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Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans



Sudarshan Singh Rathore, Aishwarya Mathivanan, Abirami Ravindran, Ponnusami Venkatachalam and Jayapradha Ramakrishnan
 
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ABSTRACT

Background and Objective: Cryptococcus neoformans (C. neoformans) is an emerging opportunistic fungal pathogen, which usually causes infection in immunocompromised hosts. Limited antifungal drugs are available to manage cryptococcal meningitis, which requires new effective class of drugs with less toxicity. The aims of the study were to enhance the production of antifungal compound from a new strain of marine Nocardiopsis synnemataformans AF1 against C. neoformans using statistical approach, to determine the molecular weight of the purified compound and to evaluate the effect of antifungal compound against the major virulence factors of C. neoformans, namely capsule and melanin. Materials and Methods: A new strain was screened and isolated from marine sediments based on antagonistic assay against C. neoformans. The antifungal compound was produced using Oyster shells as the substrate. The solid state cultural conditions were selected by one factor, time method and optimized by using response surface methodology. The antifungal compound was extracted using xylene and purified using HPLC. The purified fraction showing inhibitory action against the C. neoformans capsule growth and melanized cells were studied using MALDI-TOF MS. The minimum inhibitory concentration was determined using dilution method. Cytotoxicity was observed by HepG2 cell line. One-way ANOVA and t-test performed to test statistical significance for multiple comparisons. Results: The optimized condition to produce the antifungal compound from the new strain AF1 are found to be, 50% initial moisture content, 2% yeast extract, Oyster shells with the particle size of 16 and temperature at 40°C. The antifungal compound exhibits a significant reduction in C. neoformans cells, capsule size (30.18%) and melanized cells (99.3%). The MIC for the purified compound is estimated to be 200μg mL–1. The MALDI-TOF MS estimated the molecular weight as 242 Da. Conclusion: The results of this study show that the new strain N. synnemataformans AF1 isolated from marine environment exhibited potential antagonistic activity against C. neoformans.

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Sudarshan Singh Rathore, Aishwarya Mathivanan, Abirami Ravindran, Ponnusami Venkatachalam and Jayapradha Ramakrishnan, 2017. Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans. Journal of Biological Sciences, 17: 157-170.

DOI: 10.3923/jbs.2017.157.170

URL: https://scialert.net/abstract/?doi=jbs.2017.157.170
 
Received: January 05, 2017; Accepted: March 17, 2017; Published: April 15, 2017


Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Cryptococcus meningitis is an opportunistic neurological disease in immunocompromised individuals. It has emerged as a leading cause of morbidity and mortality in HIV patients1. The etiologic agent, Cryptococcus neoformans, is an encapsulated opportunistic fungal pathogen emerging as a major threat to immunocompromised patients. However it rarely affects healthy individuals2. Lungs serve as a primary route for C. neoformans infections and cause cryptococcal meningitis when left untreated. Compared with other types of meningitis, cryptococcal meningitis accounts for 45% of deaths3. Recently, Centre for Disease Control estimated approximately one million new cases of cryptococcal meningitis occurring each year, resulting in 625,000 deaths worldwide. Advances in medical techniques such as organ transplantation, cancer therapies with the use of immunosuppressive drugs, diabetes mellitus and immature birth increases the incidence of cryptococcal infections 4,5. The severity of infection is due to the presence of multiple virulence factors such as an outer coat capsule, melanization, production of phospholipase, mannitol, urease and proteinases6. Among these, capsule and melanin are most important virulence factors. Encapsulated C. neoformans resists phagocytosis and causes destructive immune responses7. Many of the resistance factors studied in Cryptococcus sp., are concerned with preventing damage by Reactive Oxygen Species (ROS). The capsule appears to be protective against ROS and other antimicrobials and protection is proportional to the size of capsule8. Similarly, melaninzed C. neoformans can potentially bind and neutralize cationic antimicrobial peptides and are most resistant to amphotericine-B and caspofungin9,10.

