Isolation Of An Extract From The Soft Coral Symbiotic Microorganism Salinispora Arenicola Exerting Cytoprotective And Anti-Aging Effects Part 1

Abstract: Cells have developed a highly integrated system responsible for proteome stability, namely the proteostasis network (PN). As loss of proteostasis is a hallmark of aging and age-related diseases,  the activation of PN modules can likely extend the health span. Here, we present data on the bioactivity of an extract (SA223-S2BM) purified from the strain Salinispora arenicola TM223-S2 that was isolated from the soft coral Scleronephthya lewinsohni; this coral was collected at a depth of 65 m from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel). Treatment of human cells with SA223-S2BM-activated proteostatic modules decreased oxidative load and conferred protection against oxidative and genotoxic stress. Furthermore, SA223-S2BM enhanced proteasome and lysosomal-cathepsins activities in Drosophila flies and exhibited skin protective effects as evidenced by effective inhibition of the skin aging-related enzymes, elastase, and tyrosinase. We suggest that the SA223-S2BM extract constitutes a likely promising source for prioritizing molecules with anti-aging properties.

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Keywords: aging; antioxidant responses; marine microorganism; proteostasis network; Salinispora arenicola

1. Introduction

Aging is characterized by a time-dependent decline in functional capacity and stress resistance, as well as by increased mortality and morbidity rates [1]. Aging is not just a  passive process, as it is regulated by evolutionary conserved molecular and cellular signaling events [2,3]. Organisms are constantly challenged by both internal (metabolic-related)  and external (diet- or environmental-derived) stressors, which can lead to the gradual accumulation of damaged biomolecules and eventually compromise cellular homeostasis [4,5]. Proteins are involved in all pivotal cellular functions and therefore, proteome homeostasis (proteostasis) is critical for cellular functionality and survival [6].

Proteome integrity is maintained by the proteostasis network (PN), which extends to all subcellular compartments and is regulated at the cellular, tissue, and organismal levels to ensure organismal health and longevity [7]. Sustaining proteostasis requires cells to coordinate and regulate the functions of synthesis, folding, sorting, transport,  and degradation of all polypeptides [8,9]. These cellular functions are mostly carried out by three major interconnected branches of PN, namely the network of molecular chaperones, the ubiquitin-proteasome-(UPP), and autophagy-lysosome-(ALP) proteolytic pathways [10,11].

Molecular chaperones ensure proper protein folding and conformational maintenance,  and they also cooperate with the degradation pathways [12]. UPP is the main site of protein synthesis quality control and is involved in the recycling of normal short-lived proteins and of non-repairable misfolded or unfolded proteins [6], while ALP is responsible for the recognition and removal of protein aggregates and damaged organelles [13]. Loss of proteostasis is a hallmark of aging [14], as well as a major risk factor for many age-related human pathologies, including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases [15]. Therefore, PN activation is considered a promising approach for delaying the onset and/or progression of aging and age-related diseases [16–18].

Aging of the skin is characterized by structural and functional changes in (among others) extracellular matrix components, such as elastin [19]. Elastase is the enzyme involved in the degradation of elastin, resulting in the development of wrinkles and loss of elasticity [20]. Tyrosinase is a key enzyme in melanin synthesis, which is responsible for the pigmentation of (among others) human skin [21,22]. However, the excessive activation of tyrosinase is associated with several skin disorders, such as hyperpigmentation and melanoma[22,23]. Therefore, elastase and tyrosinase inhibition constitute a prominent approach against skin aging.

Natural products (NPs) have a long history—well documented, before the birth of modern science and technology, in various cultures—of treating human diseases [24–26]. Thus, NPs can serve as highly enriched starting materials and scaffolds for drug development; indeed, over half of all drugs are either NPs, NPs-derived, or NPs-inspired [27–29]. The marine world, which harbors the most biodiversity, is largely unexplored and therefore may be the vastest resource to discover novel NPs [30]. Among the organisms that live in marine environments, soft corals are promising providers of bioactive NPs and are known to host many diverse microorganisms [31,32].

