IJCRR

IJCRR - 3(7), July, 2011

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ISOLATION AND CHARACTERIZATION OF LOLDUVIN-S: A NOVEL ANTIMICROBIAL PROTEIN FROM THE INK OF INDIANS QUID LOLIGO DUVAUCELI

Author: Smiline Girija, J.Vijayshree Priyadharshini, Pandi Suba K., Hariprasad G., Raghuraman R.

Category: General Sciences

Abstract:Objectives: Bioactive compounds from the marine habitat have been always represented as the greatest unexploited source of potentially active pharmaceutical agents. Squid ink, being reported for its various therapeutic applications has not been extensively studied for its bioactive molecules. Thus an attempt has been made in this study to isolate and characterize a novel antimicrobial protein from the ink of Loligo duvauceli. Methods: The fresh squids were decapitated and the ink sac was dissected to collect the ink. Melanin free ink was obtained by ultracentrifugation. The ink was then subjected to SDSPAGE to analyze the proteins present in it and a new protein with a molecular weight of approximately 60 Kda was selected as the target protein. Native PAGE was then performed to isolate the protein in an original form and was eluted using passive gel elution method. The eluted protein was then subjected to an enzyme linked coupled assay for the L-amino oxidase [LAO] activity. The Km and Vmax values for the L-amino acids utilized as substrates for the assay were determined by Lineweaver Burk Plots. The eluted protein was sterilized by syringe filtration after which the antimicrobial potential of the protein was checked by agar well diffusion method against the drug resistant pathogens such as ESBL, MRSA and the pathogenic yeast C.albicans. The MIC value of the protein was determined by Microbroth dilution method.
Results: A new protein of approximately 60 Kda was successfully isolated and was characterized to exhibit the L-amino acid oxidase activity. The protein was scored to possess a promising antibacterial and antifungal activity against the test pathogens. The MIC value was determined as 25 \?g/ml for ESBL producing strains of E.coli and K.pneumoniae. MIC value for the methicillin resistant S.aureus and C.albicans was deduced as 12.5 \?g/ml. This new protein exhibiting a LAO activity was further named as Lolduvin - S. Conclusion: This study was concluded by stating that Lolduvin-S exhibits the LAO activity and had been reported to possess a promising antimicrobial activity against the dreadful drug resistant pathogens. Lolduvin-S could be employed in near future as a novel therapeutic agent to treat various systemic ailments.

Keywords: Antimicrobial, drug resistant pathogens, Microbroth dilution method.

Full Text:

INTRODUCTION

Natural bioactive substances have the least quantum of side effects when compared with the synthetic products. Although most antibiotics were derived from terrestrial life, it is the marine world that may provide the pharmaceutical industry with the next generation of medicines. At present, a number of marine natural products are in the market or in clinical trials. Due to the huge diversity of marine bioactive compounds with respect to their chemical structure, mode of action and applicability, the extreme concern has been increased towards the screening of marine natural products for their biomedical potential1 . In spite of incredible evolution in medicinal field, some deadly diseases are not curable. The emergence and spread of antimicrobial resistance is now threatening to undermine our ability to treat infections and save lives. Drug resistant bacteria such as extended spectrum beta lactamase (ESBL) producing strains of Escherichia coli, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA) continue to cause a large number of infections and deaths particularly in developing countries2 . Thus a novel therapeutic compound from an alternate source with unique metabolic and physiological capabilities producing different kinds of metabolites must be employed for future therapeutic scenario globally. Squids under cephalopod family, is known to produce a black pigmented ink as a defensive ploy against its predators. This ink has already been reported to exhibit various therapeutic values3 . The crude ink extracts from various species of cephalopods have already been studied for its antimicrobial potential against biofilm bacteria, Staphylococcus aureus4 , clinical bacterial isolates and pathogenic yeasts5 , preservative property6 , antioxidant values7 and anti-retroviral activity8 . The ink has also been studied for its various bioactive proteins possessing cytolytic property9 , antitumour fractions10 and antimicrobial potential11. With these review background this study has been aimed at isolating and characterizing a new and novel protein from the ink of the Indian squid Loligo duvauceli and to study its antimicrobial potential against the drug resistant pathogens such as MRSA and ESBL producing strains of E.coli and K.pneumoniae.