The current treatment strategy for cryptococcal meningitis includes amphotericin B in combination with flucytocine for a period of 2 weeks, followed by fluconozole for a minimum of 8-10 weeks. However a long term therapy leads to fluconozole resistance in C. neoformans, which is another emerging problem11. Furthermore, fluconozole intake for a prolonged period of time can lead to other yeast infection such as candidiasis. Towards the end of these antibiotics therapy, protein abnormalities may persist for years, which would cause adverse effects including nephrotoxicity12.

Although the lipid based formulations of amphotericin B has excellent efficiency in reducing nephrotoxicity, its cost and inability to pass through the blood brain barrier are major setbacks13. Thus, the primary motive of this present study was to find an alternative antifungal and there are no reports to study marine bioactive compounds specifically to treat cryptococcal infections14,15. Present study approach in finding a specific compound that targets capsule and melanin synthesis is of great importance in treating the cryptococcal infections effectively.

Bioactive compounds are produced by both solid state and submerged fermentation. However, solid state fermentation is easily controlled and it is preferred since expenditure on media, scale up and downstream processing are less16. Response Surface Methodology is an efficient tool for media optimization and analysis of complexity during the interaction of various factors17. In the present study, used oyster shell (major marine waste product) as a novel substrate for antifungal production. This is the first report on utilizing oyster shells as substrate for the production of antifungal compounds.

This study describes the isolation and taxonomy of anticryptococcal producing strain, optimization by RSM approach, purification of bioactive molecule and determination of molecular weight by MALDI ToF MS. The compound was then evaluated for its activity against the two major virulence factors of C. neoformans (Capsule and melanin).

MATERIALS AND METHODS

All the studies were carried out during the 2015-2016 academic session in SASTRA University campus. All reagents were purchased commercial grade from suppliers (Himedia and SRL) and used without further purification.

Test culture: Cryptococcus neoformans 14116 was purchased from Microbial Culture Collection Centre, Chandigarh. The strain was maintained on potato dextrose agar slants at 4°C and 15% glycerol stocks at -80°C.

Isolation of marine actinomycetes: The marine sediment samples were collected from Pamban near Rameshwaram at Gulf of Mannar in the month of January. Actinomycetes sp., were isolated using zobell marine agar, actinomycetes isolation agar, starch agar and starch casein nitrate agar supplemented with fluconozole (50 μg mL–1) and gentamycin (10 μg mL–1) to inhibit the growth of fungi and bacteria, respectively18. The plates were incubated for 21 days at 28°C. The isolates obtained from each media were stored in 15% glycerol at -80°C.

Screening of antagonistic activity against C. neoformans: The antifungal activity was determined using the protocol of Ramakrishnan et al.19 to select a potential strain. Briefly, the spore suspensions of individual isolates were spot inoculated on Muller Hinton agar plates and incubated at 30°C for 3 days. The cells were killed by chloroform vapors. The plates were subsequently over laid with peptone yeast extract agar swabbed with C. neoformans. The resulting clear Zone of Inhibition (ZOI) was measured after 2 days of incubation. The experiment was repeated thrice. Mean diameter of ZOI and standard deviations were calculated. The strain AF1 which exhibited the maximum antagonistic activity against C. neoformans was selected for taxonomical investigation.

Morphology and taxonomy of antifungal producing strain: The spore morphology of AF1 was studied using Scanning Electron Microscope. The morphology was studied by examining gold-coated dehydrated specimen using the Japan make JEOL (JSM 5610 LV).

