Discovery and demonstration of functional type IV pili production and post- translational modification by a medically relevant Acinetobacter species DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Christian Michael Harding Graduate Program in Biomedical Sciences. The Ohio State University 2015 Dissertation Committee: Daniel Wozniak, Ph.D., Advisor Amal Amer, M.D., Ph.D. Larry Schlesinger, M.D. Kevin Mason, Ph.D. Copyright by Christian Michael Harding 2015 Abstract Acinetobacter nosocomialis is a member of the Acinetobacter calcoaceticus- baumannii complex, which are Gram-negative opportunistic pathogens of increasing relevance worldwide. Medically relevant Acinetobacter species form biofilms, are resistant to desiccation, and easily acquire antibiotic resistance genes, all of which contribute to its ability to cause disease. Despite many reports on the epidemiology and antibiotic resistance phenotypes of Acinetobacter, there are limited reports characterizing the virulence mechanisms of these important nosocomial pathogens. Type IV pili (Tfp) are transenvelope protein complexes that can act as surface appendages mediating many bacterial processes. Analysis of the genomes of fully sequenced medically relevant Acinetobacter strains reveals the presence of genes that encode proteins predicted to be involved with the biogenesis of Tfp. Furthermore, many medically relevant Acinetobacter species have been shown to exhibit twitching motility and natural transformation, two classical Tfp-associated phenotypes. Therefore we utilized mutagenesis strategies to selectively delete genes encoding proteins predicted to be involved in Tfp biogenesis and probed for Tfp functionality. In our analysis we determined that A. nosocomialis strain M2 did produce functional Tfp, which were required for natural transformation and twitching motility. During the course of our studies we also identified that the major pilin subunit, PilA, of the Tfp fiber was ii post-translationally modified. Subsequently we determined that PilA was glycosylated by the pilin-specific oligosaccharyltransferase, TfpO, at the carboxy- terminal serine. Lastly, we demonstrated that many Acinetobacter species encode two functional oligosaccharyltransferases, one devoted exclusively to pilin glycosylation and the other to general protein glycosylation. This study is the first to describe the production of functional Tfp production by a medically relevant Acinetobacter species and also demonstrated that the pilin glycosylation system shares a common pathway with the general protein glycosylation pathway. iii Dedication To Robert S. Munson, Jr. iv Acknowledgments First and foremost, I want to thank my family. Simon and Ian, you are the best brothers and I’m so proud of you. Dad, thanks for supporting me anytime I needed help and even when I didn’t. Mom, thanks for being eternally loving. Ashley, I love you endlessly and could not have done this without you. I want to thank my undergraduate mentor, Pamela Riggs-Gelaso, Ph.D., for giving me my first opportunity to work in a research lab. You believed in me and that provided me with the confidence I needed to pursue graduate school. Thanks to my committee, Amal Amer, Larry Schlesinger, and Dan Wozniak for not sugar coating my project and its problems. I will not forget the simple advice of focus and the impact that it had. I want to thank Beth Baker, Michael Carruthers, Erin Grundy, Alistair Harrison, and Estevan Santana for teaching me to clone. These are skills I will never forget. Lastly, I want to thank Robert Munson for molding me into a scientist, a critical thinker, and an honorable man. You truly will always be a mentor to me. v Vita 2009 ........................................................ B.S. Biology, College of Charleston 2009-2010 ............................................... Chemical Stock Room Manager, College of Charleston 2010 to present ...................................... Graduate Research Associate, Department of Pediatrics, The Ohio State University Publications Harding CM, Tracy EN, Carruthers MD, Rather PN, Actis LA, Munson RS, Jr. 2013. Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility. mBio 4(4):e00360-13. doi:10.1128/mBio.00360-13. Carruthers MD, Harding CM, Baker BD, Bonomo RA, Hujer KM, Rather PN, Munson RS, Jr. 2013. Draft genome sequence of the clinical isolate Acinetobacter nosocomialis strain M2. Genome Announc. 1(6):e00906-13. doi:10.1128/genomeA.00906-13. vi Harding CM, Nasr MA, Kinsella RL, Scott NE, Foster LJ, Weber BS, Fiester SE, Actis LA, Tracy EN, Munson RS, Jr, Feldman, MF. 2015. Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins. Molecular Microbiology. DOI: 10.1111/mmi.12986. Fields of Study Major Field: Biomedical Sciences Specialization: Microbial Pathogenesis vii Table of Contents Abstract .................................................................................................................. ii Dedication ............................................................................................................. iv Acknowledgements ............................................................................................... v Vita ........................................................................................................................ vi Table of Contents ................................................................................................ viii List of Tables ......................................................................................................... xi List of Figures ....................................................................................................... xii Chapter 1 : Introduction ......................................................................................... 1 1.1 Acinetobacter background ........................................................................... 1 1.2 A. baylyi ADP1 Introduction ......................................................................... 3 1.3 Acinetobacter medical relevance ................................................................ 4 1.4 Mechanisms of antibiotic resistance ........................................................... 6 1.5 Virulence mechanisms in Acinetobacter ..................................................... 8 1.5.1 Virulence introduction ........................................................................... 8 1.5.2 Type IV pili biogenesis and functions .................................................. 10 1.5.3 Acinetobacter and type IV pili associated phenotypes ........................ 13 1.5.4 General O-glycosylation systems ....................................................... 16 1.5.5 O-glycosylation in Acinetobacter ......................................................... 18 viii 1.5.6 Pilin glycosylation ................................................................................ 19 1.6 Summary .................................................................................................. 22 Chapter 2 : Draft genome sequence of the clinical isolate Acinetobacter nosocomialis strain M2 ........................................................................................ 23 2.1 Abstract ..................................................................................................... 23 2.2 Genome announcement ............................................................................ 23 2.3 Nucleotide sequence accession number ................................................... 25 Chapter 3 : Demonstration of type iv pili production by Acinetobacter baumannii and their role in natural transformation. twitching motility and surface-associated motility ................................................................................................................. 26 3.1 Introduction ................................................................................................ 26 3.2 Methods ..................................................................................................... 30 3.3 Results ....................................................................................................... 43 3.4 Discussion ................................................................................................. 61 Chapter 4 : Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type iv pilin, and the other one dedicated to O-glycosylation of multiple proteins .................................................................... 73 4.1 Introduction ................................................................................................ 73 4.2 Methods ..................................................................................................... 77 4.3 Results ....................................................................................................... 96 4.4 Discussion ............................................................................................... 131 ix Chapter 5 : Discussion ...................................................................................... 147 5.1 Research findings ...................................................................................