Reconstitution and Mechanistic Studies on the Staphylococcal agr Quorum Sensing Circuit

221 Pages·2016·27.2 MB·English

by  Boyuan Wang

Rockefeller University
Digital Commons @ RU
Student Theses and Dissertations
2016
Reconstitution and Mechanistic Studies on the
Staphylococcal agr Quorum Sensing Circuit
Boyuan Wang
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RECONSTITUTION AND MECHANISTIC STUDIES ON THE
STAPHYLOCOCCAL agr QUORUM SENSING CIRCUIT

A Thesis Presented to the Faculty of
The Rockefeller University
in Partial Fulfillment of the Requirements for
the degree of Doctor of Philosophy

by
Boyuan Wang
June 2016
© Copyright by Boyuan Wang 2016
RECONSTITUTION AND MECHANISTIC STUDIES ON THE
STAPHYLOCOCCAL agr QUORUM SENSING CIRCUIT

Boyuan Wang, Ph.D.
The Rockefeller University 2016

Quorum sensing (QS) plays a central role in virulence induction in the commensal
pathogen, Staphylococcus aureus. This bacterium secretes an auto-inducer peptide (AIP),
a small, cyclic peptide containing a thiolactone linkage as an indicator of its population
density, and up-regulates virulence gene expression in response to high extracellular AIP
levels. We have investigated two key biochemical events in S. aureus QS and revealed
several underlying regulatory mechanisms. The first such event, formation of the high-
energy thiolactone in AIP, is unusual in that it occurs directly through proteolysis of the
precursor peptide, AgrD, without free-energy input from ATP hydrolysis. We showed
that this proteolysis is, in line with the thermodynamic prediction, unfavorable and
strongly reversible in vitro. As a consequence, rapid degradation of the concomitantly
released C-terminal fragment of AgrD is required to power efficient AIP production in
vivo. This observation provides a novel connection between protein homeostasis and QS
in S. aureus. The second study focused on the AIP-sensing receptor histidine kinase
(HK), AgrC, whose auto-phosphorylation exhibits several remarkable properties in our
reconstitution system based on nanometer-scale lipid-bilayer discs (nanodiscs).
Activation of this receptor by its native activator, for instance, requires a membrane
environment enriched of anionic lipids mimicking the electrostatic property of the S.
aureus cell membrane. This strong dependence on lipid composition might explain why
homologous QS systems exist only in low-GC Gram-positive bacteria, or Firmicutes,
whose cell membranes are predominantly highly anionic. AgrC also binds to ATP at an
exceptionally weak affinity, likely due to its distinct adenine-binding pocket conserved
only in a small subfamily of HK receptors existing also exclusively in Firmicutes. The
low affinity to the nucleotide cofactor likely enables AgrC to sense the energy condition
of the bacterium and shut down the QS regardless of the population density when energy
starvation drives down the cellular ATP level. Even more intriguing is the plasticity of
AgrC auto-kinase activity when bound to different ligands. This behavior contrasts with
the generally accepted two-state model of HKs. To understand the plasticity of AgrC, we
systematically perturbed the conformation of the AgrC kinase domain using a fusion
protein strategy. We demonstrated that the conformational state of a helical linker
preceding the kinase domain exercises rheostat-like control over the kinase activity.
Using full-length AgrC embedded in nanodiscs, we showed that binding of activator and
inhibitor peptides results in twisting of the linker in different directions. These findings
provide the first view on molecular motions triggered by ligand binding on a membrane-
bound receptor HK. The smooth input-response landscape of the AgrC kinase domain
also sheds new light on the mechanism of HK evolution through domain shuffling.
ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Professor Tom W. Muir, for
providing me the opportunity to work in his lab and, specifically, on the project that I am
most interested in. The progresses I have made in my graduate school would not be
possible without his warm encouragements, his incisive criticisms, his generous support,
as well as his enormous patience and forgiveness.

I would like to thank the members of my Faculty Advisor Committee, Professor Seth A.
Darst and Tom P. Sakmar for their constant support and insightful suggestions
throughout my thesis work. I am grateful to Professor Richard P. Novick, our long-term
collaborator at New York University for offering me access to his lab resources. I would
also like to thank Professor Jon Clardy from Harvard Medical School for attending my
thesis defense as the external examiner.

My thesis research was carried out in close collaboration with several teammates in the
so-called “phosphohistidine-agr” subgroup of the Muir Lab. Peter M. Moyle and Jeffery
G. Johnson have passed down the lab’s reagent collection and intellectual legacy to me
when I get started. Rob Oslund and Jung-Min Kee have collaboratively developed, and
constantly supplied me with, a marvelous phosphohistidine-specific antibody that powers
my study on the AgrC receptor histidine kinase. It is my great pleasure to work with
Aishan Zhao, a talented and dedicated graduate student since my fourth year, and I am
happy to pass down many unfinished businesses to this trustworthy colleague and friend.
I would also like to thank Connie (Xinhui) Wang and Ashley Wang for spending their
undergraduate research with me on an agr-related problem. Stephen (Qian) Xie, a
postdoc who joined the team in 2014, also deserves recognition for his aspiration to
tackle several long-standing structural problems.

