COMPARISON OF FUNCTIONAL AND STRUCTURAL PROPERTIES OF AN OUTER MEMBRANE PORIN BETWEEN Burkholderia pseudomallei AND Burkholderia thailandensis Miss Jaruwan Siritapetawee A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry Suranaree University of Technology Academic Year 2004 ISBN 974-533-325-5 การเปรียบเทียบสมบัติเชิงหนาที่และเชิงโครงสรางของโปรตีนพอรินใน ผนังเซลลผิวนอกระหวางเชื้อ Burkholderia pseudomallei และ Burkholderia thailandensis นางสาวจารุวรรณ ศิริเทพทว ี วิทยานิพนธนี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบัณฑิต สาขาวิชาชีวเคม ี มหาวิทยาลัยเทคโนโลยีสุรนาร ี ปการศึกษา 2547 ISBN 974-533-325-5 Acknowledgements I would like to express my sincere thank to my thesis advisor, Dr. Wipa Suginta for providing guidance, support, considerable and kind help. I am deeply grateful to my co-advisor, Dr. Richard, H. Ashley, Department of Biomedical Sciences, University of Edinburgh, UK, who gave me the useful guidance, valuable advice and provided the bacterium, Burkholderia thailandensis type strain ATCC700388. I would like to thank PD. Dr. Heino Prinz, Max Planck Institute for Molecular Physiology, Dortmund, Germany who helped with MALDI-TOF MS and ESI MS and peptide mass analysis, and Prof. Dieter Naumann, Robert-Koch Institute, Berlin, Germany with FTIR measurements. I also appreciate Miss Worada Samosornsuk, Faculty of Allied Health Science, Thammasat University, Thailand, for providing essential facilities for Burkholderia pseudomallei culture and drug susceptibility tests, and Dr. Chartchai Krittanai, Institute of Molecular Biology and Genetics, Mahidol University, Thailand, who kindly provided the CD spectropolarimeter for secondary structure determination. I would like to thank Shell studentship grant and Suranaree University of Technology grant for financial support throughout the project. Special thanks are extended to all friends, scientist and technician in the School of Biochemistry for their kind help and made me happy to work. IV Finally, I would like to express my deepest gratitude to my parents and members of my family for their kind, understanding and encouragement. I also express my deepest gratitude to my dearest sister Asst. Prof. Mookhada Siritapetawee, for her kind help in preparing this thesis. Without her endless patience, this thesis will not be possible. Jaruwan Siritapetawee Contents Page Abstract in Thai .................................................................................................……. I Abstract in English ..........................................................................................….... II Acknowledgements .......................................................................................……... III Contents ........................................................................................................……...... V List of Tables ..........................................................................................….............. XII List of Figures ......................................................................................…….............XIII List of Abbreviations .........................................................................……........... XVIII Chapters I Introduction ..................................................................……....................1 1.1 Burkholderia pseudomallei .........................................…….................1 1.2 Burkholderia thailandensis……………..................…….................... 9 1.3 Mechanisms of antimicrobial resistance….……….............….......... 12 1.4 Biological membrane proteins………………………….................... 13 1.5 Outer membrane of Gram-negative bacteria and porins……..….......15 1.6 Objectives…………………………………..……….......……...........26 II Materials and Methods ...............................................……................. 28 2.1 Materials ................................................................…….................... 28 2.1.1 Bacterial strains and plasmids ………………………..…….....28 2.1.2 Chemicals ..................................................…........................... 28 VI Contents (Continued) Page 2.1.3 PCR primers .............................................................……........ 29 2.2 Methodology…............................................................…….............. 30 2.2.1 Preparation of peptidoglycan-associated proteins..…….......…. 30 2.2.2 Purification of native Omp38 protein………..…...................... 31 2.2.3 Determination of protein concentration........……................….31 2.2.4 SDS-PAGE gel electroporesis….........................…….............. 32 2.2.5 In-gel digestion and peptide mass analysis of Omp38 protein using MALDI-TOF Ms and ESI/MS ......................…….……..33 2.2.5.1 In-gel digestion…………..