For 16S rRNA gene sequencing, the genomic DNA was isolated using the procedure described by Kimura20. The gene fragments were amplified by using PCR Kit (GENEI Pvt, Ltd, India) and 517 F (5’-CCA GCA GCC GCG GTA AT-3’) and Act704R (5’-TCT GCG CATTTC ACC GCT AC-3’)21. PCR was performed using Eppendorf Mastercycler pro thermal cycler 230 V/50-60 Hz with the following profile: initial denaturation at 95°C for 4 min, 30 amplification cycles of (95°C for 1 min, annealing temperature at 50°C for 60 sec, 72°C for 1 min) and a final extension step at 72°C for 4 min. The PCR product was run and excised from 1.5% agarose gel, purified with the QIAquick PCR purification kit (QIAGEN) and sequenced using the primers 8F and U1492R as previously mentioned. Sequencing was done in Chromous Biotech, Bengaluru, India using ABI 3100 sequencer (Applied Biosystems). The sequence was edited using FinchTV (Geospiza Inc.) and BioEdit (Ibis Biosciences, Abbott Labs).

Phylogenetic analysis: Sequence similarity search of 16S rRNA sequence from AF1 strain was carried out using BLAST (NCBI). Evolutionary tree was inferred by using neighbour-joining method22. The Clustal X program was used for multiple alignments and phylogenetic23.

Media optimization
Substrate: Oyster shells were collected from Pamban sea shore and used as substrate for solid state fermentation. The shells were washed thoroughly with distilled water and dried at room temperature. The shells were then broken into pieces and passed through ISI meshes and fractions of mesh 4, 6, 8, 10, 12, 14, 16 were collected. Oyster shells of different particle sizes were then autoclaved at 121°C for 15 min for further experiments.

Lassical screening
One Factor A Time Method (OFAT): The process variables were selected like temperature, initial moisture level, particle size and effect of additional nutrients for antifungal production from AF1. Among the four variables, effect of additional nutrients and initial moisture level alone were optimized by OFAT method, remaining two variables are directly chosen for interaction studies by RSM approach24.

Effect of moisture content and additional nutrients: To study the effect of initial moisture content, various moisture levels ranging from 20-70% were employed to fermentation medium by adjusting with sea water. The fermentation process was carried out at 30°C for 13 days. In addition, effect of additional nutrients such as fermentation yeast extract (1%), beef extract (1%), (NH4)2SO4 (0.05%), NH4Cl (0.05%) were evaluated for their effect on antifungal production from AF1 strain.

Antifungal activity was monitored by well diffusion method using C. neoformans. The yield was expressed as units of activity/milliliter of crude dissolved in phosphate buffer (pH 7), where one U was defined as one mm annular clear ring around the antibiotic disc24.

Central Composite Design (CCD): In solid state fermentation, optimization of substrate particle size, initial moisture level and temperature are crucial factors to increase the yield of bioactive compound. The effects of yeast extract at different concentrations were also considered for experimental design. Based on OFAT method, parameters that had significant effect (p<0.05) on antibiotic production were selected for further experiments of Central Composite Design (CCD) and RSM. The software Minitab was used for experimental design, data analyses and quadratic model building. Experiments were carried out in 32 trials and duplicates were maintained for each run. The student’s t-test and p-values were used to identify the effect of each factor on bioactive compound production. A 24 factorial composite (for 4 factors) experimental design resulting in 32 experiments as shown in Table 1. Minitab statistical software was used to optimize the screened variables grouped as temperature (X1), moisture (X2), nitrogen source (X3), particle size (X4). In developing regression equation, the test variables were coded according to the equation25:

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
(1)

where, xi is independent variable coded value, Xi is independent variable real value on the centre point and ΔX is step change value.

Table 1:
RSM study design by using four independent variables showing anticryptococcal activity in term of capsule and melanin production
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Where: T: Temperature, MC: Moisture content, NS: Nitrogen source, PS: Particle size

The response variable was fitted by second order model in order to correlate the response variable to the independent variables. The general form of the second degree polynomial equation is:

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
(2)

where, Y is the measured response, % reduction units. βo is the intercept term, βi, βij and βii are the measures of effect of variables xi, xij and xi2, respectively. The variable xij represents the first-order interaction between xi and xj. Fermentation was initiated by using 50 g of oyster shells of 16 mesh size and 44 mL of sea water was added to produce an initial moisture content of 45%. After autoclaving, 5 mL of seed culture was added and fermentation was carried out for 13 days at 35°C.

