Scientific Papers

Superior anti-pulmonary viral potential of Natrialba sp. M6-producing surfactin and C50 carotenoid pigment with unveiling its action modes | Virology Journal


Isolation, enrichment and identification of Haloalkaliphilic Natrialba sp. M6

Collect water and sediment samples from different sites in El-Hamra lake, Wadi El-Natrun known for their haloalkaline conditions. Use sterile containers to ensure the integrity of the samples, a culture medium suitable for the growth of haloalkaliphilic archaea was followed (g/L): casamino acids, 5; KH2PO4, 1; MgSO4·7H2O, 0.2; NaCl, 200; trace metals, 1 mL; and Na2CO3, 18. Te trace metal solution contained (g/L) ZnSO4·7H2O, 0.1; MnCl2·4H2O, 0.03; H3BO3, 0.3; CoCl2·6H2O, 0.2; CuCl2·2H2O, 0.01; NiCl2·6H2O, 0.02; and Na2MoO4·H2O, 0.03 as described by [8, 9]. The medium should have high salt concentration (NaCl) and a pH range of 9–11. Adjust the pH using sodium carbonate (Na2CO3). Inoculate a small volume of the collected water and sediment samples into the prepared enrichment culture medium. Incubate the cultures at the desired temperature (typically around 37 °C) for several days to allow the growth of haloalkaliphilic archaea. Transfer a small volume of the enriched culture to a fresh batch of the culture medium to promote the growth of haloalkaliphilic archaea. Repeat this subculturing process several times to obtain a pure and distinct culture of haloalkaliphilic archaea. For testing the isolates ability for the production of biosurfactants, pre-culture of the archaeal isolates was prepared and the cell free supernatant adjusted for the following methods: emulsification index, oil spreading technique, hemolytic activity, and surface tension measurement [9]. The most promising biosurfactant producing isolate was chosen and molecularly identified using molecular techniques (DNA sequencing).

Recovery of the Sur

The culture supernatant of Natrialba sp. M6, is subjected to centrifugation at 15,000 rpm for 30 min. Centrifugation separates the cells and other debris from the supernatant, allowing for the extraction of Sur. To facilitate the extraction process, the cell-free culture supernatant is acidified by adding concentrated hydrochloric acid (HCl) until the pH reaches 2, acidification helps to modify the ionic state and solubility of the biosurfactant, aiding its subsequent extraction. After acidification, the treated supernatant is stored at 4 °C overnight. This incubation period allows the Sur to precipitate and facilitates its separation from the remaining components of the culture supernatant.

The Sur residue in the precipitate was desiccated, weighted and dissolved in known volume from 0.1 M sodium bicarbonate [9, 15].

Recovery of the Pig

The Pig produced by Natrialba sp. M6 can be extracted by the centrifugation of 50 ml of the culture broth at 10,000 rpm for 30 min at 4 °C. The supernatant was spun down, and 50 ml of distilled water was added to the clean pellets and then stored at 4 °C overnight to rupture the cells. A mixture of the solvents methanol-acetone (3:7 v/v) containing 0.1% butylhydroxytoluene (BHT) as antioxidant was used. This step was repeated until both the pellets and the solvents were becoming colorless, and then again centrifuged. The extracted Pig in solvent was stored at 45 °C overnight until solvent completely evaporated, and the Pig was collected, weighted and wrapped with aluminium foil to prevent light damage. The colored Pig solution was scanned by their absorbance in the wave length region ranged 200–700 nm. the highest wave length of the absorption was (λ350nm) by using coefficient value of absorption of 266,030 [8].

HPLC identification of the extracted Sur

HPLC system equipped with a Chromolith high performance RP-18 (100 * 4.6 mm, 5 µm) column was maintained and operated at 25 °C. The mixture of mobile phase consisting of a 3.8 mM TFA solution and an CAN (ratio of 20:80) were driven with a flow rate of 2.2.mL/min in an isocratic mode. The surfactin volume injection was set at 30 µL, and recorded by the detector device VDW at 205 nm. Each analysis run was done within 8 min. Methanolic surfactin standard stock has been prepared at concentration 5000 mg/L. Later, a surfactin solution series of different concentrations ranged from 10 to 1000 mg/L were prepared by the stock solution dilution with solvent methanol and then they have been stored at temperature 4ºC before using. The solution of a 3.8 mM TFA has been prepared in a 1 L deionized water volumetric flask and vortexed until the dissolution has been completed. Finally, an CAN and the TFA solution have been filtered via 0.22 µm filters of nylon and the gasses were removed before using [8].

