Brucella melitensis Rev.1’in Sferoplast Yapısı ile Gen Transferine Açık Hale Getirilmesi

Brusellozis, ülkemiz ve dünya hayvancılığına zarar veren kronik bakteriyel bir enfeksiyondur. Brucella doğada plazmidi bulunmadığından plazmid kaynaklı mutasyonlara çok açık değildir. Uzun pasajlar sonrası bakteriyel adaptasyon ile elde edilmiş mutantları bulunmaktadır. Konjugasyon, lipofectamin ve elektroporasyon ile yapılmış gen aktarım çalışmaları mevcuttur. Bu çalışmada kullanılan mutant pJQ200KS isimli intihar plazmidinde 6,7 kb (Backbone 5,4kb + 1,25kb İnsert) gibi oldukça büyük bir genoma sahiptir. Genom büyüdükçe transformasyonun zorlaşmaktadır. Sferoplast yapı, gen aktarımını kolaylaştırmada maya, mantar ve bitki hücrelerinde başarı ile kullanılmaktadır. Çalışmada Brucella’nın kompetent hale getirilerek gen aktarımının elektroporasyon ile kolaylaşması için hücreler, çeşitli yöntemlerle ile sferoplast haline getirildi ve gen aktarımında ki etkisine bakıldı. Stok Brucella melitensis Rev.1 kanlı agara pasajlandı. Kültür, Brucella broth içerisinde 37 ºC’de 24 saat boyunca 160 rpm’da inkübe edildi. Santrifüj ile 8000xg 4 ºC’de 5 dakika pelet haline getirildi, PBS ile yıkanarak besi yerinden arındırıldı. Sferoplast uyarımı için 72 saat boyunca penisilin, ampisilin ve glisin ile indüklendi. Elektroporasyon ile gen aktarımda en etkin yöntemin glisin ile uyarım metodu olduğu belirlendi. Aktarımı zor olan ve 7 kb’dan daha büyük olan plazmidlerin aktarımı için kompetent hücrenin sferoplast yapıya dönüştürülmesi metodu gelecekte yapılacak çalışmalarda araştırmacılara avantaj sağlayacağı düşünülmektedir.

Anahtar Kelimeler:

Brucella, Elektroporasyon, İntihar Plazmid, Sferoplast

Making Brucella melitensis Rev.1 open to gene transfer with the help of spheroplast structure.

Brucellosis is a chronic bacterial infection that harms livestock in our country and the world. Because of Brucella does not have a natural plasmid, it is not very open to plasmid-derived mutations. However, there are Brucella mutants obtained after long passages with bacterial adaptation. Gene transfer studies with conjugation, lipofectamine and electroporation are available. The suicide plasmid called pJQ200KS used in this study cannot be accomplished by conjugation since it does not have the λ pir gene region required for conjugation. It has been reported that as the size of the genome used for transfer increases, transformation becomes more difficult. Spheroplast structure has been successfully used in yeast, fungi and plant cells to facilitate gene transfer. There are two studies conducted for the transformation of Brucella into spheroplast structure. In studies to obtain spheroplasts from Brucella, after 24 hours of pre-culture, the spheroplast cells were exposed to the stimulation of penicillin, ampicillin and glycine for 48 hours, and penicillin and glycine were taken as the most effective stimulation method. Stock Brucella melitensis Rev.1 was passaged on blood agar. The culture was incubated in Brucella broth at 37 ° C for 24 hours at 160 rpm. 8000xg was pelleted for 5 minutes at 4 ° C by centrifugation, and the medium was cleared by washing with PBS. It was induced with penicillin, ampicillin and glycine for 72 hours for spheroplast stimulation. The structure has been made suitable for gene transfer. Since penicillin completely removes the cell wall structure of Brucella, unlike spheroplast formation, that it harms bacteria. It was determined that the most effective method in transferring with electroporation was stimulation with glycine. The method of transforming the component cell into a spheroplast structure for the transfer of plasmids that are difficult to transfer and larger than 7 kb will provide an advantage to researchers in studies.

