University of Cincinnati

UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ ASSEMBLY AND SECRETION OF PERTUSSIS TOXIN BY BORDETELLA PERTUSSIS A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Molecular Genetics, Biochemistry, and Microbiology of the College of Medicine 2003 by Amy Alison Rambow-Larsen B.S., Northern Illinois University, 1997 Committee Chair: Alison Weiss, Ph.D. ABSTRACT Bordetella pertussis is the causative agent of whooping cough. Pertussis toxin, one of the major virulence factors of B. pertussis, is an AB5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted to the periplasm where the toxin is assembled. The Ptl secretion system secretes assembled toxin past the outer membrane. In this study, we examined toxin expression, assembly, and secretion. Cultures followed a typical bacterial growth cycle. A one-hour lag phase was followed by logarithmic growth until the cultures entered stationary phase around 24 hours. Secreted toxin was first observed at 3 hours. Secretion continued throughout logarithmic growth phase, decreasing as the culture entered stationary phase. Toxin secretion occurred at a constant rate of 3 molecules/minute/cell from two to eighteen hours. More toxin subunits were produced than secreted, resulting in periplasmic accumulation. Periplasmic subunits were detected in both soluble and membrane-associated cellular fractions, with about half of the periplasmic subunits incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and increased between 2 and 24 hours, from 30 to 1000 molecules per cell. However, the initial amount of PtlF supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests translation rates exceed the rate of secretion, and that secretion, not toxin and Ptl-complex assembly is rate limiting. Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules. Assembled pertussis toxin, and most secretion component proteins are too large to diffuse through intact peptidoglycan. Therefore, we hypothesized that the Ptl system would contain a peptidoglycanase. PtlE possessed a sequence match to the active site of glycohydrolases, suggesting this protein might cleave the sugar backbone of peptidoglycan. A polyhistidine tagged PtlE fusion protein possessed peptidoglycanase activity. Fusion proteins with alanine substitutions at one or both of the putative active site residues (D53 and E62) lacked peptidoglycanase activity. B. pertussis strains expressing PtlE alleles with the amino acid substitutions were deficient for pertussis toxin secretion. Based on these results, we conclude that PtlE is a peptidoglycanase responsible for the local removal or re-arrangement of the peptidoglycan layer during Ptl- secretion complex assembly. ACKNOWLEDGEMENTS I would like to thank my advisor, Alison Weiss, for her encouragement and support; and my committee members, Michael Lieberman, Gary Dean, George Deepe, Carolyn Price, for their insight and guidance. I would also like to thank the past and present members of the Weiss lab: Michael Barnes, Christine Weingart, Kathy Craig-Mylius, Trevor Stenson, Paula Mobberly- Schuman, Shantini Gamage, Lyndsay Schaeffer, Angie Patton, Jim Hanson, Cojean Wang, and Colleen McGannon, for making the Weiss lab an intellectually stimulating and entertaining environment in which to work. I would like to thank my parents, Jo Ann and Paul Rambow for imparting to me their love of learning. Finally, my deepest gratitude goes to my husband, Lance Larsen, who encouraged me to enter into this endeavor, and relocated to Cincinnati with me. His constant love and support has been more valuable than I can say. TABLE OF CONTENTS LIST OF TABLES………………………………………………………… 2 LIST OF FIGURES………………………………………………………… 2 LIST OF ABBREVIATIONS……………………………………………… 4 INTRODUCTION……………………………………………………………. 5 Overview of Bordetella pertussis……………………………………... 5 Pertussis Toxin………………………………………………………... 7 Transcription and Translation of Pertussis Toxin…………………….. 9 Assembly of Pertussis Toxin…………………………………………. 12 Secretion via the Ptl System………………………………………….. 13 Relationship to Other Secretion Systems…………………………….. 14 Type II Secretion Systems……………………………………. 15 Type IV Secretion Systems…………………………………… 15 Agrobacterium tumefaciens VirB System……………………. 18 Overview of the VirB Translocon……………………... 20 The Pilus……………………………………………….. 22 The Membrane Bound Components…………………... 23 The Peptidogylcanase………………………………….. 