The Muir Lab is a fantastic place to do research in that everyone is willing to share
his/her knowledge and skills. I am grateful to Neel H. Shah and Manuel M. Müller for
their constant interest in listening to my preliminary research ideas and providing
constructive feedbacks. I have also consulted Miquel Vila-Perelló about intein
technologies, Beat Fierz about fluorescent spectrophotometry, Disan S. Davis about
membrane protein handling, Zack Z. Brown and Zhihua Liu about synthetic chemistry,
Glen P. Liszczak about X-ray crystallography, Galia Debelouchina about NMR
techniques and Yael David about general genetic questions.

I would like to thank the Dean’s Office at The Rockefeller University and Sarah Porter
and Megan Krause in the Department of Chemistry at Princeton University for their
administrative assistance. I am also indebted to the Muir Lab mangers, Maya Bar-Dagan
at Rockefeller and Lenka Bittova at Princeton, who have maintained all instruments and
ordered all supplies necessary for my research.

As an international student, it is a long process to acquire necessary English skills and
adapt to the lifestyle in United States. Special acknowledgement goes to Rob Oslund,
Manuel M. Müller, Galia Debelouchina and Professor Tom Muir for proofreading my

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written English and providing valuable suggestions. I would also like to thank Adam
Stevens for familiarizing me with baseball and American football and Champak
Chatterjee and Uyen T.T. Nguyen for sharing with me their opinions on American and/or
European cultures.

I am fortunate to have two friends, Bonan Li and Zhixing Chen living close to me during
most of my graduate school years. Interacting with them is full of fun and inspirations. I
am indebted to my parents for their unconditional love and support and to my cousins for
taking care of my parents during my years of absence. Finally, I would like to thank my
wife, Shasha, for organizing a comfortable home and being the communication specialist
of our family.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS v
LIST OF FIGURES ix
LIST OF TABLES xiii
Chapter One: Introduction 1
1.1 Quorum sensing in Staphylococcus aureus 1
1.1.1 Introduction to quorum sensing 1
1.1.2 Discovery of the agr locus 5
1.1.3 Basic architecture of the signaling circuit 8
1.1.4 Interspecies variation of agr 10
1.2 Biochemistry of the agr autoinduction 12
1.2.1 Translation of Agr proteins 13
1.2.2 Formation of the AIP thiolactone 15
1.2.3 Translocation and maturation of AIP 19
1.2.4 AIP-AgrC interaction 21
1.2.5 AgrA phosphorylation and transcription activation 27
1.3 Reagent development for the manipulation of agr 29
1.3.1 AIP scavenging in host defense 30
1.3.2 AgrC-targeting reagents 32
1.3.3 AgrA-targeting reagents 34
1.3.4 AIP-sequestration reagents 35
1.4 Summary: specific aims and significance 36

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Chapter Two: Bioinformatic Analysis of the S. aureus AgrC 39
2.1 Identification of close AgrC homologs from genomic databases 41
2.2 Sequence analysis of the HPK10 subfamily 43
2.3 Summary and discussions 46

Chapter Three: Reconstitution of Biochemical Events Related to AgrC 49
3.1 Functional reconstitution of AgrC to nanodiscs 49
3.2 Recapitulation of AIP-dependent regulation of AgrC kinase activity 55
3.3 The AgrC-I-AgrA phospho-relay and AgrA dephosphorylation 58
3.4 Physicochemical parameters of the AIP-I-AgrC-I interaction 61
3.5 Nucleotide specificity and kinetics of AgrC-I auto-phosphorylation 64
3.6 Summary and discussions 68

Chapter Four: Mechanisms by which AgrC-I Responds to Signals 72
4.1 Perturbing the conformation of the AgrC-I HK domain 73
4.2 Conformation-activity landscape of the AgrC-I HK domain and the molecular motion
induced in full-length AgrC-I by AIP-I or AIP-II binding 77
4.3 A constitutive mutation, R238H, modifies the conformation-activity landscape 82

4.4 Reconstitution of the AIP-II-revertible constitutive mutants 88
4.5 Summary and discussions 90
4.5.1 Auto-kinase regulation of AgrC-I 90
4.5.2 Trans-kinase regulation of AgrC-I 95
4.5.3 Mechanisms of constitutive activity 96

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Chapter Five: Key Driving Forces That Power AIP Thiolactone Formation 97
5.1 Estimation of the free energy cost associated with thiolactone formation 98
5.2 Expression and purification of recombinant AgrB and AgrD constructs 102
5.3 AgrD(1-32)-thiolactone is the major product of the AgrB-catalyzed proteolysis

of AgrD 105
5.4 AgrD processing by AgrB is a reversible process 107
5.5 AgrD(1-32)-thiolactone is stabilized by phospholipids 112
5.6 AgrD processing by AgrB is a reversible process in vivo 117
5.7 Summary and Discussion 119
5.7.1 Factors that facilitate AgrD proteolytic cyclization 119
5.7.2 Physiological implications of the ∆G0’ of thiolactone formation 120

Chapter Six: Outlook 124
6.1 The signal-response property of the AgrC-I HK domain 124
6.1.1 The trans-kinase activity and the pHis stability in GCN4-AgrC-I

chimeras 126
6.1.2 Introduction of scissoring motions to the AgrC-I interdomain linker

helices 128
6.1.3 Is the interdomain linker pair an analog-to-digital converter? 130
6.2 Degradation of the AgrD C-terminal cleavage fragment 132
6.3 Detection of AIP thiolactones from other Firmicutes species 134

Chapter Seven: Methods 137
7.1 Materials and Equipments 137
7.1.1 General Materials 137

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