Keywords:

Brucella, Electroporation, Suicide Plasmid, Spheroplast,

PDF

___

1. Siadat SD, Salmani AS, Aghasadeghi MR. (2012). Brucellosis Vaccines an Overview. In: Zoonosis. Morales JL (ed). pp. 143-166. Intech, Rijeka, Croatia.
2. Wyatt H. (2016). Lessons from the history of brucellosis. JMH. 5(1), 75-84.
3. Karagül SM. (2017). Pendik Veteriner Kontrol Enstitüsü ve 115 Yıllık Tarihi. Vet Hek Der Bülteni (14) 39-48.
4. Erganiş O. (2010). Hayvansal Aşıların Geliştirilmesinde Üniversite Kamu Sanayi İşbirliğinin Rolü. Eurasian J Vet Sci. 26 (1): 1-6.
5. Tarım Orman Bakanlığı. (2019). Brusellanın Konjuktival Aşı İle Kontrol ve Eradikasyonu Genelgesi. Erişim: http://extwprlegs1.fao.org/docs/pdf/tur196615.pdf . Erişim Tarihi: 16.08.2019
6. Gıda ve Kontrol Genel Müdürlüğü. (2012). Brusellanın Konjuktival Aşı ile Kontrol ve Eradikasyonu Projesi. Erişim:http://www.tarim.gov.tr/Documents/Mevzuat/Genelgeler/BRUCELLA. Pdf. Erişim Tarihi: 26.10.2019
7. Hur J, Xiang Z, Feldman EL, et al. (2011). Ontology-Based Brucella Vaccine Literature Indexing and Systematic Analysis of Gene Vaccine Association Network. BMC Immunol. 12(1): 49.
8. Gupta V, Verma DK, Singh SV, et al. (2007). Serological Diagnostic Potential of Recombinant Outer Membrane Protein (Omp31) from Brucella melitensis in Goat and Sheep Brucellosis. Small Rumin Res. 70(2-3): 260-266.
9. Tibor A, Decelle B, Letesson J. (1999). Outer Membrane Proteins Omp10, Omp16, and Omp19 of Brucella spp. are Lipoproteins. Infect Immun. 67(9): 4960-4962.
10. Corbel MJ. (1997). Brucellosis an Overview. Emerg Infect Dis. 3(2): 213.
11. Ficht TA, Pei J, Kahl-McDonagh M. (2010). In Vitro Mutagenesis of Brucella Species. In: In Vitro Mutagenesis Protocols. Braman J (ed). 3rd ed. pp.15-35. Springer, Stratagene, La Jolla, USA.
12. Haine V, Dozot M, Dornand J, et al. (2006). NnrA is Required for Full Virulence and Regulates Several Brucella melitensis Denitrification Genes. J Bacteriol. 188(4): 1615-1619.
13. Elzer PH, Phillips RW, Kovach ME, et al. (1994). Characterization and Genetic Complementation of a Brucella abortus high-temperature-requirement A (htrA) Deletion Mutant. Infect Immun. 62(10): 4135-4139.
14. McQuiston JR, Schurig GG, Sriranganathan N, et al. (1995). Transformation of Brucella Species with Suicide and Broad Host-Range Plasmids. In: Electroporation Protocols for Microorganisms. Nickoloff JA (ed). pp. 143-148. Springer, Totowa, New Jersey, USA.
15. Wu Q, Pei J, Turse C, et al. (2006). Mariner Mutagenesis of Brucella melitensis Reveals Genes with Previously Uncharacterized Roles in Virulence and Survival. BMC Microbiol. 6(1): 102.
16. Allen CA, Adams LG, Ficht TA. (1998). Transposon-Derived Brucella abortus Rough Mutants are Attenuated and Exhibit Reduced Intracellular Survival. Infect Immun. 66(3): 1008-1016.
17. Kahl-McDonagh M, Ficht T. (2006). Evaluation of Protection Afforded by Brucella abortus and Brucella melitensis Unmarked Deletion Mutants Exhibiting Different Rates of Clearance in BALB/c Mice. Infect Immun. 74(7): 4048-4057.
18. Quandt J, Hynes MF. (1993). Versatile Suicide Vectors which Allow Direct Selection for Gene Replacement in Gram-Negative Bacteria. Gene. 127(1): 15-21.
19. Miller VL, Mekalanos JJ. (1988). A Novel Suicide Vector and Its Use in Construction of Insertion Mutations: Osmoregulation of Outer Membrane Proteins and Virulence Determinants in Vibrio cholerae Requires toxR. J Bacteriol. 170(6): 2575-2583.