Cytotoxicity of Sur and Pig on viral host cell lines

The cytotoxicity of Sur and Pig extracts was assessed on three established cell lines, green monkey kidney epithelial (Vero), Madin-Darby canine kidney (MDCK), and Vero Clone E6 (Vero E6), which are susceptible to ADV7, H1N1, and coronaviruses, respectively. Cell viability was assessed by the crystal violet assay [16]. These permissive host cell lines were cultivated in DMEM containing 10% fetal bovine serum (FBS). After trypsinization, these cell lines (104 cells/well) were cultured in 96-well cell culture plates and placed in 5% CO2 incubator at 37 °C. The next day, suspensions of Sur or Pig at different serially-diluted were incubated with these viral host cells for 72 h, in 5% CO2 incubator. After washing the cells with phosphate buffer saline (PBS) and fixation, cells were stained with 0.03% crystal violet for 10 min and then elution solution (0.9% sodium citrate and 1N HCl in ethanol) was added. Using ELISA reader (BMG LabTech, Germany), the absorbances were measured at 540 nm. The cell viability (%) was estimated in order to calculate the effective concentrations (EC50 and EC100) at which 50% and 100% cell viability, respectively, using Graphpad Instat software.

Investigation of anti-ADV7 activity

Determination of inhibition potency on cytopathic activity of ADV7

The virus inoculum (10–5) was seeded to 96 well culture plate containing Vero cells monolayer and incubated in 5% CO2 incubator for 2 h at 37 °C. Then, the unabsorbed viruses in supernatant were removed and replaced with fresh culture medium containing serially-diluted concentrations of sur or pig. Then these cells were incubated in 5% CO2 incubator for 72 h at 37 °C. All wells (infected-untreated, healthy, treated infected cells) were stained with crystal violet stain as described above, to calculate % reduction of the infected cells lysis associated to exposure to Sur or Pig. The dose (IC50), at which 50% inhibition of virus-mediated cell lysis, was calculated by the Graphpad Prism software.

Crystal violet assay and quantitative PCR analysis for investigating the action modes

To investigate the direct virucidal effect, serially-diluted concentrations of Sur or Pig were incubated in 5% CO2 incubator for 2 h at 37 °C with 10–5 ADV7 before inoculating this treated suspension into a susceptible and permissive monolayer of Vero cells and further incubated for 2 h. Meanwhile, the anti-adsorption effect was measured by pretreating host monolayer of Vero cells with different concentrations of Sur or Pig for 2 h, then removing the supernatant and adding a suspension of infectious viruses to the monolayer for another 2 h of incubation. Concerning the anti-replicative effect, different diluted concentrations of Sur or Pig was added after incubating their host cells with ADV7 for 2 h and then supernatant removed. Then, the untreated and treated cells were stained with crystal violet, as described above, for assessing IC50 by the Graphpad Prism software.

For more accurate confirmation, the same conditions of three experiments of action modes were performed at lower IC50 (3 µg/mL), and then the treated and untreated infected host cells were collected for determining viral genome load using TaqMan-based real time PCR [17]. The used ADV7 primers were 5′-GAGGAGCCAGATATTGATATGGAATT-3′ and 5′-AATTGACATTTTCCGTGTAAAGCA-3′ with the probe 5′-6-carboxyfluorescein (FAM)-AAGCTGCTGACGCTTTTTCGCCTGA-6-carboxytetramethylrhodamine (TAMRA)-3′. Reaction mix contained Taq polymerase enzyme (0.05 U/L), the reaction buffer (250 nM probe, 0.4 mM dNTP, 400 nM reverse/forward primers and 4 mM MgCl2). PCR program was started at temperature 95 °C for 5 min next by 45 cycles at temperature 95 °C for 10 s, 55 °C for 30 s and then 72 °C for 20 s. Viral load was calculated using standard curve of the virus.