The statistical analyses of the model were performed in the form of analysis of variance (ANOVA). This analysis includes Fishers F-test, its associated probability, Correlation coefficient R and determination coefficient R2 that measures the goodness of fit of the model. Response surface and 2D contour plots described by regression model are drawn to illustrate the effect of independent variables and interactive effects of each independent variable on the response variables. The supernatant obtained from each experimental trial were extracted with equal volume of xylene and checked for anticryptococcal activity. Antifungal activity was defined in terms of highest % of reduction in capsule and melaninzed cells.

Extraction and purification of antifungal compound: Five hundred mL of optimized media was sterilized and inoculated with 5% seed culture followed by incubation at 28°C for 13 days. The fermented medium was then centrifuged at 10,000 rpm for 15 min at 4°C and supernatant was extracted thrice with equal amount of xylene (optimized solvent). The crude extract was evaporated using rotary evaporator and residual material was dried at room temperature26. The obtained colourless compound was dissolved with phosphate buffer (pH 7) and was chromatographed using Semi-Preparatory HPLC (Agilent 1260 infinity). Reversed-phase C18 column (10×250 mm) was used with a linear gradient from 1-60% MeOH and 9-40% water was performed over 25 min at a flow rate of 1 mL min–1. The elution pattern was monitored at 225 nm. Fractions were collected and antifungal activity was performed by broth dilution method in 96 well plate.

Determination of Minimum Inhibitory Concentration (MIC): MIC of crude and purified compound was estimated by broth dilution method in 96 well plate and test tubes27. Five mL of PDB media added with 50 μL of C. neoformans culture (106 cells/mL). Culture was added with crude and purified bioactive compound concentration between 0-2 mg mL–1 with 200 μg mL–1 interval and 0-500 μg mL–1 with 50 μg mL–1 interval respectively, incubated for 48 h at 37°C on a rotary shaker at 120 rpm. After 24 h, 96 well plate observed in ELISA plate reader (Sunrise, Tecan, Austria GmbH) at 620 nm. Cells were plated on potato dextrose agar plates from tubes and number of cells was measured. The lowest compound concentration with more than 50% fungal inhibition was estimated to be MIC.

Cell line toxicity: The mammalian cell lines HepG2 human hepatocyte cell line exposed to bioactive compound and time interval. The cell lines maintained at 37°C in 5% CO2 and 95% humidity incubator in DMEM media (Himedia) supplemented with 10 % FBS (Himedia) and 1X antibiotic (Himedia). Cell lines were incubated with antifungal bioactive compound concentration between 0-500 μg mL–1 with the interval of 50 μg mL–1 and maximum incubation period for 24 h. After incubation cell viability was measured using MTT assay kit (CCK003, EZcount MTT cell assay kit, Himedia) following the manufacturer’s protocol. The assay was performed in 96-well plates in triplicates and differences were tested for statistical significance by student’s t-test28.

Assay of antifungal compound on major virulence factors
Effect on melanin inhibition: Fifty μL of C. neoformans (106 CFU mL–1) fresh culture was inoculated with crude (1 mg mL–1) and pure (200 μg mL–1) bioactive compound and incubated at 24 h at 30°C in minimal broth media. After incubation, 10 μL of treated culture was plated on minimal agar medium containing glucose (15 mM), MgSO4 (10 mM), KH2PO4 (29.4 mM), glycine (13 mM), thiamine (3 μM), agar (2%) with the addition of L-DOPA (1 mM) for induction of melanization29. Plates were incubated at 30°C for 4-7 days in dark and the number of melanized cells was counted30. The effect of compound on the melanised cells and non melanised cell were compared.

