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Cloning and in planta expression of an omiganan antimicrobial peptide in tobacco and potato plants to control the growth of human bacterial pathogens

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References

1. Midura-Nowaczek K, Markowska A.

Antimicrobial peptides and their analogs:

searching for new potential therapeutics.

Perspectives in medicinal chemistry.

2014;6:73-80.

2. Guaní-Guerra E, Santos-Mendoza T, Lugo- Reyes SO, Terán LM. Antimicrobial peptides: general overview and clinical implications in human health and disease.

Clinical Immunology. 2010;135(1):1-11.

3. Zhang L-j, Gallo RL. Antimicrobial peptides. Current Biology. 2016;26(1):R14- R19.

4. Burt S. Essential oils: their antibacterial properties and potential applications in foods a review. International journal of food microbiology. 2004;94(3):223-253.

5. Oard S, Enright F. Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant cell reports. 2006;25(6):561-572.

6. Isaacson R. MBI-226. Micrologix/Fujisawa.

Current opinion in investigational drugs (London, England: 2000). 2003;4(8):999- 1003.

7. Castanho M, Santos N. Peptide drug discovery and development: translational research in academia and industry: John Wiley & Sons; 2011.

8. Nazarian-Firouzabadi F, Trindade LM, Visser RG. Production of small starch granules by expression of a tandem-repeat of a family 20 starch-binding domain (SBD3-SBD5) in an amylose-free potato genetic background. Functional Plant Biology. 2012;39(2):146-155.

9. Badr Hadad A, Nazarian Firouzabadi F, Ismaili A, Bagheri H. Cloning and expression of an omiganan pentahydrochloride (MBI226) antimicrobial peptide in tobacco (Nicotiana tabacum) confers resist to bacterial pathogens. Crop Biotechnology. 2017;7(19):1-13 (In Persian).

10.Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum. 1962;15(3):473-497.

11.Nazarian Firouzabadi F, Vincken J-P, Ji Q, Suurs LC, Buléon A, Visser RG.

Accumulation of multiple-repeat starch- binding domains (SBD2–SBD5) does not reduce amylose content of potato starch granules. Planta. 2007;225(4):919-933(In Persian).

12.Phan HT, Hause B, Hause G, Arcalis E, Stoger E, Maresch D, et al. Influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants. PLoS One.

2014;9(6):e99347.

13.Richards E, Reichardt M, Rogers S.

Preparation of genomic DNA from plant tissue. Current protocols in molecular biology. 1994:2.3.1-2.3.7.

14.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976;72(1-2):248-254.

15.Elisha IL, Botha FS, McGaw LJ, Eloff JN.

The antibacterial activity of extracts of nine

(14)

plant species with good activity against Escherichia coli against five other bacteria and cytotoxicity of extracts. BMC complementary and alternative medicine.

2017;17(1):2-10.

16.Furletti V, Teixeira I, Obando-Pereda G, Mardegan R, Sartoratto A, Figueira G, et al.

Action of Coriandrum sativum L. essential oil upon oral Candida albicans biofilm formation. Evidence-Based Complementary and Alternative Medicine. 2011;2011:1-10.

17.Bakal SN, Bereswill S, Heimesaat MM.

Finding novel antibiotic substances from medicinal plants—Antimicrobial properties of Nigella sativa directed against multidrug resistant bacteria. European Journal of Microbiology and Immunology.

2017;7(1):92-98.

18.Jain J, Tikare S, Mahuli A. Antifungal activity of ginger extract on Candida albicans: an in-vitro study. Journal of Dental Sciences and Research.

2011;2(2):18-21.

19.Osusky M, Osuska L, Hancock RE, Kay WW, Misra S. Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot.

Transgenic research. 2004;13(2):181-190.

20.Osusky M, Zhou G, Osuska L, Hancock RE, Kay WW, Misra S. Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnology.

2000;18(11):1162-1166.

21.Diaz AH, Kovacs I, Lindermayr C.

Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in

Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria.

PloS one. 2016;11(10):e0164097.

22.Fritsche TR, Rhomberg PR, Sader HS, Jones RN. Antimicrobial activity of omiganan pentahydrochloride tested against contemporary bacterial pathogens commonly responsible for catheter- associated infections. Journal of antimicrobial chemotherapy.