ELISA assessment of Sur or Pig binding to capsid protein of ADV7

Briefly, polystyrene plates were coated with recombinant antibody of ADV fiber (5 μg/ml). After overnight incubation, plates were washed and blocked with borate buffer saline containing skim dry milk then the preincubated mixture of 0.8 μg/ml of Sur or Pig and ADV was added. After 1 h incubation, wells were washed to remove unbinding mixture. Alkaline phosphatase-conjugated secondary antibody was added, followed by washing and adding p-nitrophenyl phosphate. The reaction was stopped by adding 3 N NaOH, and the absorbance was measured at wave length 405 nm.

Inhibitory effect on polymerase activity of ADV7

The inhibitory potential of Sur or pig on ADV by performing DNA polymerase activity using the method of [18]. Briefly, Sur or Pig serially-diluted concentrations were added to a reaction mixture of 7 mM MgCl2, 10 mM DTT, 25 mM Tris–HCl pH 7.8, 1 μg aphidicolin, activated DNA and 40 μM deoxynucleotides with 1 µCi radiolabeled [α-32P]dATP. After 1 h of incubation, the synthesized DNA was transferred to the disks of filter paper and then precipitated using trichloroacetic acid and then measured using a scintillation counter.

Investigation of anti-H1N1 activity

Determination of inhibiting potency on cytopathic activity of H1N1

One day before viral infection, MDCK cells were seeded at a density of 2 × 104cells/well into a 96-well culture plate. After cell attachment for 24 h. The cells were washed with PBS after removing the culture medium and then incubated with 100 µl of diluted H1N1 suspension containing CCID50 (50% of cell culture infective dose, 10–5). Then serially-diluted concentrations of Sur or Pig were added to the infected cells, exception of wells serving as positive infected (untreated) control. After 72 h incubation of culture plates at 37 °C in 5% CO2, cells were washed, fixed and stained with a 0.03% crystal violet solution as described above. The inhibitory dose at 50% cytopathic effect (IC50) was estimated by the Graphpad Prism software.

Investigation of anti-H1N1 action modes

To investigate the virucidal effect, serially-diluted concentrations of Sur were incubated with H1N1 for 1 h, in 5% CO2 incubator, with 10–5 H1N1, followed by 1 h incubation of this mixture (H1N1 + Sur) with monolayer of MDCK cells. The anti-adsorption effect was assessed by preincubating host cells with culture medium containing different concentrations of Sur for 1 h, then replacing this medium with H1N1 inoculum and incubating cells for 1 h. Regarding anti-replicative effect, serially-diluted concentrations of Sur were incubated with H1N1-infected MDCK cells for 1 h. In three plates of these three experiments, the infected medium was replaced by new culture medium that was incubated with cells for 72 h. Following that, all cells were stained with crystal violet, as demonstrated above, for estimation of IC50 by the Graphpad Prism software.

Hemagglutination inhibition assay

Hemagglutination inhibition (HI) assay was used to evaluate virucidal effect of Sur on H1N1 hemagglutinin for preventing its adsorption to erythrocytes [10, 19]. Erythrocytes were isolated by centrifugation of heparinized fresh chicken blood, washed and suspended in PBS at 1% (V/V). Firstly, safety doses of Sur and Pig were determined by incubating it with erythrocyte suspension for 2 h, centrifugating and measuring the supernatant, compared to the untreated erythrocyte wells, at 490 nm. Then, serial dilutions of safe dose (3 mg/ml) of Sur and Pig were incubated with an equal volume of H1N1 (8 hemagglutination units). After 1 h incubation, These mixtures of Sur-H1N1 or Pig-H1N1 were incubated with an equal volume of 1% chicken erythrocyte suspension in PBS in 96-well plate for 1 h before observing the aggregation of erythrocytes.

Inhibitory potential on neuraminidase activity

The activity of neuraminidase was determined according to instructions of fluorometric-blue neuraminidase assay kit (Abcam, USA). Briefly, serially-diluted concentrations of Sur or Pig were incubated with NeuroBlue indicator and neuraminidase for 100 min at 37 °C and fluorescence intensity (extension/emission) was measured at 320/450 nm using spectrofluorometer (BMG LabTech, Germany).

Investigation of anti-coronavirus activity against HCoV-229E and SARS-CoV-2

Evaluating anti-HCoV-229E potential

Determination of inhibiting potency on cytopathic activity of HCoV-229E

Vero E6 cells were seeded, into a 96-well culture plate, at a density of 2 × 104cells/well. After cell attachment (24 h), then the culture medium in 96 well plate was replaced with 100 µl of diluted HCoV-229E suspension containing CCID50 (10–3). Then serially-diluted concentrations of Sur or Pig were added to wells, excluding positive infected (untreated) control wells. After 72 h incubation of culture plates at 37 °C in 5% CO2, 0.03% crystal violet solution was added as showed above. The IC50 was calculated by the Graphpad Prism software.