Given these facts, we are currently performing an extensive high-throughput screening of NPs to identify bioactive molecules that could potentially delay cellular senescence and/or in vivo aging. In the study presented herein, we examined a methanolic extract,  namely SA223-S2BM, of the Salinispora arenicola (S. arenicola) TM223-S2 strain by applying in vitro assays, as well as cell-based and in vivo bio-guided approaches. S. arenicola TM223-S2 strain was isolated from the soft coral Scleronephthya lewinsohni (S. Levinson) (initially named as Scleronephthya lewinsohni by Verseveldt and Benayahu, 1978), which was sustainably obtained from the mesophotic Red Sea ecosystem EAPC (south Eilat, Israel) at a depth of 65 m. The SA223-S2BM methanolic extract activated cytoprotective mechanisms both in human cells and Drosophila melanogaster (D. melanogaster) flies and exerted in vitro skin protective properties. Therefore, the SA223-S2BM extract could be the source for the likely identification of molecules with potential cytoprotective and anti-aging properties.

2. Materials and Methods 

2.1. SA223-S2BM Extract’s Origin and Isolation 

2.1.1. Coral Collection

The soft coral S. Levinson was collected by an ROV H800 (ECA Robotics) operated by Sam Rothberg R/V of the Interuniversity Institute for Marine Sciences in Eilat (IUI). In situ, photography was carried out by a low-light black and white camera VS300 (ECA Robotics)  and 1CAM Alpha HD camera (SubC Imaging). Samples were obtained by the ROV arm from the mesophotic reef of Eilat, off the oil jetty terminal (29◦300 47.0900 N, 34◦350 56.7100 E), at 65 m, on 7 March 2017. The collection site is characterized by a moderate slope with rocky patches surrounded by a sandy seabed. Upon collection, the material was immediately frozen at −80 ◦C. A voucher specimen was preserved in 70% ethanol for taxonomic identification and deposited at the Steinhardt Museum of Natural History, Tel Aviv University (SMNHTAU_Co_37500).

2.1.2. Strain Isolation and Identification 

S. arenicola TM223-S2 was isolated from a 1 mL sample of S. Levinson. The collected soft coral sample was ground in sterile seawater and heated at 50 ◦C for 1 h. The resulting suspension was serially diluted, plated on selective isolation agar medium, and incubated at 28 ◦C for at least 6 weeks. The Salinispora strain was isolated, and the colony was purified on marine broth agar (MBA) (Difco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and conserved at −20 ◦C in a 10% glycerol solution.

2.1.3. Phylogeny Investigation

Phylogenic analysis was performed using a fragment of the 16S rRNA gene amplified from the genomic DNA of S. arenicola TM223-S2. The genomic DNA of the strain TM223-S2 was isolated using a DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s instructions. The 16S ribosomal RNA fragment was amplified by PCR using primers 16S F 27, AGA GTT TGA TC(AC) TGG CTC AG (Tm: 56.3 ◦C), and 16S R 1492, TAC GG(CT) TAC CTT GTT ACG ACT T (Tm: 57.5 ◦C). Amplicons were sequenced by Sanger sequencing and the sequences were aligned against the 16S ribosomal RNA database of the Targeted Loci project of NCBI using MUSCLE. The alignment was manually inspected, and gaps were removed. The evolutionary history was inferred by using the maximum likelihood method and Tamura–Nei model [33] with 1000 replicates. The initial tree for the heuristic search was obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with a superior log likelihood value. Evolutionary analyses were conducted in MEGA X version 10.1.7 [34].

