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Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 2055-2059 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 2055-2059 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.603.234 In vitro Efficacy of Antibacterial Proteins from Haemolymph of Silkworm Breeds against Bacterial Pathogens of Mulberry Silkworm, Bombyx mori L. S. Manimegalai1, B. Bebitha2 and P.Mohanraj3* 1 Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore-3, India 3 Department of Sericulture, Forest College and Research Institute, Mettupalayam, India *Corresponding author ABSTRACT Keywords Bombyx mori, Antibacterial, Proteins, Mulberry, Bacillus thuringiensis. Article Info Accepted: 20 February 2017 Available Online: 10 March 2017 Antibacterial activity of proteins eluted from immunized haemolymph collected from breeds of mulberry silkworm, Bombyx mori L., viz., Rong daizo, CSR2 and double hybrid were studied against bacterial pathogens. The breeds were immunized with bacterial pathogens, Escherichia coli (KR strain), Bacillus thuringiensis and Staphylococcus aureus . In vitro efficacy of purified antibacterial proteins from breeds of B.mori against E.coli, S. aureus and B. thuringiensis revealed that among the ten fractions of antibacterial proteins eluted from B.mori breeds, ninth fraction@ 200µl showed higher antibacterial activity in Rong daizo (3.01cm, 3.10 cm and 1.82cm) (2.71 cm, 3.01cm and 1.60 cm), CSR2 (2.98 cm, 3.05 cm and 1.81 cm) and double hybrid ( 2.89 cm, 2.98 cm and 1.79 cm ) against E.coli, S. aureus and B. thuringiensis (Table 1,2&3).Lesser inhibitory activity was recorded in seventh fraction @ 200 µl concentration of Rong daizo (2.71 cm, 3.01 and 1.60 cm ) followed by CSR2 (2.68 cm, 2.98cm and 1.59 cm) and DH (2.69 cm, 2.96 cm and 1.54 cm) respectively. Standard antibiotic streptomycin sulphate @ 200 µl concentration produced the inhibition zone of 7.12 cm, 8.12 cm and 6.32 cm. Purified antibacterial protein fractions showed higher antibacterial activity against gram negative E.coli and gram positive S.aureus and low activity against B.thuringiensis. Introduction Several self-defense proteins have been isolated from the silkworm, Bombyx mori and their amino acid sequences determined. These proteins include novel antibacterial proteins designated lebocin and moricin, Among the various defense mechanisms in B.mori, humoral responses involved phenoloxidase and immune proteins such as antimicrobial proteins (AMPs), lysozyme and lectins. In response to microbial infection, AMPs and lysozyme are rapidly produced primarily in the fat body and haemocytes and subsequently secreted into the haemolymph to eliminate invading pathogens (Tanaka and Yamakawa, 2011). The induced antibacterial protein of silkworm have shown antibacterial effect against a wide variety of gram positive bacteria such as Agrobacterium tumefaciens, Enterobacter cloacae, Escherichia coli, Serratia marcescens and gram negative bacteria such as Micrococcus ureae, Cornybacterium equi and Staphylococcus aureus (Morishima et al.,1988). Upon bacterial infection, antibacterial proteins, 2055 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 2055-2059 lectins and lysozymes rapidly appear in the haemolymph (Boman and Hultmark,1987). Cecropins are family of highly potent bactericidal peptides with 35-37 amino acid residues (Boman et al., 1991). B.mori was found to contain eleven types of cecropin ( Tu et al., 1989; Morishima et al.,1990; Hara et al.,1994). collected after 24 h of treatment into pre cooled tubes containing one per cent phenylthiourea, centrifuged at 10000 rpm at 4° C for 10 min and stored at -20° C. Haemolymph from untreated larvae served as control. Present study investigated the antibacterial activity of haemolymph of B.mori by inhibition zone assay. The haemolymph proteins obtained from Sephadex G-100 column (55x1.5cm) were used for SDSPAGE analysis. Among the ten fractions isolated, fractions seven and nine were found to have antibacterial activity and were compared with streptomycin sulphate. The