Escherichia coli'de Acinetobacter baumannii OccAB1 Porin Proteininin Klonlanması, Ekspresyonu ve Saflaştırılması

Öz

Acinetobacter baumannii, toprakta, suda, insan ve hayvan vücutlarının yüzeyleri gibi farklı habitatlarda serbest yaşayan gram negatif bir organizmalardır. Ancak zamanla patojenik bir yaşam tarzına adapte olarak tüm dünyada yoğun bakım ünitelerinde enfeksiyonlara ve ölümlere neden olmaktadır. Antibiyotik direncinde porin proteinleri bu bakteride temel rol oynamaktadır. Porinler ve dışa akış pompalarının aşırı ekspresyonu, ilaç direncini artırmada ve tedavinin etkisiz hale getirilmesinde katkıda bulunan faktörler olarak tespit edilmiştir. Bu sebeple antibiyotik geçişlerinin in vitro olarak çalışılabilmesi için öncelikli olarak bu proteinlerin saflaştırılması gerekmektedir. Saflaştırma işleminin ardından elde edilen bu porin proteini ile çift katmanlı lipit tabakalarda antibiyotiklerin geçişleri araştırılacaktır. Bu çalışmada, A. baumannii OccAB1 porin proteini klonlandı ve E. coli'de eksprese edildi. Sonuç olarak, ekspresyon vektörüne klonlanan A. baumannii OccAB1 porini, E. coli'de başarıyla eksprese edildi.

Anahtar Kelimeler

• E. coli
• A. baumannii
• Ni-NTA
• Protein saflaştırma
• OccAB1

Cloning, Expression, and Purification of Acinetobacter baumannii OccAB1 Porin Protein in Escherichia coli

Öz

Acinetobacter baumannii is a free-living gram-negative organism in different habitats such as soil, water, surfaces of human and animal bodies. However, adapting to a pathogenic lifestyle over time, it causes infections and deaths in intensive care units all over the world. Porin proteins play a fundamental role in antibiotic resistance in this bacterium. Overexpression of porins and efflux pumps have been identified as contributing factors in increasing drug resistance and inactivation of therapy. For this reason, to study antibiotic transitions in vitro, these proteins must be purified first. With this porin protein obtained after purification, the passage of antibiotics in bilayer lipid layers will be investigated. In this study, A. baumannii OccAB1 porin protein was cloned and expressed in E. coli. As a result, A. baumannii OccAB1 porin cloned into the expression vector was successfully expressed in E. coli.

Anahtar Kelimeler

• E. coli
• A. baumannii
• Ni-NTA
• Protein Purification
• OccAB1

Kaynakça

1. Abbott, I., Cerqueira, G. M., Bhuiyan, S., & Peleg, A. Y. (2013). Carbapenem resistance in Acinetobacter baumannii: laboratory challenges, mechanistic insights, and therapeutic strategies. Expert Review of Anti-Infective Therapy, 11(4), 395–409. https://doi.org/10.1586/ERI.13.21
2. Assenberg, R., Wan, P. T., Geisse, S., & Mayr, L. M. (2013). Advances in recombinant protein expression for use in pharmaceutical research. Current Opinion in Structural Biology, 23(3), 393–402. https://doi.org/10.1016/J.SBI.2013.03.008
3. Bhamidimarri, S. P., Zahn, M., Prajapati, J. D., Schleberger, C., Söderholm, S., Hoover, J., West, J., Kleinekathöfer, U., Bumann, D., Winterhalter, M., & van den Berg, B. (2019). A Multidisciplinary Approach toward Identification of Antibiotic Scaffolds for Acinetobacter baumannii. Structure (London, England : 1993), 27(2), 268-280.e6. https://doi.org/10.1016/J.STR.2018.10.021
4. Blattner, F. R., Plunkett, G., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., & Shao, Y. (1997). The complete genome sequence of Escherichia coli K-12. Science (New York, N.Y.), 277(5331), 1453–1462. https://doi.org/10.1126/SCIENCE.277.5331.1453
5. Borneleit, P., & Kleber, H.-P. (1991). The Outer Membrane of : Structure-Function Relationships. The Biology of Acinetobacter, 259–271. https://doi.org/10.1007/978-1-4899-3553-3_18
6. Choi, U., & Lee, C. R. (2019). Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in Escherichia coli. Frontiers in Microbiology, 10(APR), 1–9. https://doi.org/10.3389/fmicb.2019.00953
7. Clark, T. J., Momany, C., & Neidle, E. L. (2002). The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1. Microbiology (Reading, England), 148(Pt 4), 1213–1223. https://doi.org/10.1099/00221287-148-4-1213 Darcan, C., Özkanca, R., İdil. Ö. (2009). The role of RpoS, H-NS and AcP on the pH-dependent OmpC and OmpF porin expressions of Escherichia coli at different pH, Afrıcan Journal of Bıotechnology 8(9):1845-1854.
8. Darcan, C. (2012). Expression of OmpC and OmpF porin proteins and survival of escherichia coli under photooxidative stress in black seawater. Aquatic Biology, 17(2), 97–105. https://doi.org/10.3354/AB00458

