NORTHWESTERN UNIVERSITY The Role of the Type IV Pilus Complex in DNA Transformation in Neisseria gonorrhoeae A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree DOCTOR OF PHILOSOPHY Field of Life Sciences By Kyle P. Obergfell EVANSTON, ILLINOIS September 2017 2 Abstract The Role of the Type IV Pilus Complex in DNA Transformation in Neisseria gonorrhoeae Kyle P. Obergfell Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing, and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae genes encoding two low-molecular-mass, penicillin-binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross-linking. Loss of DacB peptidase resulted in global alterations to the PG composition, while loss of DacC affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross-linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics, and increased sensitivity to detergent mediated death. Loss of the two proteins drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper cell biology and expression of factors critical for pathogenesis. Required for gonococcal infection, Tfp mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the 3 pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface- exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and extended Tfp are not required for competence. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. Attempts to identify the protease responsible for S-pilin cleavage were unsuccessful. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation. 4 Acknowledgements As in all scientific pursuits, this work is the result of much collaboration, both in conception and execution. To everyone that has contributed to the success of this work in ways both large and small, thank you. Specifically, I would like to thank Dr. Joe Dillard and Dr. Ryan Schaub for providing plasmid pMR69 and performing and analyzing the peptidoglycan profiling experiments. The Tanner lab provided anti-PilE antibodies. Dr. Mark Anderson made the pilV::npt and comP::npt constructs, and Dr. Allison Criss isolated the P- antigenic variants used in these studies. Electron microscopy was performed at Northwestern’s Center for Advanced Microscopy and DNA sequencing was completed by The Genomics Core at the Northwestern Center for Genetic Medicine. I am grateful to the past and present members of the Seifert lab for paving the way and helping guide me along it: Dr. Paul Duffin, Dr. Laty Cahoon, Dr. Mark Anderson, Dr. Adrienne Chen, Kelly Kligne, Dr. Carl Gunderson, Dr. Ella Rotman, Dr. Jing Xu, Dr. Alice Chateau, Dr. Elizabeth Stohl, Dr. Linda Hu, Sarah Quillin, and Lauren Priniski. I owe a great debt to Kelly, Carl, and Lauren for improving my lab life on a daily basis. Thank you to Kavi Mehta and Chen Kam for advice, expanding my access to laboratory supplies, and after work drinks. I will forever be thankful for the friendship of Dr. Charlie Keenan and Liz Bacon who have made completing this difficult process immeasurably easier. Finally, none of this work would be possible without the incredible mentorship of Dr. Hank Seifert. Thank you for always giving me the leeway to make mistakes and always making sure that I learned from them. 5 List of Abbreviations (AA) – Amino acid (ATC) – Anhydrotetracycline (CFU) – Colony forming units (CRISPR) - Clustered, regularly interspaced, short palindromic repeat (DC) – Double complement (dsDNA) – Double-stranded DNA (DUS) – DNA uptake sequence (ELISA) – Enzyme-linked immunosorbent assay (G4) – Guanine quadruplex (GCB) – Gonococcal base (HGT) – Horizontal gene transfer (HLB) – Hydrophobic-Lipophilic-Balanced (HMM) – High molecular mass (HPLC) – High performance liquid chromatography (HVL) – Hyper-variable loop (HVT) – Hyper-variable tail (LMM) – Low molecular mass (LOS) – Lipooligosaccharide (mRNA) – Messenger RNA (ND) – Not determined (Ntd) – N-terminal domain 6 (Opa) – Opacity (PBP) – Penicillin binding proteins (PG) – Peptidoglycan (sRNA) – Small RNA (ssDNA) – Single-stranded DNA (SV) – Semi-variable region (TEM) – Transmission electron microscopy (TFA) – Trifluoracetic acid (Tfp) – Type IV pilus 7 Dedication To my family, both old and new, for their unwavering support. To my mother and father, Colleen and Mark Obergfell, for their constant love, my self-confidence, and their example and emphasis on hard work and the constant quest for knowledge. To my partner, best friend, and love of my life, Alexandra Kirsch, without whom none of this would have happened. Thank you for challenging and supporting me every day and being a constant source of happiness. 8 Statement of Publication Portions of the Abstract, Chapter 1, Chapter 3, and Chapter 5 have been published previously in the following reports: Obergfell KP, Seifert HS. The Pilin N-terminal Domain maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation. PLoS Genet. 2016;12(5): e1006069 Obergfell KP, Seifert HS. Mobile DNA in the pathogenic Neisseria. Microbiol Spectr. 2015;3(3):0015-2014. 9 Table of Contents Abstract .......................................................................................................................................... 2 Acknowledgements ....................................................................................................................... 4 List of Abbreviations .................................................................................................................... 5 Dedication ...................................................................................................................................... 7 Statement of Publication .............................................................................................................. 8 Table of Contents .......................................................................................................................... 9 List of Figures .............................................................................................................................. 13 List of Tables ............................................................................................................................... 15 Chapter 1: Introduction ............................................................................................................. 16 Introduction ............................................................................................................................... 16 Natural DNA transformation ..................................................................................................... 17 The Type IV pilus .................................................................................................................. 18 DNA uptake sequence ........................................................................................................... 21 Processing and recombination ............................................................................................... 24 Antigenic variation .................................................................................................................... 26 Introduction ........................................................................................................................... 26 Pilin antigenic variation ......................................................................................................... 28 Trans-acting factors important