre1Δ background. When the strains were grown in hypoxia conditions, we found that all overexpression strains in the wildtype background grew similarly to the control (S7C Fig). However, the overexpression of ERG25, ERG2, ERG11, and ERG26 all rescued the sre1Δ hypoxia growth defect albeit in varying degrees in that order (S7D Fig). The ERG25 overexpression best rescued sre1Δ growth in hypoxia, which could be attributable to the upregulation of ERG25 in the hypoxia condition that was indicated by our RNA-seq data (Fig 2D). The results support that upregulation of the EBP pathway genes generally enhances growth in hypoxia and confers resistance to fluconazole, but ERG25 is specifically required for cryptococcal tolerance to cobalt and Ni.

Ni alters the cellular lipid profile Our results above demonstrate that Ni increases transcription of ergosterol genes and overexpression of ERG25 in particular increases cryptococcal tolerance of Ni. To examine if Ni indeed impacts ergosterol levels, we extracted cellular ergosterol from wildtype and sre1Δ cells cultured on RPMI medium with or without the addition of Ni, and quantified ergosterol levels by measuring the absorbance at 282nm [62]. We found that the ergosterol content in wild type was reduced by 10% when exposed to Ni (Fig 4A). As expected, the ergosterol level in sre1Δ cells was lower under the normal growth condition with 80% of that in wildtype cells, and exposure to Ni caused a further reduction to 65% of that in untreated H99 cells. When exposed to Ni, the resulting amount of ergosterol in sre1Δ cells was similar to that in H99 cells exposed to fluconazole (Fig 4A). Filipin staining of ergosterol present in the outer leaflet of plasma membrane [60,63,64] showed that Ni exposure caused a 50% reduction in plasma membrane ergosterol in H99 based on fluorescence intensity (Fig 4B and 4C), consistent with the lower ergosterol levels in the presence of nickel measured spectrophotometrically (Fig 4B and 4C). The fluorescence intensity of sre1Δ cells was 50% of the wildtype level. Ni exposure reduced the fluorescence intensity even further to about 16% of that in H99 cells grown on RPMI (Fig 4C). Although both measurements revealed the same trend, the stronger reduction caused by the SRE1 deletion or by Ni treatment measured by fluorescence intensity of filipin staining compared to spectrometry may be due to the fact that the extracts contain other lipids in addition to ergosterol or its intermediates. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Ni causes alteration of sterol profiles. (A) Ergosterol was extracted from the indicated strains grown on RPMI (C), RPMI+250μM Ni (Ni), or RPMI+4μg/mL Flu (F). The extract was measured at 282nm. Student’s t-test was used to assess statistical significance. ** = ≤0.01, **** = ≤0.0001 (B) H99 and sre1Δ cells were grown overnight on RPMI or RPMI+250μM Ni plates. Cells were harvested and incubated for 45 minutes in 25μM filipin III at 21°C in the dark. Cells were imaged with a Zeiss Imager M2 microscope. Scale bar = 5μm. (C) Quantification of fluorescence intensities of cells prepared as in panel B. RPMI (R), RPMI+ 250μM Ni (N). Mann-Whitney test was used to assess statistical significance **** = ≤0.0001. (D) Equal dry weight of wildtype cells grown on RPMI media alone (-) or RPMI+Ni (+) media were used to extract membrane sterols from indicated strains. 5μL of ergosterol extract were spotted onto glass backed HPTLC Silica gel plates. The white arrow indicates free fatty acids band. The yellow arrow indicates the methyl sterol band. The bottom band indicates ergosterol. (E) Lipid extractions from the indicated strains were spotted onto a TLC plate. 5μL of ergosterol extract was spotted onto glass backed HPTLC Silica gel plates as in panel D. https://doi.org/10.1371/journal.pgen.1011413.g004 Thin layer chromatography (TLC) analysis also revealed altered lipid profiles when cells were treated with Ni (Fig 4D). Based on previous literature using the same procedures for extraction and TLC [65], Ni causes an increase in the intensity of two of these bands, likely the methyl sterol (white arrow) and free fatty acid band (yellow arrow), and there is a slight decrease in the ergosterol band at the bottom (Fig 4D). Previous studies in yeast have shown that mutations that reduce the activity of ERG25 caused a similar increase in the intensity of the methyl sterol and free fatty acid bands [65]. This result corroborates our hypothesis that Ni primarily targets Erg25 in the EBP pathway. In agreement with other measurements, the TLC analysis also revealed that ergosterol level was reduced in the sre1Δ mutant. The thick ergosterol band was reduced compared to the wildtype strain when grown on RPMI medium, and this band was almost undetectable when the mutant was grown on RPMI with Ni (Fig 4E). The ERG25 overexpression strain still showed a decrease in ergosterol content when plated on Ni media (Fig 4D). However, when ERG25 was overexpressed in the sre1Δ background, an ergosterol band is still observable in contrast to the sre1Δ strain on Ni.

