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Hatzios Thesis Formatted Investigations of Metabolic Pathways in Mycobacterium tuberculosis by Stavroula K Hatzios A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Carolyn R. Bertozzi, Chair Professor Matthew B. Francis Professor Tom Alber Fall 2010 Investigations of Metabolic Pathways in Mycobacterium tuberculosis © 2010 By Stavroula K Hatzios Abstract Investigations of Metabolic Pathways in Mycobacterium tuberculosis by Stavroula K Hatzios Doctor of Philosophy in Chemistry University of California, Berkeley Professor Carolyn R. Bertozzi, Chair Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis in humans, infects roughly two billion people worldwide. However, less than one percent of infected individuals are symptomatic. Most have a latent infection characterized by dormant, non- replicating bacteria that persist within a mass of immune cells in the lung called the granuloma. The granuloma provides a protective barrier between infected cells and surrounding tissue. When host immunity is compromised, the granuloma can deteriorate and reactivate the disease. In order to mount a latent infection, Mtb must survive in alveolar macrophages, the host’s primary line of defense against this intracellular pathogen. By evading typical bactericidal processes, Mtb is able to replicate and stimulate granuloma formation. The mechanisms by which Mtb persists in macrophages are ill defined; thus, elucidating the factors responsible for this hallmark of Mtb pathogenesis is an important area of research. This thesis explores three discrete metabolic pathways in Mtb that are likely to mediate its interactions with host immune cells. The first three chapters examine the sulfate assimilation pathway of Mtb and its regulation by the phosphatase CysQ. Chapter 1 provides a general overview of the transcriptional, biochemical, and molecular levels of sulfur metabolism regulation in Mtb, while Chapters 2 and 3 present a detailed analysis of how CysQ may influence sulfur transactions in the cell. Chapter 4 explores the biosynthesis of the cell wall glycolipid Polyacyltrehalose, which is exclusively synthesized by pathogenic mycobacteria and may mediate host-pathogen interactions. Finally, Chapter 5 describes a novel osmoregulatory pathway in Mtb that affects the pathogen’s response to osmotic stress and its production of the well-characterized virulence factor EspA. 1 This dissertation is dedicated to my father, who is tragically unable to witness the summit, but inspired the climb. i Investigations of Metabolic Pathways in Mycobacterium tuberculosis Table of Contents List of Figures v List of Tables vii List of Schemes vii Acknowledgements viii Chapter 1. Regulation of sulfur metabolism in Mycobacterium tuberculosis Introduction 1 Transcriptional regulation of sulfur metabolism genes 3 Biochemical regulation of sulfur metabolism 6 Molecular mechanisms of sulfur metabolism regulation 7 Summary 8 Thesis overview 8 References 8 Chapter 2. Biochemical characterization of the Mycobacterium tuberculosis CysQ Introduction 14 Results and discussion 16 Identification of CysQ homolog Rv2131c in the 16 M. tuberculosis genome Heterologous expression of Rv2131c in E. coli 16 Defining optimal conditions for PAP phosphatase activity 17 Sensitivity of Rv2131c to alkali metal cations 18 ii Electrospray ionization mass spectrometry assay 19 Steady-state kinetic assays 20 Genetic complementation of an E. coli ΔcysQ mutant 23 Conclusions 24 Materials and methods 24 References 29 Chapter 3. Exploring the role of the 3'-phosphoadenosine-5'-phosphatase CysQ in Mycobacterium tuberculosis sulfur metabolism Introduction 34 Results 35 Disruption of cysQ attenuates M. tuberculosis growth in vitro 35 CysQ does not affect the thiol composition of M. tuberculosis 36 SL1278 and Sulfolipid-1 (SL-1) biosynthesis are inhibited by the 36 disruption of cysQ Discussion 38 Materials and methods 39 References 40 Chapter 4. Biochemical characterization of the acyltransferase PapA3 and its contribution to Polyacyltrehalose biosynthesis in Mycobacterium tuberculosis Introduction 44 Results 45 Genomic analysis of the PAT biosynthetic locus 45 PapA3 is an acyltransferase that esterifies trehalose and 46 trehalose-2-palmitate Trehalose-2-palmitate and trehalose dipalmitate are 48 products of PapA3 iii Trehalose-3-palmitate is not produced by PapA3 51 PapA3 is required for PAT biosynthesis in vivo 53 PAT biosynthesis is independent of SL-1 biosynthesis 53 Discussion 54 Materials and methods 56 References 62 Chapter 5. Discovery of a novel osmoregulatory pathway in Mycobacterium tuberculosis Introduction 66 Results 67 Osmotic stress elicits a significant transcriptional response 67 from M. tuberculosis The anti-anti-sigma factor homolog Rv0516c is highly 68 induced by osmotic stress Rv0516c deletion enhances resistance to high osmolarity and 70 vancomycin PknD phosphorylation regulates Rv0516c expression under 71 osmotic stress Rv0516c influences the transcriptional response to osmotic 73 stress Expression of the virulence factor