2.1.4. Cultivation Conditions 

S. arenicola TM223-S2 spores were conserved at −20 ◦C in 10% glycerol. Before cultivation,  the strain was revived for 15 days on a 15 cm Petri plate containing MB (Difco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 2% Agar (Difco, Thermo Fisher Scientific Inc., Waltham, MA, USA). Sterile seawater (4 × 10 mL) was poured on the plate surface, and the spores and mycelium were recovered from the plate by gentle scratching of the surface with a scalpel to generate the inoculum. Liquid-state fermentation (LSF) was conducted in a 2 L Erlenmeyer flask for 20 days at 28 ◦C and 130 rpm agitation. Amberlite XAD16 resin (30 g/L) (Amberlite XAD16HP N, Certis, Lenntech, Delfgauw, The Netherlands) was added before sterilization to allow the in situ trapping of the microbial metabolites. Five milliliters of the inoculum were used to inoculate 1 L on MB containing 30 g of Amberlite XAD16 resin. Agar-state fermentation coupled with solid-phase extraction (SPE) [35] was performed on MB medium (Difco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 2% Agar (Difco, Thermo Fisher Scientific Inc., Waltham, MA, USA)  over 20 days at 28 ◦C. Sterilized Amberlite XAD16 (35 g) mixed with 35 mL of inoculum was homogeneously spread on the surface of 1 MB agar Petri plate (25 cm × 25 cm).

For LSF, the XAD16 resin was separated from the broth culture via filtration and washed with water before being eluted with ethyl acetate (100 mL) and then with methanol (100 mL). The eluates were concentrated to dryness in vacuo and solubilized in DMSO  at 10 mg/mL concentration. The resulting extract, SA223-S2LAE (extracted using ethyl acetate), was used for biological evaluations.

For SPE, the XAD16 resin was recovered by carefully scratching the agar plate surface. The recovered resin was washed with water to eliminate the biomass before being eluted with ethyl acetate (100 mL) and then with methanol (100 mL). The eluates were concentrated to dryness in vacuo and solubilized in DMSO at 10 mg/mL concentration. The resulting extracts, SA223-S2BAE (extracted using ethyl acetate) and SA223-S2BM (extracted using methanol), were used for biological evaluations.

2.2. Elastase and Tyrosinase Inhibitory Activities 

The elastase inhibitory activity of the prioritized extract was evaluated using elastase from porcine pancreas (PPE) type IV (Merck KGaA, Darmstadt, Germany) and the substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide (Merck KGaA, Darmstadt, Germany), as previously described [36]. The capacity of the extract to inhibit the catalytic action of tyrosinase in the oxidation of L-DOPA to dopachrome was determined, as previously reported [37].

2.3. Cell Lines, Cell Culture Conditions, and Cell Viability Assay

Human foreskin fibroblasts (BJ cells) were purchased from the American Tissue Culture Collection (ATCC), while human immortalized keratinocytes (HaCaT cells) were obtained from Elabscience, Inc. Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) non-essential amino acids and were maintained in conditions of 5% CO2, 95% humidity and 37 ◦C. In all experimental procedures applied, cells were subcultured by using a trypsin/EDTA solution (Thermo Fisher Scientific Inc., Waltham, MA, USA). The effect of the SA223-S2BM extract on cell viability was estimated using the MTT assay, as previously reported [38]. Cell treatment with hydrogen peroxide (H2O2) and doxorubicin (DXR) was performed as described in figure legends; H2O2 and DXR were obtained from Merck KGaA (Darmstadt, Germany).

2.4. Drosophila Lines and Longevity Assay

In this study, we used the Drosophila w1118 strain purchased from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Flies were maintained at 25 ◦C and 60% humidity on a 12 h light: 12 h dark cycle. In all presented experiments, only somatic tissues (head and thorax) of flies were analyzed, since reproductive tissues display distinct regulation of proteostatic mechanisms and aging rates as compared to somatic tissues [39].

The longevity of flies was assayed as described before [40] by using female and male flies (equal number per sex) that were cultured in vials containing (or not) the studied extract. Flies were transferred to vials with fresh food every two days and deaths were scored daily. For survival curves and statistical analysis, the Kaplan–Meier procedure and log-rank (Mantel–Cox) test were used; significance was accepted at p < 0.05.