Investigation of anti-HCoV-229E action modes

The modes of anti-HCoV-229E action were conducted as previously described [16]. To evaluate the virucidal effect, serially-diluted concentrations of Sur were incubated with HCoV-229E for 1 h before being added to monolayer of Vero E6 cells. The anti-adsorption effect was assessed by pre incubating cells for 1 h with serially-diluted concentrations of Sur, then discarding this medium and incubating with HCoV-229E inoculum for 1 h. In the anti-replicative effect, serially-diluted concentrations of Sur were incubated with HCoV-229E-infected Vero E6 cells for 1 h. In these three conditions, the infected medium was replaced by new culture medium on cells. Following 72 h, all cells were stained with crystal violet, as demonstrated above, for estimation of IC50 by the Graphpad Prism software.

Evaluating anti-SARS-CoV-2 potential by determinating SARS-CoV-2 main protease activity

3-Chymotrypsin protease assay was performed using 3CL Protease, Untagged (SARS-CoV-2) assay kit (BPS Bioscience, USA). Briefly, serially-diluted concentrations of Sur or Pig were pre-incubated with 3CL protease (0.5 µg/ml) for 30 min, then the reaction was started by the addition of 3CL protease substrate (40 µM). After 2 h of incubation, fluorescence intensity was measured at excitation 360 nm and emission 460 nm.

Determinaton of scavening activity against radical species-mediated lung damage

The anti-radical potentials of Sur and Pig were detected against superoxide anion, hydroxyl radical and nitric oxide using colorimetric assays. Superoxide anion radical (O2·¯) scavenging activity was evaluated, according to method described by Ravishankara et al. [20] by incubating serially-diluted concentrations of Sur or Pig with a reaction mixture of 67 mM phosphate buffer (pH 7.8), EDTA, 1.5 mM nitroblue tetrazolium, 1.5 mg% NaCN, and 0.12 mM riboflavin for 15 min. Then the absorbance was measured at 530 nm. Hydroxyl radical scavenging (OH) activity of Sur or Pig was detected by incubation their serially-diluted concentrations with 9 mmol/L salicylic acid, 9 mM FeSO4 and 9 mM H2O2 for 1 h at 37 °C. The absorbance was then measured at 510 nm [21]. Nitric oxide radical (NO) scavenging activity was quantified using Griess reagent [22]. The Graphpad Prism software estimated the concentrations (IC50) of Sur and Pig at which free radicals were scavenged by 50%.

Molecular docking studies

Structures acquisition and preparation

The available three-dimensional crystal structures of H1N1 Influenza virus neuraminidase and SARS-CoV-2 3CL protease were obtained from the Protein Data Bank (PDB, www.rcsb.org) PDB IDs: (PDB ID: 6HP0 and PDB ID: 7VTH [23], respectively) handled with the Molecular Operating Environment (MOE) software package version MOE 2019.102, Chemical Computing Group, Montreal, Canada Unwanted ligands and residues were removed. The structures of surfactin and C50 carotenoid bacterioruberin were prepared and refined employing the default “Structure preparation” MOE setting, then energy minimized employing Amber10: EHT force field with reaction-field electrostatics (an interior dielectric constant of 1 and an exterior dielectric of 80) using an 8–10 Å cutoff distance.

Docking simulations

The prepared surfactin and C50 carotenoid bacterioruberin were docked into the ligand binding sites by the Molecular Operating Environment (MOE) software package version MOE 2019.102, Chemical Computing Group, Montreal, Canada [14]. using Alpha HB and the Triangular matcher algorithm as scoring and placement functions creating the top 100 non-redundant poses of the lowermost binding energy conformers. Docking was showed with induced fitting protocol. Results were estimated as S-scores with RMSD value < 2.5 Å. The molecular interactions graphical representations were inspected and generated.

Statistical analysis

The collected data was statistically analyzed using Tukey-Post Hoc multiple comparison one-way ANOVA and unpaired t-tests (SPSS 16). All data were demonstrated as mean ± standard error of the mean (SEM). A P value of ≤ 0.05 was considered significant.



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