20. Gay P, Coq DL, Steinmetz M, et al. (1985). Positive Selection Procedure for Entrapment of Insertion Sequence Elements in Gram-Negative Bacteria. J Bacteriol. 164(2): 918-921.
21. Golovliov I, Sjöstedt A, Mokrievich A, et al. (2003). A Method for Allelic Replacement in Francisella tularensis. FEMS Microbiol Lett. 222(2): 273-280.
22. Kaniga K, Delor I, Cornelis GR. (1991). A Wide-Host-Range Suicide Vector for Improving Reverse Genetics in Gram-Negative Bacteria: Inactivation of the blaA Gene of Yersinia enterocolitica. Gene. 109(1): 137-141.
23. Ortiz-Martín I, Macho AP, Lambersten L, et al. (2006). Suicide Vectors for Antibiotic Marker Exchange and Rapid Generation of Multiple Knockout Mutants by Allelic Exchange in Gram-Negative Bacteria. J Microbiol Methods. 67(3): 395-407.
24. Smith LD, Heffron F. (1987). Transposon Tn5 Mutagenesis of Brucella abortus. Infect Immun. 55(11): 2774-2776.
25. Cassataro J, Velikovsky CA, Barrera Sdl, et al. (2005). A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection Against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response. Infect Immun. 73(10): 6537-6546.
26. Lai F, Schurig GG, Boyle SM. (1990). Electroporation of a Suicide Plasmid Bearing a Transposon into Brucella abortus. Microb Pathog. 9(5): 363-368.
27. Soler-Lloréns P, Gil-Ramírez Y, Zabalza-Baranguá A, et al. (2014). Mutants in the Lipopolysaccharide of Brucella ovis are Attenuated and Protect Against B. ovis Infection in Mice. Vet Res. 45(1):72.
28. Martin H. (1983). Protoplasts and Spheroplasts of Gram-Negative Bacteria (with Special Emphasis on Proteus mirabilis). In: Protoplasts 1983. Potrykus I, Harms CT, Hinnen A, Hütter R, King P, Shillito RD (eds). pp. 213-225. Basel, Switzerland.
29. Burgers PM, Percival KJ. (1987). Transformation of Yeast Spheroplasts without Cell Fusion. Anal Biochem. 163(2): 391-397.
30. Thierbach G, Schwarzer A, Pühler A. (1988). Transformation of Spheroplasts and Protoplasts of Corynebacterium glutamicum. Appl Microbiol Biotechnol. 29(4): 356-362.
31. Gheibi A, Khanahmad H, Kardar GA, et al. (2019). Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain. Adv Biomed Res. 8:37.
32. Kung SH, Retchless AC, Kwan JY, et al. (2013). Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa. Appl Environ Microbiol.79(5):1712-1717.
33. Hines WD, Freeman BA, Pearson GR. (1964). Production and Characterization of Brucella Spheroplasts. J Bacteriol. 87(2): 438-445.
34. Hines WD, Freeman BA, Pearson GR. (1964). Fine Structure of Brucella suis Spheroplasts. J Bacteriol. 87(6): 1492-1498.
35. Potter H, Heller R. (2018). Transfection by Electroporation. Curr Protoc Mol Biol. 121(1): 1-13.
36. Cheng Q, Shi X, Zhang Y. (2020). Reprogramming Exosomes for Immunotherapy. In: Cell Reprogramming for Immunotherapy. Katz SG, Rabinovich PM (eds). pp. 197-209. Springer, New Haven, USA.
37. Nguyen AW, Le K, Maynard JA. (2020). Engineering Antibodies on the Surface of CHO Cells. In: Genotype Phenotype Coupling. Zielonka S, Krah S (eds) pp. 397-422. Springer, Darmstadt, Germany.
38. Hynes MF, Quandt J, O'Connell MP, et al. (1989). Direct Selection for Curing and Deletion of Rhizobium Plasmids Using Transposons Carrying the Bacillus subtilis sacB Gene. Gene. 78(1): 111-120.
39. Scupham AJ, Triplett EW. (1997). Isolation and Characterization of the UDP-glucose 4′-epimerase-Encoding Gene, galE, from Brucella abortus 2308. Gene. 202(1-2): 53-59.
40. Halling SM, Detilleux PG, Tatum FM, et al. (1991). Deletion of the BCSP31 Gene of Brucella abortus by Replacement. Infect Immun. 59(11): 3863-3868.