2.5. Measurement of Reactive Oxygen Species (ROS)

Cells or flies' tissues were incubated with the CM-H2DCFDA dye (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a concentration of 10 µM in PBS for 30 min in the dark (37 ◦C and 25 ◦C, respectively). Following dye removal, cells or flies’ tissues were incubated with PBS for 15 min in the dark (37 ◦C and 25 ◦C, respectively), lysed in lysis buffer [150 mM NaCl, 1% (v/v) NP-40, 50 mM Tris, pH 8.0] and, then centrifuged at 14,000 g for 15 min (4 ◦C). Afterward, protein content was measured with the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the produced fluorescence was measured in a Spark® multimode microplate reader (Tecan Trading AG, Männedorf, Switzerland) at excitation and emission wavelengths of 490 and 540 nm, respectively. Hydrogen peroxide (H2O2) was used as a positive control, as it is widely known to cause oxidative damage/stress [41].

2.6. RNA Extraction, cDNA Synthesis,s, and Quantitative Real-Time PCR (Q-RT-PCR) Analysis 

Total RNA was extracted from cells using RNAiso plus (Takara Bio Inc., Shiga, Japan)  and quantified with a BioSpec-nano spectrophotometer (Shimadzu Inc., Kyoto, Japan). Subsequently, 1 µg RNA was converted to cDNA using the FastGene Scriptase II cDNA Kit (NIPPON Genetics, Düren, Germany). To carry out Quantitative Real-Time PCR analyses,  the 5x HOT FIREPol® EvaGreen® qPCR Supermix (Solis BioDyne, Tartu, Estonia) and PikoRealTM Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA)  were used. Specific primers for the human genes studied were as described before [42]. The HMBS gene was used as a normalizer.

2.7. Immunoblotting Analysis and Measurement of Proteasome and Cathepsins Activity 

Immunoblotting studies were conducted as previously described [43]. Primary and horseradish peroxidase-conjugated secondary antibodies were applied for 1 h at room temperature and immunoblots were developed using an enhanced chemiluminescence reagent kit (ClarityTM Western ECL Substrate, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunoblots quantitation was performed by scanning densitometry and ImageJ software [44].

Proteasome and cathepsins enzymatic activities in cells or flies’ tissues were measured at a VersaFluor™ Fluorometer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using Suc-LLVY-AMC (chymotrypsin-like activity, CT-L) and Z-FR-AMC fluoropeptides (Enzo Life Sciences, Inc., Farmingdale, NY, USA), respectively, as previously described [42,45,46]. 6-bromoindirubin-30 -oxime (6BIO) was used as a positive control, as it is known to activate UPP both in human cells and Drosophila flies [42,47]; the drug Rapamycin,  was used as a positive control for ALP and cathepsins induction [48].

2.8. Antibodies Used

Primary antibodies against the β5 (sc-55009) proteasome subunit, NQO1 (sc-16464), Beclin 1 (BECN1) (sc-11427), Clusterin (CLU) (sc-6419), HSP27 (sc-13132), HSP70 (sc-59570),  and GAPDH (sc-25778), as well as the horseradish peroxidase-conjugated secondary antibodies, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibodies against 53BP1 (4937), phospho-Histone H2A.XSer139 (γH2A.X) (9718)  and phosphor-p53Ser15 (9286) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against SQSTM1/p62 (BML-PW9860) was purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA).

2.9. Statistical Analyses 

Experiments were performed at least in duplicates. MS Excel was used for the statistical analyses. Statistical significance was evaluated using Student’s t-test (t-test). Data points correspond to the mean of the independent experiments and error bars denote the standard deviation (SD); significance at p < 0.05 or p < 0.01 is indicated in graphs by one or two asterisks, respectively.

3. Results 

3.1. Identification of the Actinomycete Associated with the Soft Coral S. Levinson

The targeted microbial strain was isolated from the coral S. Levinson, which is a soft coral endemic to the Red Sea. The colonies of this coral are typically found in deep sites,  both in the upper mesophotic coral reef ecosystems (30–60 m) and in the lower mesophotic coral reef ecosystems (60–105 m) [49]. Moreover, it is an azooxanthellate soft coral that obtains its energetic requirements by filter feeding and, therefore, it is found in habitats exposed to strong water currents.