fractions isolated from infected breeds of B.mori were more effective at higher dose of 200 µl compared to 100 µl against E.coli, S.aureus and Bacillus thuringiensis in Rong Daizo (3.01±0.042, 3.10±0.04 and 1.82± 0.051), Double hybrid (2.89± 0.732, 2.98± 0.750 and1.79± 0.445) and CSR2 (2.98±0.748, 3.05±0.764 and 1.81±0.448) respectively. Step I: Haemolymph was collected from 30 immunized larvae at 24 h after treatment and diluted five times in 0.3M ammonium acetate maintaining a pH of 7.0 and applied to a Sephadex G-75 (20x1cm) column equilibrated in 0.3M ammonium acetate at a flow rate of 10ml/hr. The column was washed several times with 0.3M ammonium acetate. The bound proteins were eluted stepwise using the same buffer and the obtained fractions were observed at 280 nm (Abraham et al.,1995). Materials and Methods Purification of induced protein from silkworm antibacterial Collection of haemolymph Silkworm breeds, Rong Daizo, CSR 2 and double hybrid were reared on mulberry leaves under ambient conditions as per the standard rearing method (Krishnaswami,1978). Bacterial pathogens, Escherichia coli (KR strain), Bacillus thuringiensis and Staphylococcus aureus were used for antibacterial assay. Three day old fifth instar larvae were fed with 10 µl suspension of log phase E.coli at 1x103 cells/ml by spraying on mulberry leaves. Haemolymph samples were Purification of antibacterial protien Step II: Antibacterial proteins obtained from the step I was applied onto a Sephadex G-100 column (55x1.5cm) in 0.1M phosphate buffer ( pH 7.0) at a flow rate of 18ml/hr and eluted fractions were observed in UVspectrophotometer at 280 nm. Haemolymph fractions were also collected from larvae treated with saline to compare the antibacterial protein induction with response to oral administration of bacteria. The fractions collected from step I and step II were tested for its antibacterial activity. Antibacterial activity was assayed by measuring the zone of growth inhibition on thin agar plates with E.coli, S.aureus and B.thuringiensis. Serially diluted control and immunized protein samples were applied into wells on a thin agar plate seeded with bacteria. The zone of inhibition was measured and compared to that of control protein. Extracted fractions were compared with the standard antibiotic, Streptomycin sulphate. 2056 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 2055-2059 Results and Discussion Studies on in vitro efficacy of purified antibacterial proteins from Rong daizo, CSR2 and double hybrid of B.mori against E.coli, S. aureus and B. thuringiensis revealed that among the ten fractions of antibacterial proteins eluted from B.mori breeds, ninth fraction @ 200µl showed higher antibacterial activity in Rong daizo (3.01cm, 3.10 cm and 1.82 cm) (2.71 cm, 3.01cm and 1.60 cm), CSR2 (2.98 cm, 3.05 cm and 1.81 cm) and double hybrid ( 2.89 cm, 2.98 cm and 1.79 cm ) against E.coli, S. aureus and B. thuringiensis (Table 1,2&3) Lesser inhibitory activity was recorded in seventh fraction compared to ninth fraction @ 200 µl concentration of Rong daizo (2.71 cm, 3.01 and 1.60 cm ) followed by CSR2 (2.68 cm, 2.98cm and 1.59 cm) and DH (2.69 cm, 2.96 cm and 1.54 cm) respectively. Standard antibiotic streptomycin sulphate @ 200 µl concentration produced the inhibition zone of 7.12 cm, 8.12 cm and 6.32 cm. The present results are in conformity with the findings of (Hoffmann and Hetru,1992.) who reported that protein purified from different insect sources are reported to attack only grampositive bacteria.. Antibacterial activity of obtained proteins also corroborated with the findings of Morishima et al. (1988) who reported that purified antibacterial protein fractions showed higher antibacterial activity against gram negative E.coli and gram positive S.aureus and low activity against B.thuringiensis. which is well supported by the present study. Table.1 In vitro efficacy of purified antibacterial proteins (ABP) from Rong Daizo against bacterial pathogens Treatments Zone of inhibition (cm±S.D.)