METHODS

Collection and dissection of squids: Fresh squids were caught from the south west coast of Chennai and were decapitated by the fishermen. The squid was fixed in 4% formalin (4ml formalin in 96ml of water) and was identified as Loligo duvauceli by an eminent Zoologist based on the Zoological taxonomy. The dissection of the squids was performed as per the instructions of the Zoologist. After dissection of the ink sac the surface of the gland was sterilized with ethanol and was blotted with sterile cotton. The ink duct was cut with sterile scissors and the sac was gently squeezed and the excreted ink was collected in sterile brown bottles. The ink was stored at 4ºC until use.

Preparation of melanin free ink: The extracted fresh ink was subjected to centrifugation under cold condition at 15,000 rpm for 30 min to remove melanin. The supernatant was used as melanin free ink. The samples were frozen and kept at - 80°C until use.

Estimation of protein

The protein estimation was done by Lowry‘s method12 , a protein assay that is based on the reaction where the peptide bonds of proteins react with copper under alkaline conditions producing Cu+ ions that react with the Folin‘s reagent producing blue colour.

SDS Polyacrylamide gel electrophoresis:

SDS-PAGE was carried out using an acrylamide concentration of 7.5% in the resolving gel13. Stock solutions were prepared as recommended14 and stored in brown bottles at 4?C. The separating gel mix (12%) was prepared in a 25ml conical flask using acrylamide monomer 2.8ml, separating gel buffer (4x) 1.750 ml, 10 % SDS 0.070 ml, 10% APS 0.035 μl and distilled water 2.345 ml (Final volume – 7ml). The solution was de-gased for 5 min and 3 μl of TEMED was added with gentle mixing and was poured into the glass plates. 100 μl of N-butanol was added over the gel mix and the arrangement was left undisturbed for 20 min. The stacking gel mix (5%) was prepared by mixing acrylamide monomer 0.333 ml, 4x Stacking Gel Buffer 0.500 ml, 10 % SDS 0.020 ml, 10% APS 0.010 μl and distilled water 1.137 ml (Final volume – 2.0 ml). The electrophoretic run was performed as per standard protocols by adding the samples to the wells along with a molecular weight marker. Staining was done using Coomasie brilliant blue and destaining was done with ethanol and acetic acid. The gel was then analysed in the documentation unit. The molecular weight of the unknown peptides was thus determined by comparing it with the standard molecular weight marker bands.

Native Polyacrylamide gel electrophoresis:

Native – PAGE was performed without SDS using the same methodology as described above. Staining was not done. The unstained gel was then carefully removed and the target protein band portion corresponding to target protein band size was excised in sterile phosphate buffer and was eluted as follows.

Passive gel elution of the target protein:

Elution of the protein was performed by passively eluting the proteins from polyacrylamide gel pieces15. The excised gel pieces corresponding to the target protein band were placed in a clean microcentrifuge tubes. The elution buffer was prepared using Tris HCl 50 mM, NaCl 150 mM and EDTA 0.1 mM (pH 7.5). 0.5- 1ml of cold elution buffer is added so that the gel pieces were completely immersed. The gel pieces were crushed using a sterile scalpel and was incubated in a rotary shaker at 30ºC overnight. Centrifugation was done at 5000-10000g for 10 minutes and the supernatant was carefully pipetted into a new microcentrifuge tube. An aliquot of the supernatant might be tested for the presence of the same protein by subjecting it to SDS-PAGE

Enzymatic assay for L – amino acid oxidase [LAO] activity:

