The Neisseria gonorrhoeae cell division interactome and the roles of FtsA and N-terminus of FtsI in cell division and antimicrobial resistance A thesis Presented to The College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Microbiology & Immunology University of Saskatchewan Saskatoon By YINAN ZOU © Copyright Yinan Zou, August, 2018. All rights reserved Permission to Use In presenting this exhibition statement in partial fulfillment of the requirements for a Graduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this exhibition statement in any manner, in whole or in part, for scholarly purposes may be granted by the professor or the professors who supervised my exhibition statement work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis/exhibition work was completed. It is understood that any copying or publication or use of this exhibition statement or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my exhibition statement. Requests for permission to copy or to make use of materials in this thesis/exhibition statement in whole or part should be addressed to: Department Head Biochemistry, Microbiology and Immunology College of Medicine University of Saskatchewan 107 Wiggins Road Saskatoon, Saskatchewan S7N 5E5 Canada or i Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada ii Abstract Bacterial cell division is an essential biological process which is driven by the formation of a ring-like structure at the division site. Cell division proteins that form the ring-like structure vary in different bacterial species. Neisseria gonorrhoeae (Ng) encodes eight essential cell division proteins, i.e. FtsZNg, FtsANg, ZipANg, FtsKNg, FtsQNg, FtsLNg, FtsINg, FtsWNg and FtsNNg. This research investigated the nature of the N. gonorrhoeae cell division interactome by ascertaining cell division protein-protein interactions; and characterized the roles of FtsANg and the N-terminal domain of FtsINg in cell division and antimicrobial resistance in N. gonorrhoeae. Nine interactions among seven gonococcal cell division proteins were observed, using a combination of biological and biophysical methods. ZipANg did not interact with any cell division protein tested. Comparison between the gonococcal cell division interactome and two other established interactomes from Escherichia coli (Ec) and Streptococcus pneumoniae (Sp) revealed two common (FtsZ-FtsA and FtsZ-FtsK) and two unique interactions (FtsA-FtsW and FtsK-FtsN) in N. gonorrhoeae. These results show that N. gonorrhoeae forms a distinct cell division interactome. Expression of ftsANg in E. coli disrupted its cell division. Fluorescence microscopy showed that 37% of FtsANg localized to E. coli cell poles or the division site, whereas 63% of FtsANg was dispersed throughout the cytoplasm. FtsANg failed to complement an E. coli ftsA mutant strain and only interacted with FtsNEc as compared to FtsAEc. This interaction was mediated by the 2A and 2B subdomains of FtsANg. These data indicate that the function of FtsANg is species-specific. iii Three conserved residues, Arg75, Arg167 and Glu193, were identified at the N- terminal non-catalytic periplasmic region of FtsINg, which forms a conserved Arg-Arg-Glu linker structure that connects both the C-terminal and N-terminal domains. Mutations of these residues affected the interaction of FtsINg with FtsWNg. Alterations of Arg75 and Arg167 also impaired the penicillin binding capacity of FtsINg, whereas a mutation at Glu193 had no influence. Circular dichroism analysis indicated that the E193G mutant altered the secondary structure and stability of FtsINg, while both R75G and R167G mutants had no significant impact on conformation. Attempts to introduce an unmarked R167G mutation on chromosomal ftsINg was not successful since the insertional mutagenesis led to a heterodiploid genotype. This research shows that the conserved residues at the N- terminal periplasmic region of FtsINg are necessary for protein interaction and may influence antimicrobial resistance. iv Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisor Prof. Jo- Anne R. Dillon for giving me the opportunity to pursue my Ph.D degree in her laboratory at University of Saskatchewan. Her guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D study. Besides my advisor, I would like to thank the rest of my thesis committee: Prof. Miroslaw Cygler, Dr. Aaron White, and Dr. Harold Bull, for their encouragement, and insightful comments. I would also like to give my thanks to staff at Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), especially Dr. Robert Brownie, for their invaluable comments on my experiments. There is an old Chinese saying: Two heads are always better than one. I would like to thank my colleagues in Dr Dillon’s Lab: Dr. Sidharath Dev, Sumudu Perera, Kusum Sharma, Guanqun Liu, Dr. Reema Singh, Dr. Nidhi Parmar, Dr. Nurul Khan, Dr. Ali Taheri, Dr Rajinder Parti, and everyone else who has come and gone in our lab. Last but not the least, I would like to thank my family: my parents and parent-in- law, for numerous support to take care of my son, Jize Zou, during my last year of my Ph.D study. To my wife, Jin Wang, thank you for coming here and staying with me throughout my Ph.D life. I would not achieve this without your support. Thank you all and best wishes Yinan v Table of contents Permission to Use .................................................................................................... i Abstract .................................................................................................................. iii Acknowledgement .................................................................................................. v Table of contents ................................................................................................... vi List of tables ......................................................................................................... xiii List of figures ....................................................................................................... xiv List of abbreviations .......................................................................................... xvii Chapter 1 General introduction ........................................................................... 1 An overview of bacterial cell division ................................................................. 2 1.1.1. The division cell wall (dcw) cluster .............................................................. 2 1.1.2. Cell division site selection ............................................................................ 4 1.1.3. Divisome formation ...................................................................................... 7 Cell division in model organisms and essential divisome proteins .................... 10 1.2.1. Cell division in Escherichia coli ................................................................. 10 1.2.1.1. FtsZ ...................................................................................................... 10 1.2.1.2. FtsA ..................................................................................................... 12 1.2.1.3. ZipA ..................................................................................................... 17 1.2.1.4. FtsK ..................................................................................................... 18 vi 1.2.1.5. FtsQ, FtsB, and FtsL ............................................................................ 19 1.2.1.6. FtsW and FtsI....................................................................................... 20 1.2.1.7. FtsN ..................................................................................................... 21 1.2.1.8. FtsE and FtsX ...................................................................................... 22 1.2.1.9. The Z-ring regulators ........................................................................... 24 1.2.1.10. E. coli cell division interactome .......................................................... 25 1.2.2. Cell division in Bacillus subtilis ................................................................. 27 1.2.2.1. Vegetative cell division ....................................................................... 27 1.2.2.2. Sporulation........................................................................................... 30 Cell division in non-model organisms and essential divisome proteins ............ 31 1.3.1. Cell division in Chlamydiales ..................................................................... 32 1.3.2. Cell division in Streptococcus pneumoniae ................................................ 35 Current knowledge of cell division in Neisseria gonorrhoeae .......................... 40 1.4.1. Medical importance of N. gonorrhoeae .....................................................