………………………….....33 2.2.5.2 Peptide mass analysis and identification of Omp38 by MALDI-TOF MS and ESI/MS………...…….……..34 2.2.6 Production of anti-Omp38 polyclonal antibodies and immunoblot analysis...................…………..………...……….. 35 2.2.7 Preparation of genomic DNA from B. pseudomallei and B. thailandensis (Miniprep protocol) ...........................…...…. 37 2.2.8 DNA analysis by agarose gel electrophoresis .............…......... 38 2.2.9 Quantitation of DNA………..……........................……........... 39 2.2.10 Gene isolation, cloning and sequencing ..................…........... 39 2.2.10.1 Primers and PCR conditions…………..……...…….39 2.2.10.2 Ligation PCR product into the pGEM®T vector…..40 VII Contents (Continued) Page 2.2.10.3 Transformation, selection and nucleotide sequence analysis…………………………………………..….41 2.2.11 Purification of PCR products from an agarose gel..........….... 42 2.2.12 Analysis of DNA inserts by boiling miniprep……............…. 43 2.2.13 QIAGEN plasmid miniprep ........................…........................ 44 2.2.14 DNA Sequencing and amino acid sequence analysis....…...... 45 2.2.15 Subcloning of Omp38 inserts to an expression vector…….... 45 2.2.15.1 Primer and PCR conditions………………..………..45 2.2.15.2 Insert and vector preparation for restriction enzyme digestion…………..…………………………..…….46 2.2.15.3 Ligation of the Omp38 fragments to an expression vector………………………..…………………..…..47 2.2.15.4 Transformation of the recombinant plasmids into the expression E. coli host cells………….....…..…..47 2.2.15.5 Colony PCR………………………..……………..…48 2.2.16 Expression of BpsOmp38 and BthOmp38 and preparation of inclusion bodies……...………………..…………..…….....48 2.2.17 Western blotting analysis………….. ............................…..... 49 2.2.18 Purification and refolding of Omp38 expressed in E. coli cells .............……………………………………………...... 50 VIII Contents (Continued) Page 2.2.19 Protein identification and peptide mass analysis by MALDI-TOF MS and ESI/MS……..…………........……….. 51 2.2.20 Antibiotic susceptibility tests and functional study of Omp38 …………………………..………………………….. 52 2.2.20.1 Antibiotic susceptibility tests………..……………...52 2.2.20.2 Proteoliposome-swelling assays……….……………54 2.2.20.3 Effects of anti BpsOmp38 antibodies on permeability of Omp38…………..………..………..……...……...55 2.2.21 Secondary structure determination….......……....................... 55 2.2.21.1 Fourier Transform Infrared (FTIR) spectroscopy....... 55 2.2.21.2 Circular Dichroism (CD) spectroscopy …............... 56 2.2.22 Molecular weight determination of Omp38............…............ 57 2.2.23 Membrane topology and 3D structural predictions ……........ 59 2.2.24 Preliminary crystallization studies of the expressed and refolding Omp38.…………..…………………………….…. 60 III Results .......................................................................................……...... 62 3.1 Isolation and purification of outer membrane proteins from B. pseudomallei and B. thailandensis .................................……........ 62 3.2 Antibody production and Western blot analysis.......................…......68 3.3 Pore-forming activity of the native Omp38 ................................…...71 IX Contents (Continued) Page 3.4 Secondary structure determination using Fourier Transform Infrared (FTIR) spectroscopy…..………………………………..…..77 3.5 Peptide mass analysis by MALDI-TOF MS and ESI/MS……..….....78 3.6 Gene isolation, nucleotide and amino acid sequence analysis………81 3.6.1 Gene isolation and nucleotide sequencing…..…………..…….81 3.6.2 Amino acid sequence analysis…………………..………..…....84 3.7 Subclining of Omp38 DNA frgaments into the pET23d(+) expression vector ……………….......................……........................ 88 3.8 Expression of Omp38 in E. coli……………......….……….........…..91 3.9 Mass analysis and refolding of the expressed Omp38 .........…….….93 3.9.1 Mass analysis by MALDI-TOF mass spectrometry....………..93 3.9.2 Refoding of the expressed Omp38……………..…………..….95 3.10 Drug susceptibility test of B. pseudomallei and B. thailandensis.....98 3.11 Pore-forming activity of the refolded Omp38……………..……...100 3.11.1 Penetration of neutral sugars through the refolded Omp38………………………..………………………..……99 3.11.2 Permeation of antibiotics through native and refolded Omp38 porins………..……………...…………………..…106 3.11.3 Effects of anti-BpsOmp38 antibodies on pore-forming activity of the Omp38……………………..…………..…...109 X Contents (Continued) Page 3.12 Secondary structure determination using Circular Dichroism (CD) spectroscopy.……………...……………………………..….114 3.13 The Mr determination using Sephacryl S-200 HR filtration chromatography……………………..………………..…118 3.14 Membrane topology and 3D structural prediction……………......120 3.15 Preliminary crystallization of the Omp38