Campylobacter jejuni Survival Strategies and Counter-Attack: An investigation of Campylobacter phosphate mediated biofilms and the design of a high-throughput small- molecule screen for TAT inhibition DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mary R Drozd Graduate Program in Veterinary Preventive Medicine The Ohio State University 2012 Dissertation Committee: Dr. Gireesh Rajashekara, Advisor, Dr. Mo Saif, Dr. Armando Hoet, and Dr. Daral Jackwood Copyrighted by Mary Rachel Drozd 2012 Abstract In these investigations we studied 1) the ability of Campylobacter to modulate its behavior in response to phosphate actuated signals, 2) the modulation of biofilm in response to phosphate related stressors, and 3) we designed and carried out a high- throughput small-molecule screen that targets protein transport via the Twin Arginine Translocation (TAT) system. We identified that the phoX , ppk1 and ppk2 genes were key components of the phosphate response that manifested increased biofilm phenotypes, and were modulated in the presence of inorganic phosphate. We used several molecular and microbiological techniques to investigate the effect of polyP, phosphate uptake inactivation, and inorganic phosphate availability on Campylobacter’s response to phosphate stress. Additionally, we counted and measured attached biofilms, as well as measured pellicle size, biofilm shedding over the course of three days, and changes in the expression of genes known to be involved in biofilm formation phenotypes. By resolving biofilm components such as pellicles, attached cells, and shed cells we found that not only did ppk1, phoX, and ppk2 deletion affect the ability of Campylobacter to form biofilms, but biofilm components were not congruently and equally affected in each mutant. Additionally, the presence of phosphate modulated those effects both independently of and additively to gene knockouts. ii Furthermore, we observed that biofilm components were additionally affected by biofilm age: where some components had their most robust growth on day 2, biofilm shedding and pellicle growth increased the most on day 3. This growth was not uniform for all mutants, as ppk1 biofilms generally matured more quickly than wild-type cells, but the ppk2 mutant in the presence of phosphate matured more slowly. In our high-throughput small-molecule screen we designed and carried out a primary screen of small molecules to identify compounds that had anti-Campylobacter activity in the presence of 1mM CuSO4. To screen a greater number of compounds, this study was streamlined from a dual-plate study where each chemical was tested both in the presence and absence of copper sulfate. Our screen resulted in the identification of 680 small-molecule primary hits from the NSRB small-molecule library. These hits were identified from 11 different small-molecule libraries containing more than 50,000 compounds. Using database bioactivity results from past trials, the primary small- molecule positive hits were reduced to 476 targeted hits through in silica primary screens. We used Golden Triangle in silica medicinal chemistry methods to identify molecules that were likely to be less suitable due to low molecular weight, interactions with solutes, and compound stability. From there, common chemical motifs were identified among the remaining 350 molecules. From these ‘chemical families’ a representative sample of each group was chosen as likely having similar chemical activity. We chose 54 chemicals as representative of 4 chemical motifs: thiourea, benzimidazoles, oxadiazoles, and acylhydrazones. The rest of the molecules were iii selected for greatest diversity. Using these techniques, 149 compounds have been chosen that will be used as ‘cherry pick’ hits for secondary screens in the near future. iv Dedication To Jeremy Smith: my amazing husband, editor-in-chief, physics guru, and adventurous leading man. To Dr. Warren Dick, who took the time to teach me so much about Environmental Science, and to Marcus Aurelius Antoninus, in gratitude for his good advice. v Acknowledgments I would like to thank Dr. Juliette Hanson for assistance with chicken colonization studies and Dr. Jun Lin for providing chicken antimicrobial peptide, fowlicidin-1. Thank you to Dr. Fuchs for his help with chemical analysis of the small-molecule primary hits. Thank you to Dr. Rajashekara, for supporting my ideas and growth as a scientist. I am deeply grateful to Dr. Chiang, Dr. Ren and the NSRB staff that made my small-molecule project possible. Also, I am grateful for the support of the SEEDS grants committee and my collaborators as well as those who took the time and interest in being mentors for me both as professors and as part of my dissertation committee: Dr. Mo Saif, Dr. Armando Hoet, Dr. Daral Jackwood. Finally I thank our department for their commitment to science as a means of positive change. vi Vita March 1997 ....................................................George School, Newtown PA May 2001 .......................................................B.A. Molecular Biology, Classical Studies Hamilton College May 2001 to 2004 ..........................................Laboratory Specialist, University of Virginia May 2004 to July 2004……. .........................Animal Technician, Charlottesville SPCA. July 2004 to 2007 ...........................................Laboratory Specialist, Virginia Commonwealth University: Medical College of Virginia 2007 to present ..............................................Graduate Research Associate, Department of Veterinary Preventive Medicine, The Ohio State University vii Publications “Contribution of PhoX to Twin Arginine Translocation mediated Campylobacter jejuni function and resilience to Environmental Stresses” Mary Drozd, Dharanesh Gangaiah, Zhe Liu, and Gireesh Rajashekara. Research Article, PLoS ONE 6(10): e26336. “A quantitative polymerase chain reaction assay for detection and quantification of Lawsonia intracellularis.” Mary Drozd, Issmat I. Kassem, Wondwossen Gebreyes, Gireesh Rajashekara. Brief Report, JVDI, 2010 “Functional characterization of the twin-arginine translocation system in Campylobacter jejuni” Rajashekara G, Drozd M, Gangaiah D, Jeon B, Liu Z, Zhang Q. Foodborne Pathog Dis. 2009 “Nitrotyrosylation of Ca2+ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis” Gracious R. Ross, Minho Kang, Najeeb Shirwany, Anna P. Malykhina, Mary Drozd, and Hamid I. Akbarali. JPET, 2007. Thomashevski A., High A.A., Drozd M., Shabanowitz J., Hunt D.F., Grant P.A., Kupfer viii G.M. “The fanconi anemia core complex forms 4 different sized complexes in different subcellular compartments.” J Biol Chem, 2004 Fields of Study Major Field: Veterinary Preventive Medicine ix Table of Contents Campylobacter jejuni Survival Strategies and Counter-Attack: An investigation of Campylobacter phosphate mediated biofilms and the design of a high-throughput small- molecule screen for TAT inhibition .................................................................................... 1 DISSERTATION ................................................................................................................ 1 Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Publications ...................................................................................................................... viii Fields of Study ................................................................................................................... ix Table of Contents ................................................................................................................ x List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1: Introduction and Literature Review ................................................................. 1 1.1 A brief clinical history............................................................................................... 1 1.2 Clinical and Microbiological Background ................................................................ 2 1.3 Sources of Campylobacter infection ......................................................................... 3 x 1.4 Microbiological Challenges Unique to Campylobacter ............................................ 6 1.5 The Twin Arginine Translocation System ................................................................ 9 1.6 Development of Dynamic Biofilm Formation Theories ......................................... 14 1.7 Phosphate Metabolism in Campylobacter ............................................................... 19 1.8 High-throughput screening for small-molecule inhibitors ...................................... 24 Chapter 2: Contribution of TAT System Translocated PhoX to Campylobacter jejuni Phosphate Metabolism and Resilience to Environmental Stresses ................................... 29 2.1: Materials and Methods ........................................................................................... 29 2.2 Results