Effect on capsule growth: As the capsule size of C. neoformansis directly proportional to its virulence, the capsule was induced in Artificial cerebral spinal fluid medium31. C. neoformans with maximum capsule was used to study the effect of bioactive compound on capsule. After 48 h of induction period, 50 μL inoculated C. neoformans was added with 1 mg mL–1 and 200 μg mL–1 of compound and re-incubated for 24 h at 30°C. Effect of compound on capsule was evaluated by performing negative staining with India ink using Trinocular microscope (Eclipse Ci-L, Nikon). Images were taken with a camera (SLR, Nikon D5100, Camera). To calculate relative size of capsule, diameters of whole cell, including capsule (Dwc) and cell body limited by cell wall (Dcb), were measured using Image J software. The size of the capsule relative to that of the whole cell was defined, as a percentage as [(Dwc - Dcb)/Dwc]100. Fifteen cells were measured for each determination and average was calculated32.

Mass spectroscopy analysis: The purified compound was subjected to Nano Spray matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS) and the molecular weight was determined from the time of flight.

Statistical analysis: All experiments were performed in duplicates and the data analysis was done using Minitab 16 software and GraphPad Prism 6. One-way ANOVA and t-test performed to test statistical significance for multiple comparisons (p<0.05)31. All graphs were prepared with GraphPad Prism 6 and were expressed as Mean±Experiments done in duplicates.

RESULTS AND DISCUSSION

Isolation and taxonomy of potential strain: In this study 33 actinomycetes were isolated from marine sediments. The strain AF1 isolated using starch agar showed the maximum activity against C. neoformans. It displayed maximum activity against the test organism with 15 mm mean diameter of zone of inhibition.

The electron micrographs reveals the spores have smooth surface in oval shapes around 4-6 spores/round (Fig. 1). The 16S rRNA gene sequence of the strain AF1 and its comparison with the gene sequences against the GenBank database revealed that the organism form a distinct phylogenetic line in the N. synnemataformans. The isolate was closely related to the type strain of Nocardiopsis strain UTMC 2171, sharing a homology of 99%. The 16S rRNA sequence analysis support the classification of the isolate AF1 as a new strain of Nocardiopsis UTMC 2171 (Fig. 1).

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Fig. 1:
(a) Scanning Electron micrograph of Nocardiopsis synnemataformans AF1 grown on Starch agar at 30°C for 14 days and (b) Neighbor-joining tree based on 16S rDNA gene sequences showing relationship between the strain Nocardiopsis synnemataformans AF1 (accession number KJ 716227) and other Nocardiopsis sp

The accession number KJ 716227 was obtained from NCBI. A recent review by Mayer et al.33, demonstrated a slight decrease in the discovery of marine antifungal products and during the last decade, only two molecules described the novel mechanism of action. Very limited researchers are attempted to find bioactive molecules against Cryptococcus sp. Recently, Das et al.5, identified a mangrove Nocardiopsis sp., demonstrating 12 mm zone of inhibition against C. neoformans. In the last decade some bioactive compounds, kahakamides AV, 2 neosidomycin, also a novel cyclic tetrapeptide, polyketides, thiopeptide have been discovered from marine Nocardiopsis strains34. None of these compounds have been proven to exhibit anticryptococcal activity. A thorough literature search evident the absence of reports on finding compounds against C. neoformans. Specific molecules are needed to treat C. neoformans as they are different from other pathogenic yeast on many aspects such as capsule as a shield layer, melanin production, its ability to grow at 37°C and can disseminate from lungs to central nervous35. Hence the bioactive compound from the marine N. synnemataformans AF1 would be a suitable drug candidate to treat cryptococcal infections.

Classical screening
one factor a time method
Effect of initial moisture content: The optimum initial moisture content for the production of antifungal compound using Oyster shells as the substrate was estimated. The results presented in Fig. 2 clearly indicated that the maximum antifungal production was recorded at 50% with the yield of 245 U mL–1. A similar report on maximum tetracycline production from N. synnemataformans at 65% moisture level using pineapple peel as substrate36. Furthermore, maximum neomycin production from Nocardiopsis by solid state fermentation at 70% moisture level using agricultural wastes such as apple pomace, cotton seed meal, soy bean powder and wheat bran in a parallel study37. These results evident that the optimum moisture level may differ widely of the same organism growing on different substrates38.

