The Physiological Significance of (p)ppGpp in Enterococcus faecalis by Anthony Ottavio Gaca ...

268 Pages·2013·7.59 MB·English

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
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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.
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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
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governed by (p)ppGpp, and highlighted the critical but still underappreciated role of basal
(p)ppGpp levels in cell homeostasis and stress tolerance.
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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.
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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
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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
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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
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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