LAO activity of the protein was performed by an enzyme coupled assay16. Purified protein was prepared for the assay in 50 mM/L phosphate buffer and 150 mM/L KCl. The reaction mixture was prepared as follows: Tris HCl 0.1 mM/L (pH 7.6), Horseradish peroxide 10 μg, O-dianisidine 0.2 mM/L and L-amino acids 2 mM/L. The test sample was mixed with 100 μl of the reaction mixture. Reaction was performed at room temperature for 1-60 min. The activity was monitored by absorbance at 436 nm. The increase in absorbance was transformed into molar concentration of the end product o-dianisidine (8.31 x 103 mol/L). The Km and Vmax values of the Lamino acids were determined by Lineweaver-Burk plots.

Antimicrobial bioassay:

The antimicrobial assay was performed by Nathan‘s agar well diffusion method17 with the drug resistant strains that included the extended spectrum beta lactamase (ESBL) producing strains of E.coli and K.pneumoniae, methicillin resistant S.aureus (MRSA) and Amphotericin B resistant C.albicans isolated from various clinical specimens from the patients visiting the outpatient unit of Meenakshi General Hospital (Data not given). The control strains include E.coli (ATCC 25922), K.pneumoniae (ATCC 10031), S.aureus (ATCC 25923) and C.albicans (ATCC 10231).

A minimum of four colonies of the test organisms were touched with a sterile loop and transferred into Sterile Mueller Hinton broth and C.albicans into Saboraud‘s dextrose broth under aseptic conditions and was incubated for two hours at 37ºC. After incubation the density of each microbial suspension was adjusted equal to that of 106 c.f.u/ml (standardized by 0.5 MacFarland standards) and was used as the inoculum.

Agar well diffusion assay

Fifty microlitres (50μl) of inoculum of each test and control organism was spread as lawn cultures onto sterile Mueller Hinton agar plates using L-rods to achieve a confluent growth. The agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile agar borer in the inoculated agar plates. The eluted protein was subjected to syringe membrane filtration prior to the bioassay and an aliquot of the same was streaked onto nutrient agar plates and was incubated at 37 C for 24 hrs for sterility checking. A 50 μl volume of the eluted protein in phosphate buffered saline was propelled directly into the wells of the inoculated specific media agar plates for each test organism. The plates were then allowed to stand for 10 minutes for diffusion of the protein to take place and incubated at 37ºC for 24 hrs. Sterile DMSO served as Negative control. Erythromycin 30 µg (for bacteria) and Amphotericin B 100 U (for yeast) were included as positive controls. After incubation the plates were observed for the zone of inhibition around the wells and the zone size was measured using an antibiotic sensitivity measuring scale (Himedia). The antimicrobial efficacy was graded based on the zone diameter as high activity (> 15 mm), moderately active (10- 14 mm), trace activity (5-9 mm) and no activity (< 4 mm)18 . Determination of MIC value for the protein MIC value for the purified protein was determined by Microbroth dilution method19. Serial dilutions of the eluted protein were done in a 96 well microtitre plate with sterile phosphate buffer. The dilution factor was 5, 2.5, 1.25, 0.625, 0.312 and 0.156 μg/100 μl. To each dilution 100 μl of the culture broths of the test strains and control strains were added in their respective wells and the plate was incubated at 37 C for 24 hrs. After incubation the spectrophotometric analysis was performed and the OD values were recorded. The MIC value was also confirmed with Microbial Spot Checker board method20 where 3 μl of each dilution was spotted onto Mueller Hinton agar plates and incubated at 37 C for 24 hrs. After incubation the spot showing the complete absence of microbial growth indicates the minimum inhibitory concentration value.