Oyster shells have been reported to be one of the most serious pollutants of both marine environment and soil. Recycling of oyster shells thus been recognised as necessary and they have subsequently been used in many applications including, fertilizer, sludge conditioner, eutrophication control, desulphurization sorbents39 and waste water treatment. Our attempt in finding its relevance as cost effective medium for the cultivation of AF1 and production of its bioactive compound were achieved.

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
Fig. 2(a-f):
Response surface plots showing interactive effects of various factors on antifungal activity (% reduction) of Nocardiopsis synnemataformans AF1 (a) Nitrogen source and Temperature (b) Nitrogen source and Moisture (c) Particle size and moisture (d) Particle size and Temperature. Selection of variables by OFAT method (e) Optimization of initial moisture content for antifungal production and (f) Optimization of additional nutrients for antifungal production
 
The experiment has done twice with duplicates at each time. Significant difference shown by *(p<0.05). Bar represents mean and SD for triplicate samples

Effect of additional nutrients: The effect of nitrogen sources on the production of bioactive compound are shown in Fig. 2. The maximum antifungal yield (260 U mL–1) was obtained with the addition of yeast extract (1% w/v). Lower yields were obtained with the addition of ammonium sulphate (130 U mL–1). Thus, yeast extract was chosen as the source of nitrogen for further experiments. The fermentation without any additional nutrients yielded 240 U mL–1, which confirms the significance of additional nutrients to the oyster shells fermentation medium. In general, oyster shells are rich in calcium carbonate, other essential minerals and organic matter. For more than 3 decades till date, researchers exploit the calcium carbonate enriched medium for isolation of actinomycetes40. From these results, oyster shell medium enriched with yeast extract would be a suitable cost effective medium for the production of bioactive compound from N. synnemataformans. Moreover it can be an appropriate cultivation and production media for other actinomycetes also.

Antifungal activity of compound produced by OFAT method: While screening for the anticryptococcal activity of the crude compound obtained by the classical optimization (yeast extract -1%, moisture -50%, oyster shells -16 mesh size, temperature 35°C), the yield obtained was 260 U mL–1. The effect of the compound on the Crytococcous capsule growth and on melanized cells shows the reduction of 8 and 48%, respectively.

Central composite regression design
Statistical approach: While analyzing the effects of various factors (temperature, moisture, particle size and yeast extract) and their interactions in enhancing the antibiotic production. The goodness of fit of the model based on RSM checked by applying multiple regression analysis on the experimental data, experimental results of the CCD design were fitted with second-order polynomial equation. The results of regression analyses are shown in Table 2. The student’s t-test and p-values were used as a tool to check the significance of each coefficient that also indicated the interaction strength between each independent variable. The larger magnitude of t-value and smaller the p-value, more significant is the corresponding coefficient41. It can be seen from the degree of significance.

The R2 value is always between 0 and 1.The closer the R2 value to 1, stronger the model and better it predicts the response17. In this case coefficient of determination R2 = 0.9658 and 0.9850 for capsule and melanin respectively Table 2. The R2 also indicates only 7 and 1% of total variations was not explained by the model. The value of adjusted determination coefficient was also high (Adjusted R2 = 93.58%, 97.18% respectively, which supports that the model generated was more suitable for optimization.

ANOVA for the response surface square model suggests that the interactions between moisture and temperature had a higher influence on antifungal activity and hence the yield (Table 3).

With larger magnitude of t-value and smaller the p-value, the corresponding coefficient would be more significant. A p-value of less than 0.05 indicates that the model terms were significant and regression model having coefficient of determination R2 value higher than 0.9 were considered as having a very high correlation27.

From multiple regression analyses it was observed that second order polynomial equation could explain antifungal production regardless of the significant of the antifungal production.