9. Darcan, C., & Aydin, E. (2012). Fur- mutation increases the survival time of Escherichia coli under photooxidative stress in aquatic environments. Acta Biologica Hungarica, 63(3), 399–409. https://doi.org/10.1556/ABIOL.63.2012.3.10
10. Darcan, C., Özkanca, R., & Flint, K. P. (2003). Survival of nonspecific porin-deficient mutants of Escherichia coli in black seawater. Letters in Applied Microbiology, 37(5), 380–385. https://doi.org/10.1046/j.1472-765X.2003.01418.x
11. Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27(3), 297–306. https://doi.org/10.1016/J.BIOTECHADV.2009.01.008
12. Dupont, M., Pagès, J. M., Lafitte, D., Siroy, A., & Bollet, C. (2005). Identification of an OprD homologue in Acinetobacter baumannii. Journal of Proteome Research, 4(6), 2386–2390. https://doi.org/10.1021/PR050143Q/ASSET/IMAGES/PR050143Q.SOCIAL.JPEG_V03
13. Esterly, J. S., Griffith, M., Qi, C., Malczynski, M., Postelnick, M. J., & Scheetz, M. H. (2011). Impact of carbapenem resistance and receipt of active antimicrobial therapy on clinical outcomes of Acinetobacter baumannii bloodstream infections. Antimicrobial Agents and Chemotherapy, 55(10), 4844–4849. https://doi.org/10.1128/AAC.01728-10
14. He, S., Wu, X., Ma, B., & Xu, Y. (2021). High specific immobilization of His-tagged recombinant Microbacterium esterase by Ni-NTA magnetic chitosan microspheres for efficient synthesis of key chiral intermediate of d-biotin. Bioprocess and Biosystems Engineering, 44(10), 2193–2204. https://doi.org/10.1007/S00449-021-02595-7
15. Huang, C. J., Lin, H., & Yang, X. (2012). Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. Journal of Industrial Microbiology and Biotechnology, 39(3), 383–399. https://doi.org/10.1007/S10295-011-1082-9
16. Joshi, H., & Jain, V. (2017). Novel method to rapidly and efficiently lyse Escherichia coli for the isolation of recombinant protein. Analytical Biochemistry, 528, 1–6. https://doi.org/10.1016/J.AB.2017.04.009
17. Lee, C. R., Cho, I. H., Jeong, B. C., & Lee, S. H. (2013). Strategies to Minimize Antibiotic Resistance. International Journal of Environmental Research and Public Health 2013, Vol. 10, Pages 4274-4305, 10(9), 4274–4305. https://doi.org/10.3390/IJERPH10094274
18. Lee, C. R., Lee, J. H., Park, M., Park, K. S., Bae, I. K., Kim, Y. B., Cha, C. J., Jeong, B. C., & Lee, S. H. (2017). Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options. Frontiers in Cellular and Infection Microbiology, 7(MAR). https://doi.org/10.3389/FCIMB.2017.00055
19. Mostachio, A. K., Levin, A. S., Rizek, C., Rossi, F., Zerbini, J., & Costa, S. F. (2012). High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil. International Journal of Antimicrobial Agents, 39(5), 396–401. https://doi.org/10.1016/J.IJANTIMICAG.2012.01.021
20. Nikaido, H. (2003). Molecular Basis of Bacterial Outer Membrane Permeability Revisited. Microbiology and Molecular Biology Reviews, 67(4), 593–656. https://doi.org/10.1128/MMBR.67.4.593-656.2003/FORMAT/EPUB
21. Nikaido, H., & Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiological Reviews, 49(1), 1–32. https://doi.org/10.1128/mmbr.49.1.1-32.1985
22. Nurjayadi, M., Setiyoto, T., Jinan, S. F., Hardianto, D., Sulfianti, A., Agustini, K., & El-Enshasy, H. A. (2021). Purification of Fim-C-Salmonella typhi recombinant protein with Ni-NTA resin as raw material for typhoid disease detection kit. Journal of Physics: Conference Series, 1869(1). https://doi.org/10.1088/1742-6596/1869/1/012033
23. Obara, M., & Nakae, T. (1991). Mechanisms of resistance to β-lactam antibiotics in Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy, 28(6), 791–800. https://doi.org/10.1093/JAC/28.6.791
24. Overton, T. W. (2014). Recombinant protein production in bacterial hosts. Drug Discovery Today, 19(5), 590–601. https://doi.org/10.1016/j.drudis.2013.11.008
25. Pagès, J. M., James, C. E., & Winterhalter, M. (2008). The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nature Reviews Microbiology 2008 6:12, 6(12), 893–903. https://doi.org/10.1038/nrmicro1994
26. Park, Y. K., Jung, S. I., Park, K. H., Kim, S. H., & Ko, K. S. (2012). Characteristics of carbapenem-resistant Acinetobacter spp. other than Acinetobacter baumannii in South Korea. International Journal of Antimicrobial Agents, 39(1), 81–85. https://doi.org/10.1016/J.IJANTIMICAG.2011.08.006