RESULTS

Centrifugation of the crude ink at 15,000 rpm for 15 min yielded melanin free ink. Using Lowry‘s method, a standard curve of absorbance was obtained as a function of initial protein concentration and it was used to determine the unknown protein concentration. Thus the total protein in the melanin free ink was determined as 65.5 mg/ml. SDS PAGE showed a major protein band with a molecular weight of 60 KDa. Two weak bands were also obtained and their molecular weights were determined as 100 KDa and 80 KDa (Figure 1). The 60 Kda band was the target protein of the study as it has not been reported earlier. The protein was successfully isolated by Native Gel Electrophoresis (Figure 2) and was eluted by passive gel elution technique. L-amino acid oxidase (LAO) assay performed showed that when the purified protein and 2 mM/L amino acids were incubated for 1 min, L-lysine and LArginine proved to serve as excellent LAO substrates. Km and Vmax values of lysine were 33-87 µmol and 1.93-2.71 µmol/sec respectively and Km and Vmax values for arginine is 25-125 µmol and 1.56-3.30 µmol/sec. The reactions were completed within 30 secs at room temperature for lysine and arginine at a concentration of 0.02-2 mM/L. Thus in this study we report a novel protein with LAO activity from the ink of Loligo duvauceli which has been named as Lolduvin-S. The protein after membrane filtration yielded no growth on nutrient agar plate; thus it was sterile and was used for bioactivity assays. Lolduvin-S had scored to possess high antibacterial activity against the drug resistant test strains (Table 1). The zone size ranges from a mean value of 20 mm for E.coli (ESBL), 21 mm for K.pneumoniae (ESBL), 22 mm for S.aureus (MRSA) and 20 mm for C.albicans. The zone size was compared with the control strains (Figure 3). The MIC value was determined as 25 µg/ml for E.coli and K.pneumoniae, 12.5 µg/ml for S.aureus and C.albicans. The previous dilution that showed the visible decrease in the number of colonies was determined as the bacteriostatic dose and was deduced as 12.5 µg/ml for E.coli and K.pneumoniae, 6.25 µg/ml for S.aureus and C.albicans. Thus a novel protein called Lolduvin-S has been isolated and characterized to possess a potent LAO activity from the ink of Loligo duvauceli. The protein had also a promising antimicrobial activity against the dreadful resistant pathogens like ESBL and MRSA organisms.

DISCUSSION

The marine ecosystem offers rich niche for marine flora and fauna. The protein structures, metabolic pathways, reproductive systems, sensory and defense mechanisms developed by marine microorganisms and other lifeforms are distinctive in nature. During the last few decades several novel compounds in point of view of potential drug development have been isolated from marine habitat with various therapeutic applications21. The antimicrobial property of the squid ink is already a known fact and the ink has been found to posses a promising antimicrobial effect in our previous studies. The proteins present in various species of gastropods and cephalopods have been characterized earlier22. This study is thus undertaken to characterize a novel antimicrobial protein from the ink of Loligo duvauceli. The melanin free ink has been used in various studies for the analysis of the protein and it holds suitable for various protein analysis The total protein estimated in this study from the squid ink suggests the availability of the bioactive protein for various pharmaceutical applications. Determination of the molecular weight of the proteins is best achieved by the bioinstrumentational tool such as the SDS PAGE. The electrophoretic separation of the proteins from other species of squid has been achieved with 2.5% and 7.5% gel resolution23. In this study we employed 12.5% gel that is suitable for the separation of the protein from Loligo duvauceli‘s ink. Apart from the major protein band at 60 Kda, two other weak bands at 100 Kda and 80 Kda have also been observed. This shows the presence of tyrosinase24 and peroxidase25 respectively which has been already reported from the ink of various cephalopods and squids. The 60 Kda protein in Loligo duvauceli has not been reported anywhere earlier and thus it has been considered as the target protein. Native PAGE separates the protein in a pure form without denaturation though effective and high amount of proteins can be obtained with gel filtration chromatography methods26. Due to high cost, gel filtration chromatography is not done for the isolation of the proteins: thus the passive gel elution protocol suggested by the Pierce technologies, USA is followed for the same. It is a simple and rapid protocol for the elution of target protein. The elution protocol is slightly modified by performing the elution under cold condition. The protein eluted is rechecked by subjecting the protein for SDS-PAGE and by obtaining the same band at 60 Kda. A similar type of protein called Escapin with a molecular weight of 60 Kda has been reported from the ink of a sea hare under the Phylum Mollusca earlier27 . Escapin is known for its L-amino oxidase activity which is a hydrogen peroxide induced destruction of the target cells (such as micro-organisms) in the presence of Lamino acids. With this view an attempt to check the LAO activity of Lolduvin-S protein is done by Enzyme linked coupled assay. Lolduvin-S has been found to possess an enzymatic L-amino acid oxidase activity. Hydrogen peroxide is sufficient to cause bacteriostasis and does so with a concentration dependency and threshold similar to that of amino acids as substrates for the enzymatic activity. Both lysine and arginine are substrates for LAO activity from the squid ink. It shows strong and rapid activity in this study when using arginine or lysine as substrates, with reactions being completed within 30 secs at room temperature. A similar protein has been reported to be bacteriostatic and bactericidal from the purple gland of the sea hare Aplysia dactylomela28 and the substrates determine the bactericidal or bacteriostatic effects. Another protein called achacin possesses a bacteriostatic effect that seems to be mediated through its oxidation of the L– amino acids lysine and/or arginine and the consequent subsequent production of hydrogen peroxide29. Another antimicrobial protein has been named as escapin from the Aplysia sp., which has been cloned and characterized. Thus this study reports the new protein Lolduvin-S with a potent LAO activity from melanin free ink of Loligo duvauceli. Lolduvin-S from the melanin free ink sample has been found to be more effective against the Gram positive bacteria, gram negative bacteria and the pathogenic yeast.

