Quantitative PCR Analysis of the Bartonella henselae carD Gene during Bacterial Stress Jacob Simmonds A thesis submitted to the Victoria University of Wellington in fulfilment for the degree of Master of Science Victoria University of Wellington Te Whare Wānanga o te Ūpoko o te Ika a Māui 2016 Abstract The Bartonella genus is comprised of arthropod-borne, intracellular bacterial pathogens that colonise the mammalian bloodstream. A large number of mammalian species are hosts for one or more Bartonella species, as either reservoir or incidental hosts. Bartonella species are only able to invade and replicate in host red blood cells in the reservoir host, and to be taken up by an associated haematophagous arthropod vector to complete transmission and the bacterial life cycle. Humans are the reservoir hosts for B. quintana and B. bacilliformis, and are incidental hosts for more than 16 additional zoonotic Bartonella species, including B. henselae, which is normally carried by cats. B. henselae infection, usually acquired through cat scratches or bites, can result in several clinical manifestations, with varying degrees of severity; the most common of these is cat scratch disease, where symptoms commonly range from enlarged lymph nodes to severe fever. Although usually a mild illness, B. henselae infection can occasionally lead to severe symptoms, affecting neurological and other major organ systems. During their life cycle the Bartonellae must adapt to various toxic host environments; such adaptation is mediated by several bacterial stress pathways, which modify bacterial transcription. However, many gaps remain in the understanding of B. henselae stress response pathways. The object of this study, the carD gene, was identified as a possible component of the Bartonella stress response. The carD gene has been shown to be critical for stress defence in other bacterial species, including Mycobacterium tuberculosis, Thermus thermophilus, and Myxococcus xanthus. Our study aimed to investigate whether carD played as significant a role during the B. henselae response to stresses as it does in other bacterial genera. We first attempted to perform growth comparisons between a B. henselae carD mutant strain and a wild type strain during exposure to stress conditions; however, our mutagenic carD plasmid interfered with bacterial growth of Escherichia coli cultures, which hindered transformation and generation of a B. henselae carD mutant. As an alternative, we investigated the expression of B. henselae carD under stress conditions, comparing carD expression during stress against a non-stressed B. henselae control, using quantitative PCR. We found no significant difference of expression of the carD gene between the control and any of our conditions, although a trend of increased carD expression was found in several stress conditions. We believe that these findings merit further study into the role of carD in the B. henselae stress response. i Acknowledgments First and foremost, I would like to thank my supervisor, Dr Joanna MacKichan, without whom this would not be possible. Thank you for all the support you have given me over the last two years and a half, for helping me in the lab with your advice and experience, and for playing a massive role in editing this thesis. I would also like to thank Dr David Ackerley, Dr Jeremy Owen (pointing me to the Mycobacterial CarD paper, it proved immensely helpful), and to Dr Jane Koehler (for advice, providing Bartonella strains, and plasmids). Thanks to all the members of both the Ackerley and Day Lab for the advice and materials (and top notch food at lab meetings); to Jack and Matt S (for bacterial strains and plasmids), to Alistair (qPCR advice), to Jen and Euan (for assistance and materials), and to especially Varun, who took time out of writing his doctoral thesis, to help not only with my initial qPCR but then following contamination issues. Thanks to all the members of Alan Macdiarmid, for any help and companionship, notably to Matt R and Dana (for help with qPCR and materials). I would like to thank both the members of the MacKichan lab who have been awesome lab mates; Raphael, Gabby, Alvey, Callum, Haydn, Kurt, and especially Vaughan. Thank you also the occupants of KK702 (and honorary members) for keeping me sane when times got tough, and for distracting me from my masters when needed it; Sonja, James, Vaughan, Kyle, Jasmine (J3), Jack, Jen, Melz, Euan, Izzie, Rory, Eddie, and Mitch. Thank you to all my friends and family who have supported me not only through undergraduate, but also through my studies. Thanks to my sister Harnah and to Dad. Thanks to Mcfly, Rachel, Rowan, Sam, and Nguyen for keeping me heartily entertained. Lastly, thank you to Jasmine (J2), for waiting patiently for me to finish this thesis. ii Table of Contents Abstract ............................................................................................................................ i Acknowledgments .............................................................................................................ii Table of Contents ............................................................................................................. iii List of Figures ................................................................................................................... vi List of Tables ................................................................................................................... vii Abbreviations used in this thesis ...................................................................................... vii Chapter 1. Introduction .................................................................................................... 1 1.1. Human-Specific Bartonella Species ..................................................................... 3 1.1.1. Bartonella bacilliformis ................................................................................... 3 1.1.2. Bartonella quintana ........................................................................................ 4 1.2. Zoonotic Bartonella Species ............................................................................... 5 1.2.1. Groups at Risk of Bartonella Infection ............................................................ 6 1.3. Bartonella henselae ........................................................................................... 8 1.3.1. History of B. henselae and Cat Scratch Disease .............................................. 8 1.3.2. Bartonella henselae, Felis catus, and Ctenocephalides felis ........................... 9 1.3.3. Cat Scratch Disease ....................................................................................... 10 1.3.4. Prevalence of Bartonella henselae ............................................................... 11 1.4. Pathology and Host Cell Interactions of Bartonella ............................................ 14 1.4.1. Bartonella Life Cycle ..................................................................................... 14 1.4.2. Pathology and Mechanism of Infection of B. henselae ................................ 16 1.5. Bartonella Stress Response ............................................................................... 18 1.5.1. Bartonella General Stress Response ............................................................. 19 1.5.2. Bartonella Stringent Response ..................................................................... 20 1.6. CarD ................................................................................................................. 23 1.7. Research Aims .................................................................................................. 25 Chapter 2. Methods ......................................................................................................... 27 2.1. Oligonucleotide Primers .................................................................................... 27 2.2. Bacterial Strains ................................................................................................ 28 2.3. Plasmids ........................................................................................................... 29 2.4. Growth Media .................................................................................................. 29 2.4.1. Liquid Media ................................................................................................. 29 2.4.2. Solid Media ................................................................................................... 30 2.4.3. Media Supplements ...................................................................................... 31 2.5. Culture Conditions of Bacteria ........................................................................... 32 2.6. Molecular Biology Protocols .............................................................................. 34 2.6.1. Isolation of Genomic DNA ............................................................................ 34 2.6.2. Polymerase Chain Reaction (PCR) ................................................................ 35 2.6.3. Agarose Gel Electrophoresis ......................................................................... 36 2.6.4. Isolation of Plasmids ..................................................................................... 36 2.6.5. Purification and Quantification of DNA products ......................................... 37 2.6.6. Size-Specific Purification of DNA products by Gel Electrophoresis