EspA is induced by 74 osmotic stress Rv0516c and PknD regulate EspA expression and secretion 74 Discussion 75 Materials and methods 78 References 82 iv List of Figures Figure 1-1 Sulfur-containing metabolites from M. tuberculosis 1 Figure 1-2 Sulfate assimilation pathway of M. tuberculosis 2 Figure 1-3 Feedback regulation of RsrA activity and SigR (σR)-mediated 5 transcription by MSH in S. coelicolor Figure 1-4 Modification of the active site cysteine of type I sulfatases by 6 formylglycine-generating enzyme (FGE) Figure 2-1 Sulfate assimilation pathways of E. coli and M. tuberculosis 14 Figure 2-2 Multiple sequence alignment of enzymes from the 16 phosphomonoesterase protein family Figure 2-3 pH, metal, and Mg2+ dependence of Rv2131c PAP 17 phosphatase activity Figure 2-4 Inhibition of Rv2131c PAP phosphatase activity by Li+, Na+, 18 and K+ Figure 2-5 Reaction progress curve for determining the linear range of 20 Rv2131c PAP phosphatase activity Figure 2-6 Sample mass spectra from the kinetic study of Rv2131c with 21 PAP as the substrate Figure 2-7 Steady-state kinetics of Rv2131c with PAP as the substrate 22 Figure 2-8 Complementation of ΔcysQ sulfite auxotrophy in E. coli 23 Figure 3-1 The sulfate assimilation pathway of M. tuberculosis 34 Figure 3-2 Disruption of cysQ attenuates M. tuberculosis growth in vitro 35 Figure 3-3 CysQ does not affect the biosynthesis of mycothiol, hydrogen 36 sulfide, or coenzyme A as determined by the monobromobimane method Figure 3-4 Loss of CysQ decreases biosynthesis of the sulfated 37 glycolipids Sulfolipid-1 and SL1278 v Figure 4-1 PAT and SL-1 share related structures and biosynthetic 44 gene clusters Figure 4-2 Purification of PapA3 from maltose-binding protein (MBP) 46 using immobilized metal affinity chromatography Figure 4-3 PapA3 is an acyltransferase that sequentially palmitoylates 47 trehalose in vitro Figure 4-4 Linear-ion trap MSn of synthetic T2P is consistent with that of 49 the product ion at m/z 615.32 in the reaction of PapA3 with trehalose Figure 4-5 Linear-ion trap MSn of the product ion at m/z 853.54 in the 50 reaction of PapA3 with trehalose Figure 4-6 Linear-ion trap MSn of the diacyl product ions from the reaction 51 of PapA3 with trehalose and T2P is consistent with 2,3-dipalmitoylation of trehalose Figure 4-7 T3P is not a substrate for PapA3 52 Figure 4-8 Linear-ion trap MSn of the monoacyl product ion from the 52 reaction of PapA3 with trehalose is consistent with that of synthetic T2P and not that of synthetic T3P Figure 4-9 PAT biosynthesis requires papA3, but not stf0, in vivo 54 Figure 4-10 Proposed PAT biosynthetic pathway 55 Figure 5-1 Rv0516c is induced by osmotic stress 69 Figure 5-2 Rv0516c enhances sensitivity to osmotic stress and vancomycin 71 Figure 5-3 Rv0516c is an anti-anti-sigma factor homolog whose expression 72 under osmotic stress is mediated by PknD phosphorylation Figure 5-4 Rv0516c deletion enhances transcription of some of the most 73 highly induced genes in the osmotic stress regulon of M. tuberculosis Figure 5-5 Osmotic stress, Rv0516c, and PknD regulate expression of 74 the virulence factor EspA Figure 5-6 Model of the Rv0516c osmoregulatory pathway 76 vi List of Tables Table 1-1 Sulfur metabolism genes from M. tuberculosis induced by 4 various conditions of environmental stress Table 2-1 Michaelis-Menten parameters for Rv2131c 22 Table 4-1 Substrate specificity of PapA3 48 Table 5-1 Genes showing greatest upregulation in M. tuberculosis strain 68 CDC1551 upon exposure to osmotic stress List of Schemes Scheme 4-1 Synthesis of 3-O-palmitoyl-α,α-D-trehalose 57 vii Acknowledgements I have many people to thank for their guidance, support, and encouragement these last five years. My advisor, Dr. Carolyn Bertozzi, has been a stalwart supporter of my career since I joined the lab. She gave me the scientific freedom to explore offbeat ideas, even those truly peripheral to the group’s research, and provided the resources to help guide me down the rabbit hole. Those research experiences have done so much to bolster my confidence as a scientist. I think most people, myself included, confront feelings of insecurity during graduate school, often questioning whether or not they can excel in their chosen field. Carolyn has always made me feel like I can excel as a scientist, and for that I am truly grateful. I admire her positive attitude, her exceptional ability to communicate complex ideas, and knack for mitigating seemingly epic research disasters into minor misfortunes. She has been a tremendous role model, and I have learned so much during my time in her lab. I would like to thank the members of the Bertozzi group, past and present, for their enduring scientific and emotional support over the years. I would especially like to thank the denizens of room 817, Mike Boyce, Jessica Seeliger, and Gaby de Almeida, for always offering a sympathetic ear during episodes of grad school catharsis and for providing such a positive work environment, not to mention a hip soundtrack for the daily grind.
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