In an earlier study, a protein in low concentration with potent L-amino acid oxidase activity showing a broad antibacterial activity against Gram-positive and Gram-negative bacteria by H2O2 generation has been reported from the skin mucous of the rock fish30. In correlation with this, the MIC value is determined at low concentration range of 6.25µg/ml. A similar protein has also been isolated from two purple pigmented bacteria from Mediterranean sea whose enzymatic activity is potent in generating the hydrogen peroxide in the presence of Lamino acids31 . A BLAST search suggests that the cDNA encoded a novel antibacterial protein sharing identity with a number of L-amino acid oxidases is effective against MRSA strain32. In correlation with these reports, this study states that the new protein Lolduvin-S from Loligo duvauceli‘s ink possess a promising antibacterial property against the drug resistant pathogens. The bioactive protein Lolduvin-S also scores to possess a high antifungal activity against C.albicans with a MIC value of 12.5 µg/ml. The same property has been reported from novel proteins from snake venoms33. Four glycoproteins with similar activity have been studied from a shell less gastropod sea hare34. A similar protein called Aplysianin-A with antifungal activity has been isolated, cloned and characterized from the purple fluid released from a sea hare with LAO activity35. The antifungal activity of nine amphibian skin secretions has been reported to possess a similar protein with LAO activity36 . Several cell surface proteins with LAO activity have been isolated from marine phytoplanktons with potent antifungal activity37. In relation with these reports, Lolduvin-S from Loligo duvauceli‘s ink possesses a potent antifungal activity. In conclusion, Lolduvin–S, a new and novel protein from the ink of the Indian squid Loligo duvauceli with a potent antimicrobial and LAO activity, can be considered in near future as a valuable pharmaceutical antimicrobial agent from the marine habitat. However further characterization and structural analysis of the same is under progress to acquire a detailed understanding of this novel protein. Lolduvin-S being found to be bactericidal against the ESBL and MRSA pathogens, it can definitely aid in the control and emergence of drug resistant strains globally.

ACKNOWLEDGEMENT

We are thankful to Dr.Mehar Sultana.,M.Sc.,M.Phil.,Ph.D., Professor, Department of Zoology, Presidency College, Chennai, Tamilnadu for speciating the squid selected for our study.

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