27. Peränen, J., Rikkonen, M., Hyvönen, M., & Kääriäinen, L. (1996). T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. Analytical Biochemistry, 236(2), 371–373. https://doi.org/10.1006/ABIO.1996.0187
28. Rasooli, I., Abdolhamidi, R., Jahangiri, · Abolfazl, Darvish, S., & Astaneh, A. (2020). Outer Membrane Protein, Oma87 Prevents Acinetobacter baumannii Infection. International Journal of Peptide Research and Therapeutics, 26(3), 2653–2660. https://doi.org/10.1007/s10989-020-10056-0
29. Rice, L. B. (2008). Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. The Journal of Infectious Diseases, 197(8), 1079–1081. https://doi.org/10.1086/533452
30. Rice, L. B. (2010). Progress and challenges in implementing the research on ESKAPE pathogens. Infection Control and Hospital Epidemiology, 31 Suppl 1(S1), S7–S10. https://doi.org/10.1086/655995
31. Santajit, S., & Indrawattana, N. (2016). Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Research International, 2016. https://doi.org/10.1155/2016/2475067
32. Segura, A., Bünz, P. v., D’Argenio, D. A., & Ornston, L. N. (1999). Genetic analysis of a chromosomal region containing vanA and vanB, genes required for conversion of either ferulate or vanillate to protocatechuate in Acinetobacter. Journal of Bacteriology, 181(11), 3494–3504. https://doi.org/10.1128/jb.181.11.3494-3504.1999
33. Smith, M. A., Weaver, V. B., Young, D. M., & Ornston, L. N. (2003). Genes for chlorogenate and hydroxycinnamate catabolism (hca) are linked to functionally related genes in the dca-pca-qui-pob-hca chromosomal cluster of Acinetobacter sp. strain ADP1. Applied and Environmental Microbiology, 69(1), 524–532. https://doi.org/10.1128/AEM.69.1.524-532.2003
34. Srinivasan, V. B., Vaidyanathan, V., & Rajamohan, G. (2015). AbuO, a tolc-like outer membrane protein of Acinetobacter baumannii, is involved in antimicrobial and oxidative stress resistance. Antimicrobial Agents and Chemotherapy, 59(2), 1236–1245. https://doi.org/10.1128/AAC.03626-14
35. Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology, 219(1), 37–44. https://doi.org/10.1016/0022-2836(91)90855-Z
36. Sugawara, E., & Nikaido, H. (2012). OmpA is the principal nonspecific slow porin of Acinetobacter baumannii. Journal of Bacteriology, 194(15), 4089–4096. https://doi.org/10.1128/JB.00435-12
37. Tokuda, H. (2014). Biogenesis of Outer Membranes in Gram-Negative Bacteria. OUP, 73(3), 465–473. https://doi.org/10.1271/BBB.80778 Uppalapati, S. R., Sett, A., & Pathania, R. (2020). The Outer Membrane Proteins OmpA, CarO, and OprD of Acinetobacter baumannii Confer a Two-Pronged Defense in Facilitating Its Success as a Potent Human Pathogen. Frontiers in Microbiology, 11, 2441. https://doi.org/10.3389/FMICB.2020.589234/BIBTEX
38. Vashist, J., Tiwari, V., Kapil, A., & Rajeswari, M. R. (2010). Quantitative profiling and identification of outer membrane proteins of beta-lactam resistant strain of Acinetobacter baumannii. Journal of Proteome Research, 9(2), 1121–1128. https://doi.org/10.1021/PR9011188
39. Vashistt, J., Tiwari, V., Kapil, A., & Moganty, R. R. (2011). Differential expression of outer membrane proteins in early stages of meropenem-resistance in Acinetobacter baumannii. Journal of Integrated OMICS, 1(2), 280. https://doi.org/10.5584/JIOMICS.V1I2.67
40. Verma, S. K., Gautam, V., Balakrishna, K., & Kumar, S. (2009). Overexpression, purification, and immunogenicity of recombinant porin proteins of Salmonella enterica serovar Typhi (S. Typhi). Journal of Microbiology and Biotechnology, 19(9), 1034–1040. https://doi.org/10.4014/JMB.0812.675
41. Vila, J., Martí, S., & Sánchez-Céspedes, J. (2007). Porins, efflux pumps and multidrug resistance in Acinetobacter baumannii. The Journal of Antimicrobial Chemotherapy, 59(6), 1210–1215. https://doi.org/10.1093/JAC/DKL509
42. Zahn, M., Bhamidimarri, S. P., Baslé, A., Winterhalter, M., & van den Berg, B. (2016). Structural Insights into Outer Membrane Permeability of Acinetobacter baumannii. Structure (London, England : 1993), 24(2), 221–231. https://doi.org/10.1016/J.STR.2015.12.009