The Physiological Significance of (p)ppGpp in Enterococcus faecalis by Anthony Ottavio Gaca Submitted in the Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Supervised by Professor José Lemos Department of Immunology, Microbiology, and Virology School of Medicine and Dentistry University of Rochester Rochester, NY ii BIOGRAPHICAL SKETCH Academic History Anthony graduated from Michigan State University in with a Bachelors of Science degree in Microbiology. While attending MSU he worked as a research assistant in the laboratory of Dr. James Tiedje identifying and characterizing novel bacterial isolates able to utilize phenolic pollutants as carbon sources. In the fall of 2008, Anthony was admitted into the IMV PhD program at the University of Rochester. In 2009, he joined the laboratory of Dr. José Lemos to begin his thesis work studying stress responses in the nosocomial pathogen Enterococcus faecalis. In 2011, Anthony was awarded the Master of Science degree from the University of Rochester after successfully completing his qualifiying examiniation. Publications Gaca AO, Jessica JK, Abranches J, Lemos JA. Basal levels of (p)ppGpp in Enterocuccus faecalis: The magic beyond the stringent response. mBio 4(5): e00646-13, 2013. Abranches J, Tijerina P, Avilés-Reyes A, Gaca AO, Kajfasz JK, Lemos JA. The cell wall-targeting antibiotic stimulon of Enterococcus faecalis. PLoS ONE 8(6): e64875, 2013. Gaca AO, Abranches J, Kajfasz JK, Lemos JA. Global Transcriptional Analysis of the stringent response in Enterococcus faecalis. Microbiology 158(8):1994-2004, 2012. Kajfasz JK, Mendoza JE, Gaca AO, Koselny KA, Giambiagi-deMarval M, Wellington M, Abranches J, Lemos JA. The Spx regulator modulates stress responses and virulence in Enterococcus faecalis. Infect Immun 80(7): 2265-75, 2012. de Oliveira NE, Abranches J, Gaca AO, Laport MS, Damaso CR, Bastos Mdo C, Lemos JA, Giambiagi-deMarval. clpB, a class III heat-shock gene regulated by CtsR, is involved in thermotolerance and virulence of Enterococcus faecalis. Microbiology 157(3):656-65, 2011. iii ABSTRACT The stringent response (SR) is a conserved stress response in bacteria that shifts cells from a growth mode to a survival mode. In the opportunistic pathogen Enterococcus faecalis, (p)ppGpp production, the SR effector molecule, is controlled by the bifunctional synthase/hydrolase Rsh and the monofunctional synthase RelQ. Previous characterization of strains lacking rsh, relQ, or both in E. faecalis revealed that Rsh is responsible for activation of the SR and that alterations in (p)ppGpp production have a major impact on the organism’s stress survival and virulence. In this study, transcriptional profiling of ∆relQ/∆rsh mutants under stringent and non-stringent conditions revealed that Rsh is responsible for the repression of genes involved in macromolecular biosynthesis and activation of stress adaptation pathways, but also showed that RelQ was required for full and timely SR induction. A subset of stress-associated genes regulated by (p)ppGpp was shown to specifically interact with the global metabolic regulator CodY, integrating (p)ppGpp into a larger and complex stress response network. Notably, the transcriptional profile of the (p)ppGpp0 (∆rsh∆relQ) strain and physiological assays revealed that a complete absence of (p)ppGpp leads to severe dysregulation of central carbon metabolism and guanosine homeostasis. Surprisingly, the ∆rsh strain, which also lacks the ability to mount the stringent response but has low levels of (p)ppGpp, was able to maintain cell homeostasis. Thus, while both ∆rsh and (p)ppGpp0 strains cannot use (p)ppGpp to respond to stresses, fitness of the (p)ppGpp0 strain appears to be further impaired by an unbalanced metabolism. In fact, we showed that the previously described association of (p)ppGpp with antibiotic survival does not relate to the SR but rather to basal (p)ppGpp pools. These findings indicate that (p)ppGpp levels well below those achieved during the stringent response contribute to cell homeostasis. Finally, enzymatic characterization of RelQ and Rsh enzymes revealed that both are capable of synthesizing pGpp, a potentially new regulatory molecule. Collectively, this study further confirmed the significance of (p)ppGpp to E. faecalis pathophysiology, identified a novel and complex stress response network iv governed by (p)ppGpp, and highlighted the critical but still underappreciated role of basal (p)ppGpp levels in cell homeostasis and stress tolerance. v CONTRIBUTORS AND FUNDING SOURCES This work was supervised by a dissertation committee consisting of Professors José Lemos (advisor), Scott Butler, Martin Pavelka, and Steven Gill of the Department of Immunology, Microbiology, and Virology and Professor Joshua Munger of the Department of Biochemistry and Biophysics. The microarray analyses in Appendices I, II, and III were conducted with the assistance of Jessica Kajfasz and Jacqueline Abranches of the Department of Microbiology and Immunology. Antibiotic survival assays shown in Figure 20 were performed by James Miller of