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Mining the Pseudomonas Virulence Factor Pathway for Novel Small Molecules

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Mining the Pseudomonas Virulence Factor Pathway for Novel Small Molecules MINING THE PSEUDOMONAS VIRULENCE FACTOR PATHWAY FOR NOVEL SMALL MOLECULES Ashley Marie Kretsch A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry. Chapel Hill 2019 Approved by: Bo Li Matthew Redinbo Marcey Waters Jeffery Dangl Robert Nicholas © 2019 Ashley Marie Kretsch ALL RIGHTS RESERVED ii ABSTRACT Ashley Marie Kretsch: Mining the Pseudomonas virulence factor pathway for novel small molecules (Under the direction of Bo Li) Bioactive small molecules often play an important role in bacterial virulence. Identification and exploration of these molecules will develop an understanding of new mechanisms for infection and potentially identify novel targets to inhibit virulence. Analysis of bacterial genomes identifies a large number of gene clusters that encode for small molecule- synthesizing enzymes (biosynthetic gene clusters), many of which are unexplored. Through genetic manipulation of these clusters, we can discover their small molecule products and determine their biological roles. We have identified a biosynthetic gene cluster conserved in over 300 strains of Pseudomonads. Many of these strains engage in pathogenic or symbiotic relationships with a range of human, animal, and plant hosts. We have shown that deletions within this gene cluster, named the Pseudomonas virulence factor (pvf), reduce fly infection by the pathogen Pseudomonas entomophila. In particular, the small molecule products of the pvf pathway are suggested to play a role in bacterial signaling and activation of virulence. Through overexpression of the pvf cluster in its native P. entomophila strain, we have discovered and characterized a number of small molecules that the pvf gene cluster is responsible for. These molecules include a class of pyrazine compounds, some of which are new to biology. We use fly infection and promoter-reporter assays to determine the biological roles of these molecules in virulence and cell-to-cell signaling of P. entomophila. Through proteomic analysis of the secretome, we have identified over 500 proteins that are differentially iii expressed between wildtype and pvf deletion strains, many which are known to be important to virulence and competition. To identify the active molecule responsible for pvf signaling activity we developed an optimized bioactivity-guided extraction and purification method. We can compare active purified fraction from spent media extracts of wildtype and pvf deletion strains using nuclear magnetic resonance (NMR) and liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) to identify the active pvf molecule. By characterizing the structures and functions of bioactive small molecules from pathogens, our work has the potential to develop an understanding for the production of these compounds and reveal novel and useful antimicrobial targets. iv To Chance Crompton. Your confidence in your career inspired me to pursue mine. Wish you were here to celebrate our accomplishments together. Rest in Peace v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Bo Li, for her mentorship and guidance throughout my graduate schooling. Your trust in my work meant I was able to pursue a project I absolutely loved, gain independence and creativity, and push through the days where accomplishments seemed impossible. Most importantly, you taught me the best leaders are those who lead by example; your own work ethic and commitment inspired me to achieve my goals. I would like to thank my family, particularly my parents. From a young age they saw my potential and gave me every opportunity imaginable to be challenged. They sat through countless hours of sporting events, took me to gifted student summer camps, and travelled the world with me. They soaked up so many tears are the hard days, and celebrated on the good days. As the only female computer scientist when she started working, my mother has been nothing but inspiration to pursue an academic career. I am also honored to be one of three successful sisters, all in different fields, but all with the same tenacity (and stubbornness). My grandparents and aunt have been a huge support system during my time in Chapel Hill, even driving through the middle of the night to let me be surrounded by love and compassion. Spending holidays with them has been a great gift in graduate school. I am so blessed to be raised by a powerhouse of women: my mom, my grandmothers, and my great-grandmothers. I would like to thank my lab mates and support system in Chapel Hill, particularly my classmates, the First Five: Andy Chan, Erinn O’Neill, Zach Dunn, and Kevin Santa Maria. Starting in a brand-new lab takes a lot of hard work and great minds, and I am thankful for all vi the support along the way. Crossfit Homeward has kept me healthy and happy, and provides goals to achieve when lab work is slumping. I wouldn’t even be in graduate school without the immense support from my best friends. My chemistry classmates and lab mates from Mudd: Emma, Chance, Scotty, Bethany, Bryan, and Christian. My Stag and Athenas: Joey, Amy, Jenni, Devin, Brad, Heather, Rachelle, and Bridget. My wild friends Marco, Sarah, Michelle, Milo, Braden, Crystal and Alli. A special thanks to my best friends Julia, Josiah, and Grant: I would not be here today if it wasn’t for your love and friendship. Last but not least, I would like to thank my fiancé, Steffen Good. No one joins a graduate program to find their life partner, but I am very lucky I did. Thank you for your unwavering support, your knowledge, and all the times you made me laugh when I wanted to cry. Graduate school was so much better when it included homecooked meals, Impractical Jokers, gym dates, and lots of carpools to lab together. vii TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................................... xi LIST OF SCHEMES ................................................................................................................................ xii LIST OF FIGURES ..................................................................................................................................xiii LIST OF ABBREVIATIONS ................................................................................................................. xviii Chapter 1 Introduction. ........................................................................................................................... 1 1. 1 The new Gold Rush for antibiotics and signaling molecules by genome mining in microbes .................................................................................................................. 1 1.2 Genome mining in Burkholderia and Pseudomonas to identify the Pseudomonas virulence factor .......................................................................................... 11 1.3 P. entomophila as a model to study the pvf cluster .............................................. 14 Chapter 2 Discovery of novel pyrazine N-oxides by overexpressing the Pseudomonas virulence factor pathway ...................................................................................................................... 18 2.1 Introduction ...................................................................................................................... 18 2.2 Materials and Methods................................................................................................... 21 General methods ................................................................................................... 21 Inducible pvf expression in native and heterologous Pseudomonas strains ....................................................................................................................... 21 Comparative metabolomics ................................................................................ 22 Metabolite purification and structural characterization .............................. 24 Chemical synthesis of PNO A (2) and PNO B (3) .......................................... 26 in vitro reconstitution of PvfC activity ............................................................. 28 2 Precursor feeding with [ H1−8]DL-valine ........................................................... 30 Expression of partial pvf cluster and metabolomics ................................... 31 viii P. entomophila and (d)PNOs biological assays ............................................ 32 pH stability of dPNO ............................................................................................. 33 2.3 Results ............................................................................................................................... 34 Identification of several related metabolites from induced expression of pvf ................................................................................................... 34 Structural elucidation of (d)PNOs ..................................................................... 45 Biosynthesis ........................................................................................................... 47 Biological activity of (d)PNOs ............................................................................ 52 2.4 Discussion ........................................................................................................................ 55 Chapter 3 The pvf signaling
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