* by ABP from Rong Daizo E.coli S.aureus B.thuringiensis i. 100 2.30±0.032 2.90±0.013 1.41± 0.033 ii. 200 2.71±0.008 3.01±0.014 1.60± 0.012 i. 100 2.81±0.014 2.92±0.008 1.55± 0.008 ii. 200 3.01±0.042 3.10±0.04 1.82± 0.051 i. 100 3.51±0.005 3.61±0.008 3.21±0.005 ii. 200 7.12±0.015 8.12±0.017 6.32±0.008 ABP eluted fractions (µl) A1. Fraction -7 A2. Fraction -9 B. Streptomycin Sulphate *Values are the diameter of zone of inhibition (mean of three replications) in centimeter ± standard deviation 2057 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 2055-2059 Table.2 In vitro efficacy of purified antibacterial proteins (ABP) from CSR 2 silkworm breed against bacterial pathogens Zone of inhibition (cm±S.D.) * by ABP from CSR 2 E.coli S.aureus B.thuringiensis Treatments ABP eluted fractions (µl) Fraction -7 i. 100 ii. 200 Fraction -9 i. 100 ii. 200 Streptomycin Sulphate i. 100 ii. 200 2.28± 0.570 2.68 ±0.674 2.87± 0.720 2.98±0.748 1.39± 0.348 1.59±0.398 2.76 ±0.693 2.98±0.748 2.89±0.720 3.05±0.764 1.53±0.383 1.81±0.448 3.51±0.005 7.12±0.015 3.61±0.008 8.12±0.017 3.21±0.005 6.32±0.008 *Values are the diameter of zone of inhibition (mean of three replications) in centimetre ± standard deviation Table.3 In vitro efficacy of purified antibacterial proteins (ABP) from Double hybrid of Bombyx mori against bacterial pathogens Zone of inhibition (cm±S.D.)* by ABP from Double hybrid ABP eluted fractions (µl) Bacterial pathogens A1.Fraction 7 E.coli S.aureus B.thuringiensis i. 100 2.27± 0.565 2.86± 0.712 1.41± 0.348 ii. 200 2.69± 0.667 2.96± 0.737 1.54± 0.386 A2.Fraction 9 E.coli S.aureus B.thuringiensis i. 100 2.74± 0.680 2.86± 0.715 1.51± 0.373 ii. 200 2.89± 0.732 2.98± 0.750 1.79± 0.445 B. Streptomycin Sulphate i. 100 3.51±0.005 3.61±0.008 3.21±0.005 ii. 200 7.12±0.015 8.12±0.017 6.32±0.008 Treatments * Values are the diameter of zone of inhibition (mean of three replications) in centimeter ± standard deviation The present results on antibacterial activity of antibacterial protein isolated from B.mori is in line with the findings of Pandia rajan et al.( 2011) who reported that cocoon shell extract had antimicrobial activity against E. coli, Bacillus cereus, S. aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. Abraham et al. (1995) reported induction of antibacterial activity in the haemolymph of silkworm by injection with live cells of E.coli which was purified as antibacterial protein by Sephadex G-100 column chromatography, exhibited antibacterial activity against both E.coli and M. luteus. The present result on antibacterial activity of ABP against bacterial pathogens were supported with the findings of Sharma et al. (2000) who reported the effectiveness of 2058 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 2055-2059 proteins in inhibiting the growth of P. aeruginosa (AC-3) and also attributed that low molecular weight proteins were found to be effective against them; the diameter of the inhibition zone was found to be 3.5 cm. In conclusion, proteins extracted from haemolymph of mulberry silkworm breeds, Rong daizo, CSR2 and double hybrid immunized with bacterial pathogens, Escherichia coli (KR strain), Bacillus thuringiensis and Staphylococcus aureus showed antibacterial activity through inhibition zone assays. Antibacterial activity was found to be higher against E.coli and S.aureus compared to B.thuringiensis for all the breeds studied. The lower efficacy might be due to higher virulence of B.thuringiensis. Thuss exploring the mechanism of induction of antibacterial proteins upon immunization of silkworm breeds with E.coli and S.aureus may pave way for development of disease tolerant breeds. 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Purification and characterization of peptide from Bombyx mori, Sco. China Ser. B., 32: 1072 – 1081. How to cite this article: Manimegalai, S., B. Bebitha and Mohanraj, P. 2017. In vitro Efficacy of Antibacterial Proteins from Haemolymph of Silkworm Breeds against Bacterial Pathogens of Mulberry Silkworm, Bombyx mori L. Int.J.Curr.Microbiol.App.Sci. 6(3): 2055-2059. doi: https://doi.org/10.20546/ijcmas.2017.603.234 2059