From the phylogenetic analysis based on the 16S rRNA sequence, the isolate TM223-S2  was found to belong to the Salinispora genus (Figure 1).

Since S. arenicola can be distinguished from S. Pacifica and S. tropica based on the 16S  rRNA sequence [50], we named this isolate S. arenicola TM223-S2 with a Genbank accession number MW446171. The strain was cultivated as reported in the Materials and Methods Section and extracted respectively by ethyl acetate and methanol. Preliminary biological investigation indicated that the extract from the liquid culture, SA223-S2BAE, and the ethyl acetate extract from the solid culture, SA223-S2LAE, were devoid of any significant biological activity (Figure S1). Therefore, only the methanolic extract from the solid culture associated with solid-phase extraction, SA223-S2BM, was used in this study.

3.2. The Extract SA223-S2BM Shows no Toxicity in Human Cells and Activates Key Components  of the PN

Firstly, we examined the effect of SA223-S2BM on the viability of normal skin BJ  fibroblasts and human-immortalized HaCaT keratinocytes by performing the MTT toxicity assay. For this purpose, cells were incubated for 24 h with increasing concentrations of the extract and, as shown in Figure 2A1, A2, the extract does not exert a cytotoxic effect on either of the two cell lines examined at concentrations up to 10 µg/mL; at higher concentrations (above 10 µg/mL), the extract reduces cell viability mostly in HaCaT cells.

We then studied the effect of the extract on cellular pathways, which ensure proteome stability of the cell. We observed that short-term (24 h) exposure of BJ fibroblasts to the studied extract significantly induced the expression of the PSMB7 proteasome gene (at a  concentration of 10 µg/mL), while in parallel, it tended to increase the expression of the PSMB6 gene at the same concentration (Figure 2B1). In addition, the extract induced the expression of the 20S proteasome gene, PSMB6, and of the 19S proteasome gene, RPN11  at a concentration of 1 µg/mL in HaCaT cells (Figure 2B2). Furthermore, treatment of BJ  fibroblasts with the extract for 24 h led to a mild induction of the expression levels of the 20S β5 proteasome subunit at the concentration of 1 µg/mL (Figure 2C). In support of our findings, exposure to the extract significantly increased the chymotrypsin-like proteasome activity both in BJ fibroblasts and HaCaT keratinocytes after 24 h incubation (Figure 2D1, D2);  the different results of the examined concentrations of the extract among BJ and HaCaT cells can be attributed to cell type-specific effects.

Additionally, Q-RT-PCR expression analyses revealed that treatment of BJ fibroblasts with the extract for 24 h induced the expression levels of ALP-related genes, named CTSB, CTSL, CTSD, and SQSTM1, at a concentration of 10 µg/mL (Figure 3A1). Further, we observed that exposure of HaCaT cells to the extract for 24 h led to the induction of the expression levels of genes involved in ALP functionality (CTSB, CTSL, CTSD, and SQSTM1) (Figure 3A2).

In line with these findings, we noted upregulation of the autophagy-related proteins BECN1 and SQSTM1/p62 after exposure of BJ fibroblasts to the extract at the concentration of 1 µg/mL (Figure 3B1); increased expression levels of the proteins BECN1 and SQSTM1/p62 were, also, observed in HaCaT keratinocytes at both studied concentrations (Figure 3B2). In accordance, we found that the extract enhanced the lysosomal cathepsins enzymatic activity in both BJ and HaCaT cells (Figure 3C1, C2); again, the different results of the examined concentrations of the extract among BJ and HaCaT cells can be attributed to cell type-specific effects.

Furthermore, we examined the effect of the extract on key molecular chaperones' expression levels. Thus, immunoblotting analyses revealed that the extract induced the expression levels of the molecular chaperones CLU (at both studied concentrations) in BJ  cells (Figure 3D1), as well as of HSP27 (1 µg/mL) and HSP70 (at both studied concentrations)  in HaCaT cells (Figure 3D2).

【For more info:george.deng@wecistanche.com / WhatApp:8613632399501】