Table 2:
Analysis of variance for the model regression representing reduction of capsular growth and melanized cells
Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
T: Temperature, MC: Moisture content, NS: Nitrogen source, PS: Particle size, Cc: C. neoformans (capsule) and Cm: C. neoformans (melanin), C. neoformans (capsule) S = 1.44160 PRESS = 137.374, R-Sq = 96.58% R-Sq(pred) = 85.86% R-Sq(adj) = 93.58%, C. neoformans (melanin), S = 1.88198 PRESS = 266.224, R-Sq = 98.50% R-Sq(pred) = 92.93% R-Sq(adj) = 97.18%

Table 3: ANOVA for Significant Interactions between moisture and temperature
Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
(3)

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
(4)

where, Yc and Ym correspond to the effects on the capsule and melanized cells, respectively.

The interaction studies from 2D contour plot shown in Fig. 2 reveal that all factors (moisture, temperature, yeast extract, particle size), had significant effects (p<0.05) on the antibiotic production. The best interactions are between moisture of 40% temperature of 40°C, yeast extract of 2% and Oyster shells with the particle size of 16. The smaller particle size of oyster shell provides large surface area which helps to mix the substrate with the microorganisms, other nutrients and uniform distribution of temperature that supports the microbes to enhance the production of antibiotics. Ellaiah et al.42 convey that the increase in higher substrate moisture in solid state fermentation results in sub optimal product formation due to reduced mass transfer process such as diffusion of solutes and gas to cell during fermentation. Also, the decrease in moisture results in reduced solubility minimizes heat exchange, oxygen transfer and low availability of nutrients to the culture, leading to decrease in productivity42. This supports our results at 45% initial moisture level using oyster shell.

Further validation experiments were also carried out to verify the adequacy and accuracy of the model on the result suggested that the predicted value agreed with experimental values. The enhanced antifungal activity was obtained with statistically optimized production medium exhibiting 20.18 and 99.3% reduction of capsule growth and melanized cells, respectively. Figure 3 clearly reveals the significant reduction (p<0.05) in capsule growth and melanized cells, respectively. The antifungal compound production yield was increased by 60 U mL–1 in statistically optimized condition when compared to conventional method.

Extraction and Purification of antifungal compound: The supernatant which were obtained by growing the strain AF1 in the optimized medium for a period of 13 days were extracted with xylene and the extract was evaporated to dryness resulting in a yellow oily residue (2.6 g). The crude extract was purified by using semi preparatory HPLC. The HPLC analysis yield 20 fractions and the compounds corresponding to the first peak starts from 3.5-14.4 min (Fig. 4). The active peak was identified by performing antifungal assay by disc diffusion method. A zone of clearance of 26 mm for HPLC peak 10th with a RT of 8.7-8.9 min was observed on assay plates. However, the other peaks shows no antifungal activity.

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
Fig. 3(a-f):
Effect of bioactive molecule from Nocardiopsis synnemataformans AF1 on capsule growth and melanized cells. India ink analysis of crytptococcal yeast cells was done for capsule size: (I) Capsule inhibition: (a) Effect of crude (1 mg mL–1) and pure (200 μg mL–1) compound on capsule thickness (%), (b) Induction of capsule in MOPS medium, (c) Reduction in capsule size-cells treated with 200 μg mL–1 of compound. (II) Melanin inhibition, (d) Effect of crude (1 mg mL–1) and pure (200 μg mL–1) compound melanin producing cells, results analyzed by CFU mL–1 count, (e) Growth of cryptococcal cells producing dark pigmentation of colonies-without treatment and (f) Growth of cryptococcal cells in the treated with bioactive molecule with the addition of 200 μg mL–1 showing significant reduction of melanin production. The experiment has done twice with duplicates at each time
  Significant difference shown by *p<0.05. Bar represents mean and SD for triplicate samples

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Fig. 4: HPLC profile of Nocardiopsis synnemataformans AF1
 
(*) represents active peak shows anticryptococcal activity; (A) Zone of inhibition (26 mm) of active peak on Mueller Hinton Agar

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
Fig. 5:
AF1 antifungal compound minimum inhibitory concentration and cytotoxicity study against HepG2 cell line. Antifungal AF1 compound MIC50 was observed at 200 μg mL against C. neoformans and more than 75% viability of HepG2 cells was observed up to 500 μg mL–1
  Bar represents mean and SD for triplicate samples

Also the purified compound showed significant reduction (p<0.05) in capsule growth and melanin pigment production. Thus, the compound produced during the study would be a potential molecule against C. neoformans.