2008;61(5):1092-1098.

23.Melo MN, Dugourd D, Castanho MA.

Omiganan pentahydrochloride in the front line of clinical applications of antimicrobial peptides. Recent patents on anti-infective drug discovery. 2006;1(2):201-207.

24.Melo MN, Castanho MA. Omiganan interaction with bacterial membranes and cell wall models. Assigning a biological role to saturation. Biochimica et Biophysica

Acta (BBA)-Biomembranes.

2007;1768(5):1277-1290.

25.Sader HS, Fedler KA, Rennie RP, Stevens S, Jones RN. Omiganan pentahydrochloride (MBI 226), a topical 12-amino-acid cationic peptide: spectrum of antimicrobial activity and measurements of bactericidal activity.

Antimicrobial agents and chemotherapy.

2004;48(8):3112-3118.

26.Subbalakshmi C, Sitaram N. Mechanism of antimicrobial action of indolicidin. FEMS microbiology letters. 1998;160(1):91-96.

27.Nawrocki KL, Crispell EK, McBride SM.

Antimicrobial peptide resistance mechanisms of gram-positive bacteria.

Antibiotics. 2014;3(4):461-492.

(15)

28.Sabat A, Kosowska K, Poulsen K, Kasprowicz A, Sekowska A, van den Burg B, et al. Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus.

Infection and immunity. 2000;68(2):973- 976.

29.Subczynski WK, Wisniewska A. Physical properties of lipid bilayer membranes:

relevance to membrane biological functions.

Acta Biochimica Polonica 2000;47(3):613- 626.

(16)

Cloning and in planta expression of an omiganan antimicrobial peptide in tobacco and potato plants to control the growth of human bacterial pathogens Nazarian Firouzabadi F1,2*, Badrhadad A3, Sheikhian A4

1. Razi Herbal Medicines Research Center, Lorestan University of Medical Science, Khorramabad, Iran [email protected]

2. Professor, Agronomy and Plant Breeding Department, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.

3. Ph.D. Student, Agronomy and Plant Breeding Department, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

4. Associate Professor, Department of Immunology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran

Received: 17 Oct 2018 Accepted: 3 Dec 2018

Abstract

Background

: Antimicrobial peptides are one of the vital components of innate immunity in plants and animals. The identification and introduction of novel and effective peptide molecules to plants is a cost-effective way both to improve the resistance of crop plant species to pathogens, and to produce peptides for pharmaceutical applications by using recombinant DNA technology.

Material and Methods

: An expression construct containing the omiganan (MBI-226) antimicrobial gene sequence was cloned and used for tobacco and potato Agrobacterium tumefaciens-mediated transformation. Following tissue culture, Polymerase chain reaction analysis (PCR) confirmed that some kanamycin resistant plants are transgenic. A number of transgenic plants, along with a non-transgenic control, were selected. Total protein was extracted from the transgenic plants, and the non-transgenic control, and was used for antimicrobial activity assay against some human pathogens, including; Escherchia coli, Staphylococcus epidermis, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Bacillus cereus, Candida albicans using the disc diffusion method.

Results

: Total protein extract from transgenic plants was significantly (P<0.05) able to inhibit the majority of bacteria growth, whereas non-transgenic total protein extract did not inhibit human pathogens growth. There was a significant difference (P<0.05) between transgenic lines with respect to omiganan peptide activity. In contrary to gram-negative bacteria, omiganan peptide did not have a significant effect (P>0.05) on human gram-positive pathogenic bacteria.

Conclusion

: The total protein from the omiganan-expressing peptide had a strong antibacterial activity against some human bacterial pathogens. By expression and purification of the omiganan peptide, the peptide could be used as an antibiotic to destroy pathogenic bacteria. This approach could open an opportunity to produce antibacterial peptides in plants for pharmaceutical applications.

Keywords:

Antimicrobial peptides, Agrobacterium tumefaciens, Pathogen, Recombinant protein, Tobacco.

*Citation: Nazarian Firouzabadi F, Badrhadad A, Sheikhian A. The cloning and in planta expression of an omiganan antimicrobial peptide in tobacco and potato plants to control human bacterial pathogens growth. Yafte. 2019; 20(4):14-29.

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