Department of Microbiology and Immunology. Lastly, enzyme inhibition assays shown in Figure 19 were conducted by Kuanqing Liu under the supervision of Jue D. Wang of the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA. All other work conducted for the dissertation was completed independently by the student. Anthony’s graduate studies were supported by the NIH/NIDCR Training Program in Oral Sciences, grant T90DE021985. vi TABLE OF CONTENTS Biographical Sketch ii Abstract iii Contributors and Funding Sources v Table of Contents vi List of Tables ix List of Figures x Glossary xii Chapter 1: Introduction 1-33 1.1 The enterococci 2 1.2 The stringent response 4 1.3 (p)ppGpp metabolism: Structure and regulation of RSH 5 enzymes 1.4 Pleiotropic effects of the stringent response on cellular 12 physiology 1.5 Control of cellular metabolism by (p)ppGpp contributes to 21 antibiotic tolerance 1.6 Other Gram-positive global metabolic regulators 23 1.7 The intersection of global nutrient sensing regulators in stress 28 tolerance and virulence 1.8 The stringent response mediates stress tolerance and 31 virulence in E. faecalis Chapter 2: Materials and Methods 34-51 2.1 Bacterial strains and growth conditions 35 2.2 CodY deletion mutant construction 38 2.3 E. faecalis-macrophage co-culture 40 vii 2.4 Galleria mellonella infection 40 2.5 RNA Extraction 41 2.6 Microarray experiments 41 2.7 Microarray data accession number 42 2.8 Real-time quantitative PCR 43 2.9 Detection of (p)ppGpp accumulation patterns 46 2.10 Detection of intracellular guanine nucleotides 46 2.11 Measurement of intracellular pyruvate and fermentation end 46 products 2.12 H O measurements 47 2 2 2.13 Purification and enzymatic assays of HprT and Gmk 48 2.14 Antibiotic time-kill kinetics 49 2.15 Overexpression and purification of 6x His-tagged Rsh, RelQ, 59 and CodY 2.16 In vitro synthesis of (p)ppGpp by RelQ and Rsh 50 2.17 CodY gel shift assays 50 Chapter 3: Global transcriptional analysis of the stringent response 52-74 in Enterococcus faecalis 3.1 Abstract 53 3.2 Introduction 53 3.3 Results 56 3.4 Discussion 72 Chapter 4: CodY and (p)ppGpp control stress tolerance and virulence 75-94 in Enterococcus faecalis 4.1 Abstract 76 4.2 Introduction 76 viii 4.3 Results 80 4.4 Discussion 89 Chapter 5: Basal levels of (p)ppGpp regulate cell homeostasis in 95-120 Enterococcus faecalis 5.1 Abstract 96 5.2 Introduction 96 5.3 Results 99 5.4 Discussion 114 Chapter 6: Characterization of RelQ enzymatic activity from 121-133 Enterococcus faecalis 6.1 Abstract 122 6.2 Introduction 122 6.3 Results 125 6.4 Discussion 131 Chapter 7: Perspectives and Future Directions 134-149 7.1 Summary 135 7.2 Perspectives and Future Directions 136 7.3 Closing Remarks 149 References 150-169 Appendix I: Microarray data for mupirocin treated stringent (OG1RF and 170-214 ∆relQ) strains Appendix II: Microarray data for mupirocin treated relaxed (∆rsh and 215-233 ∆rsh∆relQ) strains Appendix III Microarray data for control, untreated cells 234-256 ix LIST OF TABLES Table 1 Bacterial strains and plasmids used in this study 36 Table 2 Primers for bacterial strain construction and protein overexpression 39 Table 3 qRT-PCR primers for confirmation of microarray analysis 44 Table 4 Primers for the synthesis of probes used in gel shift assays 51 Table 5 Real-time PCR validation of microarray trends 62 Table 6 Amino acid biosynthesis, transport, stress, and virulence-associated 70 genes positively regulated by (p)ppGpp in OG1RF and/or ∆relQ strains after mupirocin treatment Table 7 Virulence-related and stress genes regulated by (p)ppGpp 81 containing potential CodY regulatory sequence(s) Table 8 Quantitative real-time PCR validation of microarray trends 105 x LIST OF FIGURES Chapter 1: Figure 1 (p)ppGpp structure, synthesis, and hydrolysis 6 Figure 2 Domain organization of (p)ppGpp metabolic enzymes 10 Figure 3 (p)ppGpp synthesis in Gram-positive and Gram-negative bacteria 11 Figure 4 The intersection of the (p)ppGpp, CcpA, and CodY 30 Chapter 3: Figure 5 The ∆relA∆relQ mutant showed reduced survival in macrophage and 57 reduced virulence in Galleria mellonella Figure 6 Venn diagrams of differentially expressed genes in OG1RF and 59 ∆rsh/∆rel strains treated with mupirocin for 15 min and 30 min compared to control samples of each strain. Figure 7 Heat map of genes involved in transcription, translation, and DNA 61 replication repressed by mupirocin treatment in OG1RF and ∆relQ Figure 8 Time-course accumulation of (p)ppGpp in E. faecalis after mupirocin 64 treatment Figure 9 Genes of the GTP biosynthetic pathway repressed by (p)ppGpp 66 Chapter 4: Figure 10 Gel shift analysis of CodY binding to putative stress and virulence 83 gene targets Figure 11 Restoration of growth in the absence of branch chain amino acids by 85 deletion codY in the (p)ppGpp0 strain Figure 12 Reintroduction of codY into the ∆rsh∆relQ∆codY restores attenuated 86 growth in the absence of isoleucine Figure 13 Deletion of codY in the (p)ppGpp0 strain fully restores virulence and 87 enhances intracellular survival in murine macrophage