MIC determination: The crude and purified compound at a final concentration of 1 mg mL–1 (data not shown) and 200 μg mL–1 was found to be the MIC50, respectively. MIC was confirmed by the absence of growth in agar plate and 96 well plate at OD600 was comparable with positive and negative control, results shown in Fig. 5.

Cell line toxicity: Cytotoxicity of antifungal bioactive compound on HepG2 human hepatocyte cell linehas evaluated by MTT assay. The multiple concentrations of antifungal compound used, results shown in Fig. 5. There was no cytotoxicity was observed for compound in HepG2 up to concentration of 500 μg mL–1, more than 80% viability observed with maximum of 24 h incubation for the cell lines with antifungal compound. As per test parametric statistical analysis, no significant difference was observed for 1 and 24 h compound treatment to HepG2 cells.

Image for - Potential Anticryptococcal Compound from Marine Nocardiopsis synnemataformans
Fig. 6:
Mass spectroscopy analysis of HPLC purified antifungal compound from Nocardiopsis synnemataformans AF1

MALDI-TOF analysis: The MALDI-TOF MS spectrum confirms the molecular weight of the purified antifungal compound to be 242.1 Da Fig. 6. Amphotericine B is the standard initial therapy to treat cryptococcal infections. The larger encapsulated strains are less likely to respond to the current therapy (AMB plus flucytocine). Although AMB function as immunomodulator, its effectiveness are insignificant to control the cryptococcal infections in severely immune-compromised host. Amphotericine B is derived from Streptomyces sp which has the molecular weight of 924 Da43. Molecular size is one of the important criteria to traverse the drugs to the CNS tissue barrier44. Hence, the isolated molecule with low molecular size from a new strain of Nocardiopsis synnemataformans might be a novel and potential antifungal molecule which requires the complete structural elucidation studies.

CONCLUSION

The results of this study show that the strain AF1 isolated from marine environment exhibited potential antagonistic activity against C. neoformans. The solid state cultural conditions were optimized by using RSM approach. The enhancement of antifungal production and antifungal activity were achieved at initial moisture level of 50%, temperature at 40°C, yeast extract of 2% and Oyster shells with the particle size of 16. The potentiality of the purified antifungal molecule from AF1 was demonstrated by its significant influence on reduction of capsule growth and melanization. At the same time, the low molecular weight of the molecule was found to be a good promise as a potential drug candidate. Also there no toxicity was observed, cell viability was more than 80% up to 500 μg mL–1 concentration in HepG2 cell line. The complete structural elucidations are in the laboratory pipeline.

SIGNIFICANCE STATEMENTS

This study discovered the potential application of oystershell as a suitable substrate to produce antifungal compound. The study highlights the use of response surface methodology an efficient tool for media optimizations and analysis of complexity during the interactions of various factors. The present study describes the isolation and taxonomy of anticryptococcal producing strain, optimization by RSM approach, purification of bioactive molecule and determination of molecular weight by MALDI-ToF MS. This is the first study to identify and produce anticryptococcal compound from marine Nocardiopsis sp. The present study will help the researchers to explore marine habitat as the potential source to discover antifungal compound.

ACKNOWLEDGMENTS

This study was financially supported by the Department of Science and Technology, New Delhi, under the Fast Tack Young Scientist Scheme. (No: SB/FT/LS-249/2012) and HPLC facility EMR scheme SR/S0/HS-0073/2012 to JP. We thank SASTRA University for providing us the infrastructure to carry out our research work. We thank to Dr. Gunasekar Varadarajan for his valuable suggestion for RSM study.

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