26 The Cytoplasmic ATPases…………………………….. 27 Studies of Pertussis Toxin Secretion…………………………………. 33 PERTUSSIS TOXIN EXPRESSION AND SECRETION………………… 34 Introduction……………………………………………………………. 34 Materials and Methods………………………………………………… 36 Results…………………………………………………………………. 43 Discussion……………………………………………………………... 57 PEPTIDOGLYCANASE ACTIVITY OF PTLE…………………………... 63 Introduction……………………………………………………………. 63 Materials and Methods………………………………………………… 66 Results…………………………………………………………………. 76 Discussion……………………………………………………………... 91 SUMMARY AND FUTURE DIRECTIONS……………………………….. 93 LITERATURE CITED……………………………………………………… 99 APPENDIX – ADDITIONAL STUDIES 1 LIST OF TABLES Table 1. Protein-protein interactions in the VirB translocon………………… 21 Table 2. Bacterial strains used in this study………………………………….. 66 Table 3. Plasmids used in this study………………………………………….. 67 Table 3. Primers used in this study…………………………………………… 72 LIST OF FIGURES Fig. 1. Pertussis toxin expression and secretion……………………………… 10 Fig. 2. Operon structure of type IV secretion systems Ptl, VirB and Tra…….. 17 Fig. 3. Model of the VirB and Ptl secretion complexes………………………. 19 Fig. 4. Model of the HP0535 protein…………………………………………. 31 Fig. 5. Standard curves for calculation of protein concentrations……………. 39 Fig. 6. Growth curve of B. pertussis BP338………………………………….. 44 Fig. 7. Accumulation of pertussis toxin in supernatant………………………. 45 Fig. 8. Toxin secretion per cell……………………………………………….. 47 Fig. 9. Periplasmic S1 and periplasmic toxin………………………………… 48 Fig. 10. Localization of cellular S1…………………………………………… 50 Fig. 11. Periplasmic S2 and S3 and pertussis toxin…………………………... 51 Fig. 12. Localization of cellular S2 and S3…………………………………… 54 2 LIST OF FIGURES continued Fig. 13. Expression and localization of PtlF………………………………….. 55 Fig. 14. Accumulation of PtlF………………………………………………... 56 Fig. 15. Model of pertussis toxin assembly and secretion……………………. 58 Fig. 16. Features of the PtlE protein………………………………………….. 77 Fig. 17. Analysis of polyhistidine-tagged PtlE expressed in E. coli BL21…… 79 Fig. 18. Analysis of polyhistidine-tagged PtlE expressed in B. pertussis BP338…………………………………………………………………………. 81 Fig. 19. Cloning of amino acid substitutions into the ptx/ptl operon…………. 83 Fig. 20. Western blot of PtlF expression in wild type and PtlE mutants……. 84 Fig. 21. Toxin expression and secretion in haploid strains (ptx/ptl mutant background)…………………………………………………………………… 87 Fig. 22. Toxin expression and secretion in merodiploid strains (wild-type background)…………………………………………………………………… 88 Fig. 23. Fusion proteins for analysis of peptidoglycanase activity in mutants.. 89 Fig. 24. Activity of polyhistidine-tagged mutant PtlE proteins expressed in E. coli BL21[DE3]……………………………………………………………….. 90 Fig. 25. Determining the location of periplasmic holotoxin………………….. 95 3 ABBREVIATIONS USED IN THIS TEXT ADP – adenine diphosphate ATP – adenine triphosphate BG – Bordet-Gengou cAMP – cyclic adenine monophosphate C-terminus – carboxyl terminus CHO Cells – Chinese Hamster Ovary Cells DNA – deoxyribonucleic acid FHA – filamentous hemaglutanin IncN – N incompatibility group IncQ – Q incompatibility group mRNA – messanger ribonucleic acid N-terminus – amino terminus OD – optical density ORF – open reading frame PBS – Phosphate Buffered Saline RNA – ribonucleic acid Sec – general secretory pathway SS broth – Stainer-Scholte broth T-DNA – Transferred DNA Ti-plasmid – tumor inducing plasmid 4 INTRODUCTION Overview of Bordetella pertussis The Gram-negative bacterium, Bordetella pertussis, is the causative agent of whooping cough. The World Health Organization estimates that there were 296,000 deaths worldwide from whooping cough in the year 2000 (148). The incidence of disease is highest in children under one year of age, and the occurrence of complications such as pneumonia, cardiac and pulmonary hypotension, encephalopathy, and seizures, is highest in infants (28, 124). In the United States, there were 62 deaths from pertussis during the four year period between 1997 and 2000. Fifty-six of these deaths (90%) were of infants under 6 months of age (28). Disease progression can be divided into three stages: catarrhal, paroxysmal, and convalescent. The catarrhal stage may last for several weeks, with symptoms resembling a cold. The disease is often not diagnosed at this

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