Two histidine residues in Erg25 metal binding motifs are important for Ni tolerance The results presented earlier indicate that Erg25 is critical for Ni tolerance in C. neoformans. Erg25 contains four conserved metal binding motifs enriched in histidine (S8 Fig). According to AlphaFold, several histidine residues are predicted to associate in a histidine-rich pocket present in Erg25 [66,67]. These histidine residues are from three of the four metal binding motifs and are not simply histidine residues proximal to each other in the primary sequence. In these regions, pairs of His residues (H187 and H272) are predicted to interact via cation-pi interactions (Fig 5A). I-TASSER, a protein structure prediction software [68–70], similarly predicts that these histidine residues are capable of interacting with a cation. We postulate that Ni binds to Erg25, and these His residues are critical for the function of Erg25. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Mutating histidine enriched pocket abolishes the ability of Erg25 to confer Ni tolerance. (A) Alphafold generated images of histidine enriched pocket of Erg25. Predicted cation-pi interaction is indicated by an orange dashed line. (B) The indicated strains were serially diluted and spotted onto RPMI and RPMI+ 250μM Ni plates. The plates were incubated for 2 days before imaging. (C) Equal dry weight of cells was used to extract membrane sterols from indicated strains. 5μL of ergosterol extract were spotted onto glass backed HPTLC Silica gel plates. (D) Alphafold generated image of Erg3 histidine enriched pocket. https://doi.org/10.1371/journal.pgen.1011413.g005 To test our hypothesis, we mutated histidine residues 187 and 272, and overexpressed the ERG25H187A H272A allele in both the wild type and the sre1Δ mutant. Although overexpression of the ERG25H187A H272A allele in wild type did not have any effect on growth on RPMI medium, it slightly reduced cryptococcal tolerance to Ni, in contrast to the much-enhanced Ni tolerance by the overexpression of the ERG25 allele (Fig 5B). Accordingly, the overexpression of the ERG25H187A H272A allele failed to rescue the growth defect of sre1Δ on Ni supplemented medium (Fig 5B). We speculate that mutations of these histidine residues prevented the binding of Erg25H187A H272A to Ni, which might have allowed Ni to bind to the native Erg25, compromising ergosterol biosynthesis. Moreover, nonfunctional Erg25H187A H272A may compete with the native Erg25 to complex with Erg26 and Erg27, further impairing the EBP pathway, rendering cells hypersensitive to Ni. Indeed, TLC analysis revealed that the ERG25H187A H272A overexpression strain had a larger reduction in ergosterol when exposed to Ni compared to the wild type (Fig 5D).

Overexpression of ERG3 or URE7 does not rescue sre1Δ growth on Ni We hypothesize that either Erg25 enzymatic function is required for nickel tolerance or Erg25 acts as a nickel sink because of its metal binding pocket can bind to nickel efficiently. Deletion of SRE1 reduces Erg25 abundance and thus renders the fungus sensitive to nickel. Conversely, over-production of Erg25 confers Cryptococcus nickel resistance. However, these observations do not distinguish the two aforementioned hypotheses. To that end, we decided to overexpress another protein with a similar metal binding pocket. We expect that if the “nickel sink” hypothesis is true then overexpression of this protein would also confer nickel tolerance. Erg3 possesses similar conserved metal binding motifs as Erg25 (Fig 5D) [66,67]. ERG3 is not on our DEG list because its transcript level increase in response to Ni was below 2-fold (Fig 2B). However, we found that unlike ERG25, overexpression of ERG3 did not have any obvious impact on cryptococcal growth or Ni tolerance in either the wild type or the sre1Δ mutant (Fig 5B). Consistent with ERG3 being non-consequential in conferring Ni tolerance, TLC analysis revealed a similar trend noted above regarding lipid changes in response to Ni in both wild type and in sre1Δ with or without ERG3 overexpression. Therefore, Erg3 simply possessing a similar binding pocket to Erg25 is not sufficient for sre1Δ growth rescue on Ni. We decided to test our hypotheses further with a known nickel-binding protein. Ure7 binds Ni as a chaperone protein during the activation of Ure1 [35]. Again, if the “nickel sink” hypothesis is true, we expect URE7 overexpression would allow for Ni tolerance of the sre1Δ mutant. We first confirmed that our overexpression construct indeed increased the URE7 transcript level in the wild type and we then deleted SRE1 in the URE7OE overexpression strain. We confirmed increased URE7 transcript level in both WT and sre1Δ background via RT-PCR (S9 Fig). The URE7 overexpression construct, once introduced into the ure7Δ mutant, was able to restore its urease activity assay based on CUA assay (Fig 6A), thus confirming that the overexpressed Ure7 is functional. Upon spotting strains onto RPMI media +/- 250μM Ni, we found that the URE7 overexpression failed to restore growth of the sre1Δ mutant on Ni (Fig 6B). In all, the findings indicate that in contrast to ERG25, overexpression of ERG3 or URE7 does not confer nickel tolerance. Thus, Erg25 is likely not acting simply as a Ni sink. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Overexpression of URE7 does not confer Ni tolerance to sre1Δ. (A) H99, ure7Δ, and URE7 overexpression in both strain backgrounds with cell density OD 600 = 3 were spotted onto CUA plates. The plates were incubated for three days and imaged. Urease activity is indicated by the yellow to pink color change. (B) The indicated strains were serially diluted and spotted onto RPMI and RPMI+ 250μM Ni plates. The plates were incubated for 2 days before imaging. https://doi.org/10.1371/journal.pgen.1011413.g006

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